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PowerPoint Presentation: THEORY AND INSTRUMENTATION OF INFRARED SPECTROSCOPY By T.SUJITH, Y11MPH476 1

PowerPoint Presentation: “Spectroscopy is an instrumentally aided study of the interactions between matter (sample being analyzed) and energy (any portion of the electromagnetic spectrum, EMS)” Introduction: 2 Energy of molecule = Electronic energy + Vibrational energy + Rotational energy Infrared spectroscopy is concerned with the study of absorption of infrared radiation, which results in vibrational transitions It is also called as Vibrational spectroscopy IR spectra mainly used in structure elucidation to determine the functional groups

PowerPoint Presentation: 3 Electro Magnetic Radiation:

PowerPoint Presentation: 4 Molecular effects:

PowerPoint Presentation: 5 The Range of Infrared Radiation: The IR radiation refers broadly to that region of electromagnetic spectrum which lies between the visible and microwave regions IR region may be divided into four sections: The photographic region: This ranges from visible to 1.2µ The Very Near Infrared region: Also known as overtone region and ranges from 1.2-2.5µ The Near Infrared region: This is also known as vibration region and ranges from 2.5-25µ The Far Infrared region: This is known as the rotation region. This ranges from 25-400µ

PowerPoint Presentation: 6 Principle: Molecules are made up of atoms linked by chemical bonds. The movement of atoms and chemical bonds like spring and balls (vibration) This characteristic vibrations are called Natural frequency of vibration When energy in the form of infrared radiation is applied and when, Applied infrared frequency= Natural frequency of vibration Absorption of IR radiation takes place and a peak is observed

PowerPoint Presentation: 7 What happens when a sample absorbs IR energy? stretching and bending of bonds (typically covalent bonds) E vibration increases momentarily - O - H IR ( 3500 cm - 1 ) - O — H When an analytical chemist speaks of infrared spectroscopy, he usually means the range from 2.5-25µ

This range gives important information of the vibrations of the molecules, and hence the structure of the molecule

PowerPoint Presentation: 8 Theory of Infrared Absorption Spectroscopy Correct Wavelength of Radiation: A molecule absorbs radiation when only Natural frequency of vibration = Frequency of incident radiation Electric Dipole: i)A molecule can only absorb radiation when its absorption causes a change in its electric dipole. ii)A molecule is said to have electric dipole when there is a slight positive and a slight negative electric change on its component atoms

PowerPoint Presentation: 9 MOLECULAR VIBRATIONS

PowerPoint Presentation: 10 Modes of vibrations: Stretching: change in bond distance. Occurs at higher energy: 4000-1250 cm 1 H 2 O

PowerPoint Presentation: 11 Bending: change in bond angle. Occurs at lower energy: 1400-666 cm 1 -CH 2 -

PowerPoint Presentation: Fundamental Vibrations: A molecule has as many as degrees of freedom as the total degree of freedom of its individual atoms. i)Each atom has 3 degree of freedom (x,y,z) ii)A molecule of n atoms therefore has 3n degrees of freedom. ***For Non linear molecules (e.g. H 2 O) Vibrational degrees of freedom or Fundamental Vibrations = 3n – 6 Symmetrical Stretching Asymmetrical Stretching Scissoring 12

PowerPoint Presentation: ***For linear molecule (e.g. CO 2 ) : Vibrational degrees of freedom or Fundamental Vibrations = 3n – 5 Symmetrical Stretching Asymmetrical Stretching Scissoring (bending out of the plane of the paper) Scissoring (bending in the plane of the paper) 13

PowerPoint Presentation: 14 HOOKE’S LAW: It approximates stretching frequency In this approximation, two atoms and the connecting bond are treated as a simple harmonic oscillator According to Hooke’s law, the frequency of the vibration of the spring is related to the mass and the force constant of the spring k, by the following formula, Where, k is the free constant m is the mass v is the frequency of the vibration

PowerPoint Presentation: How does the mass influence the vibration? H 2 I 2 MM =2 g/mole MM =254 g/mole The greater the mass - the lower the wave number (ύ) 15

PowerPoint Presentation: 16 Stretching Frequencies a)Frequency decreases with increasing atomic mass b)Frequency increases with increasing bond energy

IR Correlation Diagram: IR Correlation Diagram Transmittance (%) 100 80 60 40 20 0 4000 3500 3000 2500 2000 1500 1000 2.5 3.0 4.0 5.0 6.0 10.0 Frequency (cm -1 ) Region I 3600-2700 cm -1 Region II 1800-1600 cm -1 / Wavelength (microns, mm) O-H N-H C-H bond stretching alcohols phenols carboxylic acids amines amides alkynes alkenes alkanes C=O acid chlorides anhydrides esters ketones aldehydes carboxylic acids amides Fingerprint Region (below 1500 cm -1 ) C-H =C-H -C-H 17

PowerPoint Presentation: 18 IR SPECTRUM i)No two molecules will give exactly the same IR spectrum (except enantiomers) ii)Simple stretching: 1600-3500 cm -1 iii)Complex vibrations: 400-1400 cm -1 , called the “fingerprint region”

PowerPoint Presentation: 19 Instrumentation The main parts of an IR spectrometer are as follows: IR radiation sources Monochromators Sample cells and sampling of substances Detectors

PowerPoint Presentation: 20 IR radiation sources: The radiation source must emit IR radiation which must be Intense enough for detection Steady Extended over the desired wavelengths Various popular sources of IR radiation are: Incandescent Lamp: Has low spectral emissivity (b ) Nernst Glower: Glower is composed of rare earth oxides such as zirconia, yttria, thoria It is non conducting at room temperature Heated by external means to 1000-1800ºC to bring it into conducting state Provides maximum radiation at about 7100 cm 1 Disadvantages: Emits IR radiation over wide wavelength range Frequent ˉmechanical failure Its energy also concentrate in the visible and near IR regions of the spectrum

PowerPoint Presentation: 21 Parts of Nernst Glower Globar source Mercury Arc

PowerPoint Presentation: 22 (c) Globar Source: Contains a rod of sintered silicon carbide, when it is heated to 1300-1700ºC emits radiation in IR region at 5200 cm 1 Unlike the Nernst glower, it is self ˉ

starting As its temperature coefficient is positive, it can be conveniently controlled with a variable transformer Disadvantage: Less intense source than Nernst glower (d) Mercury Arc: In far IR region special high pressure mercury arc lamps are used Beckman devised the quartz mercury lamps At the shorter wavelengths, the heated quartz envelope emits the radiation whereas at longer wavelengths the mercury plasma provides radiation through the quartz

PowerPoint Presentation: 23 Monochromators: (a) Prism Monochromator: Must be constructed of materials which transmit in the infrared Sodium chloride is most common prism salt Two types i) Single pass ii) Double pass Single pass prism monochromator

PowerPoint Presentation: 24 The double pass monochromator produces more resolution than the monochromator in the radiation, before it finally passer on to the detector For 4000-650cm 1 region in ˉboth mono and double pass monochromators NaCl prisms are used Prism of lithium fluoride or calcium fluoride give more resolution in the region where the significant stretching vibrations are located (b) Grating monochromator: High dispersion can be achieved Gratings offer linear dispersion and may be constructed for a wide variety of materials The grating is essentially a series of parallel straight lines cut into a plane surface Dispersion by grating follows the law of diffraction n λ = d(sin i ± sin θ ) Where, n is the order, λ the wavelength of the radiation, d the distance between the grooves, i the angle of incidence of beam of IR radiation and θ the angle of dispersion of light of a particular wavelength

PowerPoint Presentation: 25 Grating monochromator

PowerPoint Presentation: 26 Sample cells and sampling of substances: The only common point to the sampling of different phages is that the material containing the sample must be transparent to IR radiation This condition restricts our selection to only certain salts Eg: NaCl, KBr, ThBr etc Sampling of solids i) Solids run in Solution: Solids may also be dissolved in a non-aqueous solvent Provided - There is no chemical reaction between sample and solvent - Solvent does not absorb in the studied range A drop of solution is placed on an alkali metal disk and the solvent allowed to evaporate, leaving a thin film of the sample Or the entire solution is placed in a liquid sample cell Limitation: There is no single solvent which is transparent throughout IR region

PowerPoint Presentation: 27 ii) Solid Films: If a solid is amorphous in nature the sample is deposited on the surface of a KBr or NaCl cell by evaporation of a solution of the solid Useful for rapid qualitative analysis but becomes useless for carrying out quantitative analysis iii) Mull Technique: The finely ground solid sample is mixed with Nujol(mineral oil) to make a thick paste which is then made to spread

between IR transmitting windows Limitation: Nujol has the absorption maxima at 2915, 1462, 1376, and 719 cm 1 , absorption bands of the sample happen to coincide with the absorption bands of Nujol mull ˉThis method is good for qualitative analysis but not for quantitative analysis iv) Pressed Pellet Technique: A small amount of finely ground solid sample intimately mixed with about 100 times its weight of KBr The finely ground mixture is then pressed under very high pressure in a press to form a small pellet The resulting pellet is transparent to IR radiation

PowerPoint Presentation: 28 Advantages over Mull Technique: Use of KBr eliminates the problem of bands which appear in the spectrum due to the mulling agent KBr pellets can be stored for long periods of time As the concentration of sample can be suitably adjusted in the pellets, it can be used for quantitative analysis The resolution of the spectrum in the KBr is superior to that obtained with mulls Disadvantages: It always has a band at 3450 cm 1 , from the OH group of moisture present in the ˉsample High pressure involved during the formation of pellets may bring about polymorphic changes in crystallinity in the samples This method is not successful for some polymers which are difficult to grind with KBr

PowerPoint Presentation: 29 b) Sampling of liquids: samples that are liquids at room temperature are usually put frequently with no preparation, into rectangular cells made of NaCl, KBr or ThBr and their IR spectra are obtained directly c) Sampling of gases: The gas absorption cell is similar to the cell for liquid samples To compensate for the small number of molecules of a sample that is contained in a gas, the cells are larger Multiple reflections can be used to make the effective path length Detectors : Thermal detectors are better choice except in the near infrared where photoconductivity cells are generally used Requirements for thermal detectors i) Short responsive time ii) Absorbed heat must be lost rapidly(As heat transfer is a slow process this is difficult requirement)

PowerPoint Presentation: 30 Various types of detectors used in IR spectroscopy: Bolometer : A bolometer is based upon the fact tat the electrical resistance of a metal increases approximately 0.4% for every Celsius increase of temperature Usually consists of a thin metal conductor When radiation falls on this conductor, its temperature changes As the resistance of a metallic conductor changes with temperature, the degree of change in resistance is regarded as a measure of the amount of radiation that has fallen on the bolometer The response time for a bolometer is 4mSec

PowerPoint Presentation: 31 b) Thermocouple: The thermocouple detector is based upon the fact that an electrical current will flow when two dissimilar metal wires are connected together at both ends and a temperature differential exists between two ends The end exposed to the infrared radiation is called the “hot junction”. In order to increase the energy gathering efficiency, it is usually a “black body” The

other connection, “cold junction” is thermally insulated and carefully screened from stray light A thermocouple is closed in an evacuated steel casing with a KBr window to avoid losses of energy by convection When hot junction is exposed to IR radiation which increases the temperature of the junction The temperature difference between the two junctions generates potential difference which depends on how much IR radiation falls on the hot junction The response time a thermocouple is about 60mSec

PowerPoint Presentation: 32 c) Golay cell: It consists of a small metal cylinder filled wit xenon gas. It is sealed with blackened metal plate at one end and other end by a flexible metalized diaphragm When radiation fall on the metal plate, it heats the gas in the cylinder which causes it to expand The resulting pressure increases in the gas deforms to metalized diaphragm Light from a lamp is made to fall on the diaphragm which reflects the light on to a photocell Motion of the diaphragm changes the output of cell The signal seen by the phototube is modulated in accordance with the power of the radiant beam incident on the golay cell Advantages: -Useful wavelength range is very wide - Response time is much faster than bolometer or thermocouple Disadvantage: More expensive and bulky

PowerPoint Presentation: 33 d) Photoconductivity cell : Non thermal detector of greater sensitivity It consists of a thin layer of lead sulphide or lead telluride supported on glass and enclosed into an evacuated glass envelope When IR radiation is focused on lead sulphide, its conductance increases ans causes more current to flow Response time is 0.5mSec Disadvantage: When operated at room temperature, it has a very restricted range(limited to near infrared ) *** The range can be broadened by drastic cooling e) Thermistors: Made of fused mixture of metal oxides As the temperature of the mixture increases, its electrical resistance decreases(As opposed to the bolometer) Response time is slow

PowerPoint Presentation: 34 Speed and accuracy of analysis Small sample requirement IR spectra are information rich; the peak position, intensities, widths, and shapes in a spectrum all provide useful information Can be applied to solids, liquids, gases and polymers Advantages of IR spectroscopy: Can’t be applied to single atomic entities as they don’t contain chemical bonds and hence don’t absorb IR radiation Nobel gases such as helium and argon don’t have infrared spectra Disadvantages:

PowerPoint Presentation: 35 Difficult to be applied to samples of complex composition because the spectrum will also be complex to be interpreted and it will be very hard to know which infrared bands are due to which molecules Monoatomic ions such as Pb +2 dissolved in water aren’t chemically bonded to anything and don’t have an infrared spectrum Aqueous solutions are difficult to analyze using infrared spectroscopy

PowerPoint Presentation: 36 Applications: Identification of functional group and structure elucidation Identification of drug substance Identifying the impurities in a drug sample Study of hydrogen bonding Study of polymers Ratio of cis-trans isomers in a mixture of compounds Quantitative analysis

Dealing with mixtures:: Dealing with mixtures: Library searching: In this technique, the mixture spectrum is mathematically compared to a collection of known spectra kept in a library. A number called the hit quality index (HQI) describes how similar or different the spectra are to each other. It takes place by one of two ways: 1. Spectral subtraction: This process can remove the bands of unwanted components from a spectrum. Subtraction involves taking the spectrum of a mixture and subtracting from it the spectrum of a pure compound that is present in the mixture. 37

PowerPoint Presentation: In this part we make use of a reference sample in addition to the unknown sample we are working on We obtain the spectra of the sample and the reference and then compare them Plot the two spectra using the same scale and then compare the two spectra with each others Properly performing identities: 38

PowerPoint Presentation: 39 Fourier Transform system(FTIR)

PowerPoint Presentation: 40 Dispersive IR Fourier Transform IR Many moving parts; results in mechanical slippage Only mirror moves Calibration against reference spectra is required Use of laser provides high frequency accuracy Stray light results in spurious results Stray light does not effect, since all signals are modulated A small amount of IR beam may be allowed to pass through slits A much larger beam may be used Only radiation of a narrow frequency range falls on the detector All frequencies of radiation fall on the detector Slow scan speeds Rapid scan speeds Sample is subjected to thermal effects Sample is not subjected to thermal effects Any emission of IR radiation by the sample will fall on the detector Any emission of IR radiation by the sample will not be detected Comparison of Fourier Transform and Dispersive IR

PowerPoint Presentation: 41 Conclusion Infrared spectroscopy is one of the most powerful analytical techniques which offers the possibility of chemical identification One of the most important advantage of infrared spectroscopy over other usual methods of structural analysis is that it provides useful information about the structure of molecule quickly, without tiresome evaluation method Infrared spectroscopy itself established as a valuable tool for determination of organic, and to a lesser extent, inorganic, structure IR spectrum of a chemical substance is a fingerprint for its identification

PRINCIPLES OF IR SPETROSCOPY: PRINCIPLES OF IR SPETROSCOPY PRESENTED BY, Dhanya KT, 1 st year M Pharm , Dept: of pharmaceutics, Nehru college of pharmacy, Pambady. Date of presentation:13-7-2011 1

Contents : Contents Introduction to IR spectroscopy Range of IR spectroscopy radiation Principles of IR spectroscopy Modes of vibrations 3n Degrees of freedom Factors influencing vibrational frequencies References 2

INFRARED SPECTROPHOTOMETER: INFRARED SPECTROPHOTOMETER 3

Introduction : Introduction IR spectroscopy ( vibrational spectroscopy) is connected with the study of of IR radiation, which result the vibrational transitions. IR spectra is mainly used in structural elucidation to determine the functional group 4

Slide 5: Energy of molecule = Electronic energy + Vibrational energy + Rotational energy IR spectroscopy is the study focused on the change in the vibration of molecule and absorption of energy due to vibrations. 5

Slide 6: Atoms or groups of atoms are connected by bonds. These are non rigid in nature. Due to continuous motion of the molecule they maintain vibration with frequency. Applied frequency = natural frequency of vibration. The absorption of IR take place and a peak is observed IR spectrum of a chemical substance is a finger print for its identification. 6 In any molecule,

Band positions in IR : Band positions in IR Band positions in IR may be expressed conveniently by wave number( ν ), whose unit is cm-1. The relation between wave number( ν ) wave length( λ ) and frequency ( ν ) is as follows ν = C/ λ ν / (cm-1)= ν /c = 1/ λ C= velocity of light 7

Slide 8: Band intensities of IR spectrum may be expressed either as transmittance or absorbences . Transmittance (T): It is defined as the ratio of radient power transmitted by a sample to the radient power incident on the sample. Absorbence (A): Absorbance is defined as the logaritham to the base10 of the reciprocal of the transmittance. Ie ; A= log 10 I/T 8

Range of IR spectroscopy region : Range of IR spectroscopy region The IR radiation refers broadly to that region of electromagnetic spectrum which lies between the visible and microwave region. visible NEAR Infrared MID FAR Microwave λ cm ν cm-1 Energy 7.8x10-5 to 3x10-4 12820 to 4000 10-37 Kcal/mole 3x10-4 to 3x10-3 4000 to 400 1-10 Kcal/mole 3x10-3 to 3x10-2 400 to 33 0.1-1 Kcal/mole 9

Slide 10: 10

Slide 11: The infrared region constitutes 3 parts a) The near IR (12,820-4000cm-1) b) The middle (4000-400cm-1) c) The far IR (400-33cm-1) most of the analytical applications are confined to the middle IR region because absorption of organic molecules are high in this region. Wave number is mostly used measure in IR absorption because wave numbers are larger values & easy to handle than wave length which are measured in µm. 11

Theory of IR: Theory of IR For a molecule to absorb IR radiation, it has to fulfill certain requirements : Correct wavelength of radiation : A molecule to absorb IR radiation the natural frequency of vibrations of some part of a molecule is the same as the frequency of incident radiation. Eg : Hcl 12

Slide 13: Electric dipol A molecule can only absorb IR radiation when its absorption cause a change in its electric dipol . A molecule is said to have an electric dipol when there is a slight positive and a slight negative electric charge on its component of atom. Eg :- Ethylene, Bromoethylene 13

MOLECULAR VIBRATIONS: MOLECULAR VIBRATIONS There are 3 types of vibrations. Fundamental vibrations 2. Overtone vibrations 3. Combination vibrations 14

Slide 15: There are 2 types of fundamental vibrations : Stretching vibrations Bending vibrations 1)Stretching vibrations: in this bond length is increased or decreased periodically. They are of 2 types symmetrical streching asymmetrical streching 15

Slide 16: a) symmetrical stretching : 2 bonds increase or decrease in length symmetrically. b) Asymmetrical stretching : in this one bond length is increased and other is decreased. 16

Slide 17: 2)Bending vibrations: These are also called as deformations. In this bond angle is altered. These are of 2 types In plane bending → scissoring, rocking Out plane bending → wagging, twisting 17

Slide 18: Scissoring: This is an in plane bending. In this bond angles are decreased,2 atoms approach each other. Rocking: In which bond angle is maintained ,but the bond move with in the plane . 18

Slide 19: Wagging: It is an out of plane bending. In this 2 atoms move to one side of the plane. They move up and down the plane. Twisting: In this one atom moves above the plane and the other atom moves below the plane. 19

Slide 20: Overtone vibrations: when a molecule absorbs a quantum of energy( E), corresponding to the vibration's frequency (ν) according to the relation E = h ν The overtone band is arise due to the absorption of a photone that leads to doubly excited vibrational state. 20

Slide 21: Combination vibrations Combination modes involve a simultaneous increase in the vibrational quantum number of two or more vibrational modes by the absorption of a single photon However, in order to “combine”, the vibrations must a) involve the same functional group, and b) have the same symmetry properties 21

NUMBER OF VIBRATIONAL MODES (3n degrees of freedom) : NUMBER OF VIBRATIONAL MODES (3n degrees of freedom) 22 x y z (usually the axis of highest symmetry)

Slide 23: A molecule can vibrate in many ways, and each way is called a vibrational mode. If a molecule contains ‘N’ atoms, total number of vibrational modes For linear molecule it is (3N-5) For non linear molecule it is (3N-6) 23

The Distribution of Degrees of Freedom for Polyatomic Molecules : The Distribution of Degrees of Freedom for Polyatomic Molecules Degrees of Freedom Linear Molecules Non-Linear Molecules Translational 3 3 Rotational 2 3 Vibrational 3n-5 3n-6 Total 3n 3n 24

Slide 25: Eg : 1. H 2 O, a non-linear molecule, will have 3 × 3 – 6 = 3 degrees of vibrational freedom or modes. 2. CO 2 ,linear molecule, will have 3× 3-5=4 degrees of vibrational modes. 25

Slide 26: Stretching vibrations = the number of bonds in the molecule the no of Bending vibrations = total number of vibrations - stretching vibrations = ( 3 N – 5 for a linear molecule ) - the no of stretches = ( 3 N – 6 for a non-linear molecule ) - the no of stretches 26 Vibrations in a molecule can be classified as

Slide 27: In polyatomic molecules, the actual number of modes of vibrations will be altogether different from those calculated theoretically. Reasons The over tones and combination of tones may increase the number of modes of vibrations. Some other phenomenon may reduce the number of bonds: 1.Vibrations that do not fall in the IR region cannot be observed in the spectrum. 2.Some weak vibrational bonds, if present cannot be seen in the IR 27

Slide 28: 3. Some vibrational bands, which are having the same or slight different frequencies, overlap each other and appear as one band in the IR spectrum. 4.Some vibrational bands may degenerate and appear at the same place in IR spectrum. 5.If there occurs no required change in dipol character of the molecule, no band will appear in the IR spectrum. 28

VIBRATIONAL FREQUENCY: VIBRATIONAL FREQUENCY occurs when atoms in a molecule are in periodic motion while the molecule as a whole has constant translational and rotational motion. The frequency of the periodic motion is known as a vibration frequency. The value of stretching vibrational frequency of a bond can be calculated by the application of hooke’s law. ν /c = ν = 1/2 п c[k/m1m2/m1+m2]1/2 = 1/2 п c√k /µ Where, µ →reduced mass m1&m2 →masses of the atoms k →force constant c →velocity of radiation 29

Factors influencing vibrational frequencies: Factors influencing vibrational frequencies Calculated value of frequency of absorption for a particular bond is never exactly equal to its experimental value. There are many factors which are responsible for vibrational shifts Vibrational coupling and fermi resonance it is observed in compounds containing –CH2 & -CH3. EG. Carboxylic acid anhydrides amides aldehydes 30

Slide 31: Fermi resonance Fermi resonance results in the splitting of two vibrational bands that have nearly the same energy and symmetry in IR spectroscopies . The two bands are usually a fundamental vibration and either an overtone or combination band The wave functions for the two resonant

vibrations mix, and the result is a shift in frequency and a change in intensity in the spectrum. As a result, two strong bands are observed in the spectrum, instead of the expected strong and weak bands. 31

Slide 32: 32

Slide 33: 2) Hydrogen bonding: Hydrogen bonding brings about remarkable downward frequency shifts. Stronger the hydrogen bonding, greater is the absorption shift towards lower wave length than the normal value. There is 2 types of hydrogen bonding a) inter molecular →broad bands b) intra molecular → sharp bands hydrogen bonding in O-H and N-H compounds deserve special attention. Eg: alcohols&phenols enols & chelates 33

Slide 34: 3) Electronic effects: In this the frequency shifts are due to electronic effects which include conjugation, mesomeric effect, inductive effect . a) conjugation: conjugation lowers the absorption frequency of C=O stretching whether the conjugation is due to α , β - unsaturation or due to an aromatic ring. b) mesomeric effect: a molecule can be represented by 2or more structures that differ only in the arrangement of electrons. c) inductive effect: depends upon the intrinsic tendency of a substituent to either release or withdraw electrons.

SEMINAR ON INFRARED SPECTROSCOPY: SEMINAR ON INFRARED SPECTROSCOPY By Srikanth.C.Dodda, M.Pharmacy(1 st Semester) DEPARTMENT OF PHARMACEUTICAL ANALYSIS

INTRODUCTION:: INTRODUCTION: 3 One of the most Powerful analytical techniques which offers the possibility of Chemical Identification . In Conjugation with N.M.R., Mass spectroscopy, IR is used for Structural Analysis . Gives useful information about the structure of molecule quickly, with out tiresome evaluation method. Used for various Qualitative & Quantitative Analysis .

ADVANTAGES:: ADVANTAGES: 4 Common, Inexpensive . Easy to Operate . The sample may be recovered and further utilized.

WHAT IS THE TECHNIQUE INVOLVED?: 5 WHAT IS THE TECHNIQUE INVOLVED? A Chemical substance shows marked selective absorption in the IR region . After absorption of IR radiations, the molecules of a chemical substance vibrate at many rates of vibration, giving rise to close-packed absorption bands called an IR absorption spectrum (extended over a wide wavelength range)

BAND INTENSITIES IN IR SPECTRUM CAN BE EXPRESSED:: BAND INTENSITIES IN IR SPECTRUM CAN BE EXPRESSED: 6 Transmittance(T) Absorbance(A) Ratio of radiant power transmitted by a sample to the radiant power incident on the Sample. Logarithm, to the base 10, of the reciprocal of the transmittance A=log 10 (1/T)

BAND POSITIONS IN IR SPECTRUM CAN BE EXPRESSED: : BAND POSITIONS IN IR SPECTRUM CAN BE EXPRESSED: Wave number Units: cm -1 or kilokayser ( k K ) Wave number is the reciprocal of wavelength. 7 Number of waves per centimeter in Vacuum. This quantity is denoted by v 1kK=1000K=1000 cm -1

RANGE OF IR REGION:: RANGE OF IR REGION: 8 Name of the region Range Photographic Region Visible to 1.2µ Very near IR Region 1.2 to 2.5µ Near IR Region 2.5 to 25µ Far IR Region 25 to 300-400µ An Analytical chemist gets the important info about the molecular vibrations in this region and hence he/she able to know about the structure of the molecule.

MOLECULAR VIBRATIONS:: MOLECULAR VIBRATIONS: In a polyatomic molecule, each atom is having three degrees of freedom in three directions which are perpendicular to each other. So, A molecule of “ n” atoms has “ 3n” degrees of freedom. 9

Comparison of degrees of freedom between Non-linear and linear molecules: Comparison of degrees of freedom between Non-linear and linear molecules Degrees of freedom describes Non-Linear molecule Linear molecule Rotation 3 2 Translation 3 3 Vibrational degrees of freedom (or) fundamental vibrations 3n-6 3n-5 10

TYPES OF VIBRATIONS:: TYPES OF VIBRATIONS: 11 Stretching Bending Symmetric Asymmetric In-plane Out-of-plane Scissoring Rocking Wagging Twisting

STRETCHING VIBRATIONS:: STRETCHING VIBRATIONS: Atoms move along the same bond axis. Bond length increases & decreases at regular intervals. Bond angle changes only if it is required (by the centre of gravity resisting displacement) 12

STRETCHING VIBRATIONS: STRETCHING VIBRATIONS Symmetrical stretching: In this, the bond lengths increase or decreases periodically. 13

STRETCHING VIBRATIONS: STRETCHING VIBRATIONS Asymmetrical stretching: In this, one bond length is increased and other is decreased. 14

BENDING VIBRATIONS: BENDING VIBRATIONS These are also called as deformations. In this bond angle is altered. These are of 2 types In plane bending→ scissoring, rocking Out plane bending→ wagging, twisting 15

IN PLANE BENDING:: IN PLANE BENDING: Scissoring: This is an in plane bending. In this, bond angles are decreased & two atoms approach each other. 16

IN PLANE BENDING:: IN PLANE BENDING: Rocking: In this, movement of atoms takes place in same direction . 17

OUT PLANE BENDING:: OUT PLANE BENDING: Wagging: It is an out of plane bending. In this,2 atoms move to one side of the plane. They move up and down the plane. 18

OUT PLANE BENDING:: OUT PLANE BENDING: Twisting: In this, one atom moves above the plane and the other atom moves below the plane. 19

FACTORS INFLUENCING VIBRATIONAL FREQUENCIES: FACTORS INFLUENCING VIBRATIONAL FREQUENCIES 20 Calculated value of frequency of absorption for a particular bond is never exactly equal to its experimental value. There are many factors which are responsible for vibrational shifts.

PowerPoint Presentation: Vibrational coupling: It is observed in compounds containing CH2( Methylene ) & – CH3(Methyl). Example: Carboxylic acid anhydrides Amides Aldehydes 21

PowerPoint Presentation: Hydrogen bonding: Hydrogen bonding brings about remarkable downward frequency shifts. Stronger the hydrogen bonding, greater is the absorption shift towards lower wave length than the normal value. 22

PowerPoint Presentation: Bands differ for different types of hydrogen bonding a) inter molecular→broad bands b) intra molecular → sharp bands Hydrogen bonding in O-H and N-H compounds deserve special attention. Example: Alcohols & Phenols Enols & Chelates 23

PowerPoint Presentation: Electronic effects: In this the frequency shifts are due to electronic effects which include conjugation, mesomeric effect, inductive effect. a) Conjugation: conjugation lowers the absorption frequency of C=O stretching whether the conjugation is due to α , β - unsaturation or due to an aromatic ring. b) Mesomeric effect: a molecule can be represented by 2or more structures that differ only in the arrangement of electrons. c) Inductive effect: depends upon the intrinsic tendency of a substituent to either release or withdraw electrons. 24

INSTRUMENTATION: INSTRUMENTATION The Main parts of IR spectrometer are as follows: IR Radiation Source Monochromators Sample cells Detectors 25

PowerPoint Presentation: IR RADIATION SOURCES Incandescent lamp Nernst Glower Globar Source Mercury Arc 26

INCANDESCENT LAMPS: INCANDESCENT LAMPS Here, ordinary incandescent lamp is used. glass enclosed. Disadvantages: fails in far infrared. low spectral emissivity. 27

NERNST GLOWER: NERNST GLOWER Composed of rare earth oxides such as Zirconia, Yttria & Thoria. Non conducting at room temperature. Heated externally to bring it to conducting state.(1000 to 1800°C) Provides maximum radiation at about 7100 cm -1 . 28

Disadvantages:: Disadvantages: Emits IR radiation over wide wavelength range. Frequent mechanical failure. Energy concentrated in visible & near I R region of spectrum. 29

PowerPoint Presentation: 30 NERNST GLOWER

MERCURY ARC : MERCURY ARC Special high pressure mercury lamps are used in far IR. Beckman devised the Quartz Mercury Lamps in unique manner. At Shorter wavelength ------- heated quartz envelope provides radiation At Longer wavelength -------- mercury plasma provides radiation 31

MERCURY ARC: MERCURY ARC 32

MONOCHROMATORS: MONOCHROMATORS They convert polychromatic light into mono chromatic light. They must be constructed of materials which transmit the IR. Two types: Prism Monochromators Grating Monochromators 33

PowerPoint Presentation: PRISMS a) Metal halide prisms: Prisms which are made up of KBr are used in the wavelength region from 12-25µm. Lithium Fluoride prisms are used in the wavelength region from 0.2-6µm. CeBr prisms used in wavelength region from 15-38µm. 34

PowerPoint Presentation: b ) NaCl prisms: Used in the whole wave length region from 4000-650 cm −1 . They have to be protected above 20ºC because of hygroscopic nature. 35 SINGLE -PASS MONOCHROMATOR s

PowerPoint Presentation: GRATINGS : They offer better resolution at low frequency than prisms. Consists of a series of parallel straight lines cut into a plane surface. 36

PowerPoint Presentation: 37 Animation on Gratings( Labeling is in Polish Language, Please Adjust)

SAMPLING TECHNIQUES: SAMPLING TECHNIQUES Sampling of Solids Sampling of Liquids Sampling of Gases 38

SAMPLING OF SOLIDS: SAMPLING OF SOLIDS 39 SOLIDS RUN IN SOLUTION : Solids dissolved in a aqueous solvent Placed over the alkali metal disk Solvent is allowed to evaporate Thin film of solute formed or Entire solution is run in one of the cells for liquids

PowerPoint Presentation: NOTE : This method not used because suitable number of solvents available are less. Absorption due to solvent has to be compensated by keeping the solvent in a cell of same thickness as that containing the reference beam of double beam spectrometer. 40

SOLID FILMS : SOLID FILMS Technique used for Amorphous samples. Sample is deposited on the KBr / NaCl cell by the evaporation of solution. Only useful for rapid qualitative analysis. Not useful for Quantitative analysis. 41

MULL TECHNIQUE : : MULL TECHNIQUE : Finely ground solid sample is used. Mixed with Nujol (mineral oil) Thick paste is made. Spread between I R transmitting windows Mounted in path of I R beam & The spectrum is run. Disadv antage: Nujol has the absorption maxima at 2915, 1462, 1376 & 719 cm -1 . 42

PRESSED PELLET TECHNIQUE: : PRESSED PELLET TECHNIQUE: Finely ground sample used. Potassium Bromide is mixed (100 times more). Passed through a high pressure press. Small pellet formed (1-2 mm thick, 2cm diameter). The pellet is transparent to I R radiation & is run as such. 43

PowerPoint Presentation: Advantages Pellet can be stored for long period of times. Concentration of sample can be adjusted in KBr pellet hence used for quantitative analysis. Resolution of spectrum is superior . Disadvantages Always has a band at 3450 cm -1 (moisture OH-). At high pressure polymorphic changes occur. Unsuccessful for polymer which are difficult to grind with KBr. 44

PowerPoint Presentation: Preparing a KBr Disk 45

SAMPLING OF LIQUIDS: SAMPLING OF LIQUIDS Liquid samples taken. Put it into rectangular cells of KBr, NaCl etc. I R spectra obtained. Sample thickness … such that transmittance lies between 15 – 20 % i.e., 0.015 – 0.05 mm in thickness. 46

PowerPoint Presentation: For double beam, matched cells are generally employed. One cell contains sample while other has solvent used in sample. Matched cells should be of same thickness, protect from moisture. 47

SAMPLING OF GASES: SAMPLING OF GASES Small size particles hence the cells are large. 10 cm to 1m long. Multiple reflections can be used to make the effective path length as long as 40 cm. Lacks sensitivity. 48

PowerPoint Presentation: Two Types Of Detectors Thermal Detectors Bolometers Thermocouple Golay Detectors Pyroelectric Detectors Thermistors Photon Detectors Semiconductors Photovoltaic Intrinsic Detectors Photoconductive Intrinsic Detectors DETECTORS 49

BOLOMETER: BOLOMETER It consists of thin metallic conductor , its resistance changes due to increase in temperature when IR radiation falls on it. It is a electrical resistance thermometer which can detect and measure feeble thermal radiation. The electrical resistance increases approximately 0.4% for every Celsius degree increase of temperature . 50

PowerPoint Presentation: The degree of change in resistance is regarded as the measure of the amount of IR radiation falling on it. A bolometer is made of two platinum strips , covered with lamp black , one strip is shielded from radiation and one exposed to it. The strips formed two branches of Wheatstone bridge. 51

WORKING:: WORKING: The circuit thus effectively operating as resistance temperature detector . When IR radiations falling on the exposed strip would heat it, and change the resistance, this causes current to flow, the amount of current flowing is a measure of intensity of IR radiation The response time is 4 seconds . 52

PowerPoint Presentation: 53 G Resistance strip Light Shielded Current in Current out Indicator strip hv Fixed resistor Variable resistor Schematic diagram of a bolometer-Wheatstone Bridge for IR Detection IR

THERMOCOUPLE: THERMOCOUPLE Based upon the fact that an electrical current will flow when two dissimilar metal wires are connected together at both ends and a temperature differential exists between the two ends. 54

PowerPoint Presentation: 55 Example : Bismuth & Antimony SCHEMATIC DIAGRAM OF THERMOCOUPLE

GOLAY DETECTOR: GOLAY DETECTOR It consist of a small metal detector closed by a rigid blackened metal plate (2 mm), flexible silvered diaphragm at the other end filled with Xenon gas . Its response time is 20 msec , hence faster than other thermal detectors. It is suitable for wavelengths greater then 15µ. 56

WORKING:: WORKING: The radiation falls on the blackened metal plate, this heats the gas which lead to deformation of flexible silvered diaphragm. The light from a lamp inside the detector is made to fall on the diaphragm which reflects the light on to a phototube . The signal seen by the phototube / photocell is modulated in accordance with the power of the radiation beams incident on the gas cell. 57

PowerPoint Presentation: 58 GOLAY DETECTOR

FOURIER TRANSFORM IR SPECTROMETER: FOURIER TRANSFORM IR SPECTROMETER In the FT-IR instrument, the sample is placed between the output of the interferometer and the detector. The sample absorbs radiation of particular wavelengths. An interferogram of a reference is needed to obtain the spectrum of the sample. After an interferogram has been collected, a computer performs a Fast Fourier Transform , which results in a frequency domain trace (i.e intensity vs wavenumber). 59

Advantages of Fourier transform IR over dispersive IR: Advantages of Fourier transform IR over dispersive IR Improved frequency resolution . Improved frequency reproducibility. (older dispersive instruments must be recalibrated for each session of use) Faster operation . Computer based. (allowing storage of spectra and facilities for processing spectra) Easily adapted for remote use. (such as diverting the beam to pass through an external cell and detector, as in GC - FT-IR) 60

PowerPoint Presentation: The detector used in an FT-IR instrument must respond quickly because intensity changes are rapid . Pyroelectric detectors or liquid nitrogen cooled photon detectors must be used. Thermal detectors are too slow. To achieve a good signal to noise ratio, many interferograms are

obtained and then averaged. This can be done in less time than it would take a dispersive instrument to record one scan. 61

APPLICATIONS:: APPLICATIONS: Identification of functional group and structure elucidation. Identification of Drug substance. Identifying the impurities in the drug sample. Study of hydrogen bonding (intermolecular / intramoleular). Study of polymers. Ratio of Cis-Trans isomers in a mixture of compounds. Quantitative analysis. 62

PowerPoint Presentation: CONCLUSION 63

REFERENCES: REFERENCES “INSTRUMENTAL METHODS OF CHEMICAL ANALYSIS” BY G.R.CHATWAL AND S.K.ANAND, EDITION-5 TH , PAGE NO.-2.29 TO 2.82. PHARMACEUTICAL DRUG ANALYSIS” BY ASHUTOSH KAR, EDITION-2 ND , PAGE NO.-314 TO 338. “INSTRUMENTAL ANALYSIS” BY D.A.SKOOG, F.J.HOLLER AND S.R.CROUCH, EDITION-1 ST REPRINT-2008, PAGE NO.-477. “TEXTBOOK OF PHARMACEUTICAL ANALYSIS” BY DR.S.RAVI SANKAR, EDITION 3 RD , PAGE NO.-5-1 TO 5-7. WEBSITES SUCH AS WIKIPEDIA, SLIDESHARE, AUTHORSTREAM, ETC.

UV

SPECTROSCOPY: SPECTROSCOPY “The study of response of molecule when it is exposed to certain kind of radiation”. Types: 1)Absorption Spectroscopy: The study of absorbed radiation by molecule , in the form of spectra. 2)Emission Spectroscopy: The radiation emitted by molecules can also be studied to

reveal the structure of molecule.

ULTRA VIOLET SPECTROSCOPY: ULTRA VIOLET SPECTROSCOPY Introduction: UV spectroscopy provides us information about the structure of molecules that contain double or triple bond or conjugated bonds.

This spectroscopy can differentiated between conjugated and isolated dienes. Dienes and trienes Carbonyl compounds and α : β unsaturated carbonyl comp. It also throws light on the stereochemistry

of geometrical isomerism and enable us to identify cis and trans isomers .

PRINCIPLE: PRINCIPLE UV-visible spectroscopy measure the response of a sample to ultra violet and visible range of electromagnetic radiation. Molecules and atom have electronic transition while most of

the solid have enter band transition in the UV and Visible range.

Visible: Visible The region beyond red is called infra-red while that beyond violet is called as ultra –violet. Electromagnetic Spectrum Visible The region beyond red is called infra-red while that beyond

violet is called as ultra –violet.

THE ABSORPTION SPECTRUM: THE ABSORPTION SPECTRUM The absorption of uv radiation brings about the promotion of an electron from bonding to antibonding orbital. The wavelength of radiation is slowly

changed from minimum to maximum in the given region, and the absorbance at every wavelength is recorded. Then a plot of energy absorbed Vs wavelength is called absorption spectrum. The significant

features: λ max (wavelength at which there is a maximum absorption) є max (The intensity of maximum absorption) The UV spectrum depends on solvents concentration of solution path length of the solution

Slide 8: 8 UV Spectroscopy Observed electronic transitions Here is a graphical representation Energy s* p s p* n Atomic orbital Atomic orbital Molecular orbitals Occupied levels Unoccupied levels

Different types of Excitations : Different types of Excitations σ - σ* Transition π - π* Transition n- σ* Transition n- π* Transition

Slide 10: 10 UV Spectroscopy Observed electronic transitions Although the UV spectrum extends below 100 nm (high energy), oxygen in the atmosphere is not transparent below 200 nm Special equipment to study vacuum or far UV is required Routine organic UV spectra are typically collected from 200-700 nm

This limits the transitions that can be observed: s s p n n s * p * p * s * p * alkanes carbonyls unsaturated cmpds. O, N, S, halogens carbonyls 150 nm 170 nm 180 nm √ - if conjugated! 190 nm 300 nm √

INSTRUMENTATION: INSTRUMENTATION 11 The construction of a traditional UV-VIS spectrometer is very similar to an IR, as similar functions – sample handling, irradiation, detection and output are

required Here is a simple schematic that covers most modern UV spectrometers: sample referen ce detector I 0 I 0 I 0 I log( I 0 / I ) = A 200 700 l , nm monochromator/ beam splitter optics UV-VIS sources

Slide 12: UV Spectroscopy Instrumentation and Spectra Two sources are required to scan the entire UV-VIS band: Deuterium lamp – covers the UV – 200-330 Tungsten lamp – covers 330-700 As with the

dispersive IR, the lamps illuminate the entire band of UV or visible light; the monochromator (grating or prism) gradually changes the small bands of radiation sent to the beam splitter The beam splitter sends a

separate band to a cell containing the sample solution and a reference solution The detector measures the difference between the transmitted light through the sample (I) vs. the incident light (I 0 ) and sends

this information to the recorder

Slide 13: 13 UV Spectroscopy Instrumentation and Spectra Instrumentation As with dispersive IR, time is required to cover the entire UV-VIS band due to the mechanism of changing wavelengths A recent

improvement is the diode-array spectrophotometer - here a prism (dispersion device) breaks apart the full spectrum transmitted through the sample Each individual band of UV is detected by a individual

diodes on a silicon wafer simultaneously – the obvious limitation is the size of the diode, so some loss of resolution over traditional instruments is observed sample Polychromator – entrance slit and dispersion

device UV-VIS sources Diode array

Slide 14: UV Spectroscopy Instrumentation and Spectra Instrumentation – Sample Handling Virtually all UV spectra are recorded solution-phase Cells can be made of plastic, glass or quartz Only quartz is

transparent in the full 200-700 nm range; plastic and glass are only suitable for visible spectra Concentration (we will cover shortly) is empirically determined A typical sample cell (commonly called a

cuvet ):

Slide 15: 15 UV Spectroscopy Chromophores Definition Remember the electrons present in organic molecules are involved in covalent bonds or lone pairs of electrons on atoms such as O or N Since similar functional groups will have electrons capable of discrete classes of transitions, the characteristic energy

of these energies is more representative of the functional group than the electrons themselves A functional group capable of having characteristic electronic transitions is called a chromophore ( color

loving ) Structural or electronic changes in the chromophore can be quantified and used to predict shifts in the observed electronic transitions

Slide 16: UV Spectroscopy Chromophores Substituent Effects General – from our brief study of these general chromophores, only the weak n p * transition occurs in the routinely observed UV The

attachment of substituent groups (other than H) can shift the energy of the transition Substituent's that increase the intensity and often wavelength of an absorption are called auxochromes Common

auxochromes include alkyl, hydroxyl, alkoxy and amino groups and the halogens

Slide 17: UV Spectroscopy Chromophores Substituent Effects General – Substituent's may have any of four effects on a chromophore Bathochromic shift (red shift) – a shift to longer l ; lower energy

Hypsochromic shift (blue shift) – shift to shorter l ; higher energy Hyperchromic effect – an increase in

intensity Hypochromic effect – a decrease in intensity 200 nm 700 nm e Hypochromic Hypsochromic Hyperchromic Bathochromic

Slide 18: 18 UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules - Dienes The rules begin with a base value for l max of the chromophore being observed: acyclic butadiene = 217 nm The

incremental contribution of substituent's is added to this base value from the group tables: Group Increment Extended conjugation +30 Each exo-cyclic C=C +5 Alkyl +5 -OCOCH 3 +0 -OR +6 -SR +30 -Cl, -Br

+5 -NR 2 +60

Slide 19: UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules - Dienes For example: Isoprene - acyclic butadiene = 217 nm one alkyl subs. + 5 nm 222 nm Experimental value

220 nm Allylidenecyclohexane - acyclic butadiene = 217 nm one exocyclic C=C + 5 nm 2 alkyl subs. +10 nm 232 nm Experimental value 237 nm

Slide 20: UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Group Increment 6-membered ring or acyclic enone Base 215 nm 5-membered ring parent enone Base 202 nm Acyclic dienone Base 245 nm Double bond extending conjugation 30 Alkyl group or ring residue a, b, g and higher 10, 12, 18 -OH a, b, g and higher 35, 30, 18 -OR a, b, g, d 35, 30, 17, 31 -O(C=O)R a, b, d 6 -Cl

a, b 15, 12 -Br a, b 25, 30 -NR 2 b 95 Exocyclic double bond 5 Homocyclic diene component 39

Slide 21: UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Aldehydes, esters and carboxylic acids have different base values than ketones Unsaturated system Base Value

Aldehyde 208 With a or b alkyl groups 220 With a,b or b,b alkyl groups 230 With a,b,b alkyl groups 242 Acid or ester With a or b alkyl groups 208 With a,b or b,b alkyl groups 217 Group value – exocyclic a,b

double bond +5 Group value – endocyclic a,b bond in 5 or 7 membered ring +5

Slide 22: 22 UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Unlike conjugated alkenes, solvent does have an effect on l max These effects are also described by the

Woodward-Fieser rules Solvent correction Increment Water +8 Ethanol, methanol 0 Chloroform -1 Dioxane -5 Ether -7 Hydrocarbon -11

Slide 23: 23 UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Some examples – keep in mind these are more complex than dienes cyclic enone = 215 nm 2 x b - alkyl

subs. (2 x 12) +24 nm 239 nm Experimental value 238 nm cyclic enone = 215 nm extended conj.

+30 nm b -ring residue +12 nm d -ring residue +18 nm exocyclic double bond + 5 nm 280 nm Experimental 280 nm

APPLICATIONS:: APPLICATIONS: Determination of structure of organic compound: Exam: Element, Functional group, etc. Determination of stereochemistry: Exam: Cis or Trans. Strength of Hydrogen

bond:

QUANTITATIVE ANALYSIS BY UV - SPECTROSCOPY :

QUANTITATIVE ANALYSIS BY UV - SPECTROSCOPY Aju S. Sam 1st year M.Pharm Nehru College of Pharmacy

Introduction :

Introduction LAMBERT’S LAW: “When a beam of light is allowed to pass through a transparent medium the rate of decrease in intensity with the thickness of the medium is directly proportional to intensity of

the light.” -dI/dt ∞ I It = I0 e-kt (1)

Slide 3:

BEER’S LAW “The intensity of a beam of monochromatic light decreases exponentially with increase in concentration of absorbing substances arithmetically.” It = I0e–k’c (2) On simplification and combining

(1) and (2) we get

Slide 4:

log (I0/It) = act This is called BEER-LAMBERT’S LAW. Relationship between Absorbance(A), Transmittance(T) , & Molar extinction coefficient(ε). A = εct = log (I0/It)= log (1/T) = log -T

METHODS USED FOR ASSAY :

METHODS USED FOR ASSAY USE OF CALIBRATION GRAPH: Step 1: select the λmax . Step 2: prepare series of known conc. Solutions. Step 3: set λmax in spectrophotometer. Step 4: measure absorbance.

Step 5: plot calibration curve. absorbance conc.

2. USE OF STANDARD ABSORPTIVE VALUE :

2. USE OF STANDARD ABSORPTIVE VALUE In this method standard ε value ie ε 1%1cm is used. A = ε1%1cmct e.g. ε1%1cm for methyl testosterone in B.P is 540 at 241 nm.

3.SINGLE OR DOUBLE POINT STANDARDISATION :

3.SINGLE OR DOUBLE POINT STANDARDISATION Involves measurement of sample and reference. Conc. of reference close to that of sample. C test= A test × C std A std Double point involves use of 2 reference.

one greater conc. than sample, other lower conc. than sample. C test=(Atest- Astd1)(Cstd1- Cstd2)+Cstd1(Astd1- Astd2) Astd1-Astd2

4.ASSAY USING ABSORBANCE CORRECTED FOR INTERFERENCE :

4.ASSAY USING ABSORBANCE CORRECTED FOR INTERFERENCE The concentration of absorbing component = total absorbance – absorbance of interfering substance.

5.Assay after solvent extraction of sample :

5.Assay after solvent extraction of sample Involves separation of absorbing interferents by solvent extraction, choice of pH . e.g. B.P Assay of Caffeine in Aspirin & Caffeine tablet

6.SIMULTANEOUS EQUATION METHOD :

6.SIMULTANEOUS EQUATION METHOD Total absorbance of a solution is equal to the sum of absorbance of individual components present. absorbance λ1 wave length λ2 mixture x y

Slide 11:

A1= ax1bcx+ay1bcy (at 1) A2= ax2bcx+ay2bcy (at 2) C x = A2ay1-A1ay2 ax2ay1-ax1ay2 Cy= A1ax2-A2ax1 ax2ay1-ax1ay2 e.g. B.P assay of quinine related alkaloids in Cinchona bark.

7.ABSORBANCE RATIO METHOD :

7.ABSORBANCE RATIO METHOD For a substance which obeys BEER’S LAW at all wavelengths, the ratio of absorbance at any two wavelengths is a constant value independent of concentration or path length.

In USP the ratio is referred as Q value. e.g. cyanocobalamin exhibits three λmax at 278nm, 361nm, 550nm A361/A550 = 3.30 ± 0.15 A361/A278 = 1.79 ± 0.09

DIFFERENCE SPECTROSCOPY :

DIFFERENCE SPECTROSCOPY Two samples used, one in reference beam and one in sample beam. Conc. of absorbing substance identical. But some solution parameter like pH is different. Difference

absorbance ∆A = A alk - A acid

DIFFERENTIAL SPECTROSCOPY :

DIFFERENTIAL SPECTROSCOPY

Slide 15:

0 10 20 30 40 50 60 70 80 90 100 (BLANK) RECEIVER DARK 0 10 20 30 40 50 60 70 80 90 100 HIGH ABSORBANCE 0 10 20 30 40 50 60 70 80 90 100 (BLANK) 0 10 20 30 40 50 60 70 80 90 100 TRACE

ANALYSIS 0 10 20 30 40 50 60 70 80 90 100 C1 C1>C2 C2 0 10 20 30 40 50 60 70 80 90 100 MAXIMUM PRECISION

DERIVATIVE SPECTROPHOTOMETRY :

DERIVATIVE SPECTROPHOTOMETRY Conversion of normal spectrum into its first or higher derivative spectrum. First derivative spectrum- (dA/dλ) v/s λ A λ dA/dλ λ + - λmax Cross over point (λmax)

Slide 17:

Second derivative spectrum A λ d2A/dλ2 λ + - X+Y X Y

REFERENCES :

REFERENCES A.H.BECKETT, J.B.STENLAKE.PRACTICAL PHARMACEUTICAL CHEMISTRY, 4TH EDITION, PART TWO; 2005: 275-300. WILLARD,MERRITT,DEAN,SETTLE. INSTRUMENTAL METHODS OF ANALYSIS, 7TH

EDITION;1986:159-178. GURDEEP.R.CHATWAL,SHAM.K.ANAND. INSTRUMENTAL METHODS OF CHEMICAL ANALYSIS, 5TH EDITION;2007:140-178.

INSTRUMENTATION OF ULTRA-VIOLET SPECTROSCOPY:

INSTRUMENTATION OF ULTRA-VIOLET SPECTROSCOPY Guided By : - Pinak Patel Presented By :- Anand Mandanka Dharmaj Degree Pharmacy Collage

Instrumentation :

Instrumentation Components of spectrophotometer Source Monochromator Sample compartment Detector Recorder

Slide 3:

RADIANT SOURCE WAVELENGTH SELECTOR SOLVENT PHOTO- DETECTOR READOUT SAMPLE Fig.-block diagram of instrumentation of UV-spectrophotometer

Slide 4:

Light source a)D2 Lamp b)WI Lamp Entrance slit monochromator sample Exit slit Read out amplifier detector Figure.- block diagrammatic representation of UV-spectrophotometer

Light source :

Light source Distribution of energy through spectrum is function of temperature. For Visible region- Tungsten filament lamp Use for region 350nm to 2000nm. Problem- Due to evaporation of tungsten life period decreases. It is overcome by using tungsten-halogen lamp. Halogen gas prevents evaporation of

tungsten.

Slide 6:

For ultra violet region- Hydrogen discharge lamp consist of two electrode contain in deuterium filled silica envelop. gives continuous spectrum in region 185-380nm. above 380nm emission is not

continuous. UV-Vis spectrophotometer have both deuterium & tungsten lamps. Selection of lamp is made by moving lamp mounting or mirror to cause the light fall on monochromator.

Slide 7:

Deuterium lamps:- Radiation emitted is 3-5 times more than the hydrogen discharge lamps. Xenon discharge lamp:- Xenon stored under pressure in 10-30 atmosphere. It possesses two tungsten electrode

separated by 8 cm. Intensity of UV radiation more than hydrogen lamp. Mercury arc:- Mercury vapour filled under the pressure . Excitation of mercury atom by electric discharge

Monochromator:

Monochromator Filters – a)Glass filters- Made from pieces of colored glass which transmit limited wavelength range of spectrum. Color produced by incorporation of oxide of vanadium, chromium, iron, nickel, copper. Wide band width 150nm. b)Gelatin filters- Consist of mixture of dyes placed in gelatin &

sandwiched between glass plates. Band width 25nm. c)Inter ferometric filters- Band width 15nm.

Slide 9:

Prisms- Prism bends the monochromatic light. Amount of deviation depends on wavelength. Quartz prism used in UV-region. Glass prism used in visible region spectrum. Function – They produce non

linear dispersion. They are universal and versatile ,used to get desired wavelength.

Slide 10:

Fig.-Mechanism of prism working Fig.-mechanism of working of prism .

Slide 11:

Grating- Large number of equispaced lines on a glass blank coated with aluminum film. Blaze angle Normal surface vector Normal to groove face

Types of grating:

Types of grating Two types of gratings:- Transparent grating Refraction or Diffraction grating Transparent grating:- Grating is transparent & radiation enters through one side that passes through the grating &

separation occurs at other surface.

Diffraction Grating:

13 Diffraction Grating Mechanism : The rays which are incident upon the gratings gets reinforced with the reflected rays and hence the resulting radiation has wavelength which is governed by the equation:

m λ = b (sin i ± sin r) Where λ = wavelength of light produced b = grating spacing i = angle of incidence r = angle of reflection m = order (0,1,2,3 etc)

Slide 14:

Spectroscopy requires all materials in the beam path other than the analyte should be as transparent to the radiation as possible. The geometries of all components in the system should be such as to maximize

the signal and minimize the scattered light. The material from which a sample cuvette is fabricated controls the optical window that can be used. Some typical materials are: Optical Glass - 335 - 2500 nm

Special Optical Glass – 320 - 2500 nm Quartz (Infrared) – 220 - 3800 nm Quartz (Far-UV) – 170 - 2700 nm •Keep the cuvette clean. •Don’t clean with paper products. •Store dry. •Don’t get finger prints on them.

•Store carefully and gently Sample cell (cuvette)

Detectors:

Detectors Three common types of detectors are used Barrier layer cells Photocell detector Photomultiplier Photo voltaic cells or barrier layer cells :- They are primarily used for measurement of radiation in visible region. Maximum sensitivity-550nm. It consist of flat Cu or Fe electrode on which

semiconductor such as selenium is deposited. on the selenium a thin layer of silver or gold is sputtered over the surface.

Slide 16:

A barrier exist between the selenium & iron which prevents the electron flowing through iron. Therefore electrons are accumulated on the silver surface. These electrons are produced voltage. - terminal Silver

surface selenium + terminal fig.-Barrier layer cell

Photovoltaic or Barrier layer cells:

Photovoltaic or Barrier layer cells MECHANISM: Electrons are transferred at the interface from the semiconductor to metal when the semiconductor is irradiated. The electric current produced is

proportional to the radiance power of the incident beam and to the area of photosensitive being irradiated. 17

:

18 Merits : At low level of illumination it produces photo current proportional to the radiant power received on it. It is relatively cheap and widely used in filter photometers and cheap

spectrophotometers. Demerit s: Slow response. Fatigue effect . While using it there should be no fluctuations in power and low external electric resistance.

Slide 19:

Photocell detector:- It consist of high sensitive cathode in the form of a half cylinder of metal which is evacuated. Anode also present which fixed along the axis of the tube Photocell is more sensitive than

photovoltaic cell. + - light Fig.- photocell detector

Photomultiplier Tubes:

20 Photomultiplier Tubes Mechanism: It has a number of photo emissive electrodes each charged at a successively higher potential and so arranged that the electrons ejected from the photocathode travel

successively from one electrode to the next. In each step the photocurrent is increased by the secondary emission of electrons.

Photo multiplier tube:

Photo multiplier tube

Merits:

22 Merits It is highly sensitive to light. Best suited where weaker or low radiation is received.

Recorder:

Recorder Signal from detector received by the recording system The recording done by recorder pan.

UV INSTRUMENTS-TYPES:

24 UV INSTRUMENTS-TYPES Colorimeters Spectrophotometers

Double Beam Colorimeter:

25 Double Beam Colorimeter

UV- spectrophotometer:

UV- spectrophotometer Single beam spectrophotometer:- Double beam spectrophotometer:- Advantage of double beam spectrophotometer:- It is not necessary to continually replace the blank with

the sample or to adjust the autozero. The ratio of the powers of the sample & reference is constantly obtained. It has rapid scanning over the wide wavelength region because of the above two factors.

Single Beam Spectrophotometer:

27 Single Beam Spectrophotometer

Double Beam Spectrophotometer:

28 Double Beam Spectrophotometer

Slide 29:

fig.-Schematic representation of single beam UV-spectrophotometer Fig.-schematic representation of double beam UV- spectrophotometer

INSTRUMENTAL FACTORS CAUSING DEVIATION FROM BEER’S LAW:

INSTRUMENTAL FACTORS CAUSING DEVIATION FROM BEER’S LAW Relative Concentration Error Polychromatic Radiation Measurement Speed Stray Light

Slide 31:

Polychromatic Radiation:- Spectral Slit Width(S.S.W) is a measure of extent of monochromatic radiation. Monochromator allows only specific band of radiation to pass through it and absorbs all other radiation.

Spectral slit width is a range of wavelength in nm coming out of a monochromator & passing through solution.

Slide 32:

Measurement Speed:- It is a time required to measure the entire spectrum. Stray light:- It is the light reaching to the light measuring device other than light coming from the sample When light comes out of a monochromator some of the light travels in another direction & this light may get reflected by dust or

other particles & reach the measuring device called as Stray Light.

References :

References Gurdeep R. chatwal; Sham K. Anand; Instrumental Methods Of Chemical Analysis. Y. Anjaneyulu; K. Chandrasekhar; Valli Manickam; Text book of analytical chemistry. Y. R.Sharma; Elementary organic spectroscopy. P.S.Kalsi; Spectroscopy of organic compound. B.K.Sharma;

Instrumental methods of chemical analysis.

Slide 2:

Electromagnetic Radiation: Atomic Spectra Molecular Spectra Beer Lambert Law I= Io-IT -dI/db=kI A=log Io/IT =abc Electronic Transitions Sigma to Sigma* (Methane) antibonding to Sigma*(Alcohol, Ether) Pi to

pi*(K-band, Aromatics) antibonding to pi*(R-band, Saturated ketone)

Slide 3:

Chromophore Auxochrome

Instrumentation : :

Instrumentation : Introduction Have a look at this schematic diagram of a double-beam UV-Vis. spectrophotometer;

Slide 5:

Components Sources of radiant energy (UV and visible) Collimating System (making parallel) Wavelength selector (monochromator) Sample holder Detector Signal processor and readout

Slide 6:

Collimating system Lenses, mirror, slit, Monochromator Filters Glass filters Gelatin Interference Prism Diffraction grating The beam is split into its component wavelengths by the grating or prism. By moving

the dispersing element or the exit slit, radiation of only a particular wavelength leaves the monochromator through the exit slit.

Sample Holder :

Sample Holder Cuvette : Quartz Sample Holder Quartz

Detectors : :

Detectors : Barrier layer cell (photovoltaic cell): ♣ consist of a semiconductor, such as a selenium, which is deposited on a strong metal base, such as iron. ♣ Then a very thin layer of silver or gold is sputtered

over the surface of the semiconductor to act as a second collector electrode.

iii) The photomultiplier tube : :

iii) The photomultiplier tube : Consists of Photoemissive cathode (emits electrons when struck by photons) several dynodes (emits several electrons for each electron striking them) anode. A photon of

radiation entering the tube strikes the cathode, causing the emission of several electrons. These electrons are accelerated towards the first dynode. The electrons strike the first dynode, causing the

emission of several electrons for each incident electron. These electrons are then accelerated towards the second dynode, to produce more electrons which are accelerated towards dynode three and so on. Eventually, the electrons are collected at the anode. The resulting current is amplified and measured.

Very sensitive to UV and visible radiation, fast response times. Intense light damages photomultipliers; they are limited to measuring low power radiation.

Cross section of a photomultiplier tube :

Cross section of a photomultiplier tube

Recording systems : :

Recording systems : The signal from the photomultiplier tube is finally received by the recording system. The recording is done by recorder pen. The type of arrangement is only done in recording UV

spectrophotometers. Power supply : The power supply serves triple function » Decreases the line voltage to the instruments operating level with a transformer. » Converts A.C. to D.C. with a rectifier if

direct current is required by the instrument. » smooths out any ripple which may occur in the line voltage in order to deliver a constant voltage to the source lamp & instruments.

Description of a UV spectrophotometer: :

Description of a UV spectrophotometer: A) single beam system 1.Bausch and Lamb 2.Beckman D.U.

Slide 13:

All of the light passes through the sample cell. Io must be measured by removing the sample. This was the earliest design, but is still in common use in both teaching and industrial labs. Single Beam

instrument

Double beam instrument : :

Double beam instrument :

Double beam instrument : :

Double beam instrument : The light splits into two beams before it reaches the sample. One beam is used as the reference; the other beam passes through the sample. Some double-beam instruments have

two detectors (photodiodes), and the sample and reference beam are measured at the same time. In other instruments, the two beams pass through a beam chopper, which blocks one beam at a time. The

detector alternates between measuring the sample beam and the reference beam.

Slide 16:

1. Identification 2. Structural Elucidation 3. Quantitative Applications Analysis of Organic compounds Analysis of Inorganic Compounds Mixtures of Absorbing substances Applications

Operational Methodology :

Operational Methodology Estimation of λmax Study of Beer Lambert law and determination of Linearity range. If the system is multicomponent, Study of Spectra of all components to determine absorbances at

different wavelengths, Isobestic point etc. Derivative spectrum Calculations Validation

Ultraviolet-visible spectrophotometer :

Ultraviolet-visible spectrophotometer It measures the intensity of light passing through a sample (I), and compares it to the intensity of light before it passes through the sample (Io). The ratio I / Io is called the

transmittance, and is usually expressed as a percentage (%T). The absorbance, A, is based on the transmittance: A = − log(%T)

UV VISIBLE SPECTROSCOPY - INSTRUMENTATION:

UV VISIBLE SPECTROSCOPY - INSTRUMENTATION By Pradnya Mardolkar 1 st year M. Pharm Quality Assurance Department

PowerPoint Presentation:

Essential components of spectrophotometer are :- Source of Electro magnetic radiation Monochromator Sample compartment Detector/ transducer Recorder/ display

Block diagram of a spectrometer:

Block diagram of a spectrometer

LIGHT SOURCES:

LIGHT SOURCES Should be able to meet the following conditions: Provide sufficient radiant energy over required wavelength region. Provide stable output power during the course of measurements. Supply

continuous radiation over entire wavelength used Should be free from any fluctuation

UV Light sources:

UV Light sources HYDROGEN DISCHARGE LAMP H₂ stored under pressure. Electric discharge through lamp excite hydrogen molecules, emitting radiations. High pressure cause collisions between H₂

molecules & results in pressure broadening. Stable, robust & widely used.

DEUTERIUM DISCHARGE LAMP :

DEUTERIUM DISCHARGE LAMP Deuterium used in place of hydrogen. 3 -5 X increase in emission intensity. Expensive than hydrogen lamps.

XENON DISCHARGE LAMP :

XENON DISCHARGE LAMP Contain two tungsten electrodes separated by 8mm. Lamp filled with xenon gas stored under pressure ( 10 – 30 atm ) Low voltage application produce intense arc between

electrodes & produce UV light. High intensity of light. Limitation:- uv radiation released by lamp causes generation of ozone by ionisation of the oxygen molecule.

MERCURY ARC LAMP :

Hg (vapor form) held under high pressure. Electric discharge excites atoms. MERCURY ARC LAMP

VISIBLE LIGHT SOURCES:

VISIBLE LIGHT SOURCES TUNGSTEN FILAMENT LAM P Tungsten filament in glass envelope. Useful over range of 350 to 2000nm. Lamp life limited due to- evaporation of tungsten which darkens the inside of

the lamp reducing incident light.

TUNGSTEN HALOGEN LAMP :

TUNGSTEN HALOGEN LAMP Contain small quantity of iodine inside quartz envelope which houses tungsten filament. Iodine reacts with gaseous tungsten to form tungsten iodide. Molecules strike the

filament, tungsten is re-deposited. Quartz required for high operating temperature of the lamp.

CARBON ARC LAMP :

CARBON ARC LAMP Used when more intense source of visible light is required.

MONOCHROMATORS AND FILTERS:

MONOCHROMATORS AND FILTERS Filters Device which allows878 radiation of required wavelength to pass through Wholly or partially absorb unwanted radiation.


Absorption filters Interference filters

DIFFERENCE:

DIFFERENCE Absorption filters Work by selective absorption Made of glass Contain chemicals(dyes) that absorb all radiations except that desired to pass. Color absorbed is complement of the color of filter Interference filters Narrower bandwidth of about 15nm Consist of two parallel glass plates silvered internally Separated by thin film of dielectric material ( e.g.- cryolite ). Interference of light waves

eliminate undesired radiation

Monochromators:

Monochromators Converts polychromatic beam of light into a monochromatic beam Can isolate a selected narrow band of wavelength anywhere within a comparatively wide spectral range. Consists of:-

Entrance slit Collimator 1 Prism or grating Collimator 2 Exit slit

Types of Monochromators:

Types of Monochromators Prism monochromators : Non linear dispersion- shorter wavelength dispersed to greater degree than the longer ones. Made of glass, quartz or fused silica. Glass- visible.

Quartz, fused silica- ultraviolet region.


White light, through prism disperses into polychromatic light(rainbow.) Rotation of prism to pass required wavelength through exit slit. Effective wavelength depends on- dispersive power of wavelength

material optical angle of the prism.

Types of mounting:

Types of mounting CORNU TYPE Optical angle 60° Emerging light fall on exit slit on rotation. LITTROW TYPE Optical angle 30° One surface is aluminized. Light passes through prism & emerges on same side as

that of light source.

Diffraction gratings :

Diffraction gratings Large number of parallel lines (grooves), ruled on highly polished surface such as alumina. Done with a suitable shaped diamond tool. For UV/ Visible region about 15000- 30000

lines/square inch are drawn. Incident light diffracted over range of angles. (grooves act as scattering centre)


Construction of master grating is tedious, time consuming & expensive. Identical grooves, exactly parallel, equally spaced over length of gratings (3-10cm). Replica gratings prepared by coating original

with epoxy. Made reflective by coating surface with aluminium, gold or platinum.


Law of diffraction nλ= d (sini±sinθ)


ECHELLETE GRATING Grooved to have broad faces for reflection and narrow unused faces. Efficient diffraction of radiation due to geometry CONCAVE GRATING Grating formed on concave surface

disperses radiation and focuses it on exit slit. Eliminates need of auxillary collimating/ focusing mirrors. Cost reduction Reduction in no. of optical surface increase energy throughout monochromator .


Holographic grating Pair of identical lasers used on glass surface coated with photoresist . Interference fringes from two beams sensitize photoresist ,dissolves leaving a grooved surface. Coated with

aluminium . Greater perfection with respect to shape and dimension. Give spectra free from stray radiation & ghosts.

SAMPLE CONTAINERS:

SAMPLE CONTAINERS Cells or cuvettes used to hold the sample. Shape- rectangular or cylindrical. thickness- 1 cm Cells --- transparent in the wavelength region being measured. REGION TYPE OF

MATERIAL USED FOR CELLS OR CUVETTES VISIBLE GLASS OR PLASTIC UV REGION QUARTZ OR FUSED SILICA

Radiation Transducers/ detectors :

Radiation Transducers/ detectors Properties of radiation transducers High sensitivity High signal to noise ratio Constant response over a considerable range of wavelength Exhibit fast response time Zero output

signal in the absence of illumination

Types of radiation transducers:

Types of radiation transducers Two types :- Photon transducers/ photoelectric or quantum detectors- respond to photons Heat transducers- responds to heat Types of photon transducers -Photovoltaic cells Phototubes Photomultiplier tubes Photoconductivity transducers Silicon photodiodes Charge

transfer transducers

Barrier layer or photovoltaic cell:

Barrier layer or photovoltaic cell


Advantages Requires no power supply. Disadvantages Lack of sensitivity.

:

Photoemissive tubes

Advantages: :

Advantages: More sensitive than BLC- used in UV & Visible region Output current can be amplified- used in measuring low intensity radiations.

PHOTOMULTIPLIER TUBE :

PHOTOMULTIPLIER TUBE Successive phototubes built into one envelope. Photo cathode, series of electrodes, each at more positive potential than the one before it, an anode.

http://www.authorstream.com/Presentation/Marianna-45045-Solar-Energy-Voltaic-Outline-Power-Overview-PV-Radiation-as-Education-ppt-powerpoint/


:

Disadvantages Measures low power radiations as intense light damages photo electric surface. Sensitivity limited by its dark current emission. Advantages More sensitive then phototube in UV and

visible region Ideal for measuring weak light intensity Fast response time

RECORDERS/ READ OUT SYSTEMS:

RECORDERS/ READ OUT SYSTEMS Signal from the detector is proportional to the intensity of light incident on the detector. After amplification it is displayed as percentage transmittance (%T) or as

absorbance. Common systems employed for displaying %T or absorbance are: Moving coil meter Digital display Strip chart recorder.


SINGLE BEAM SPECTROPHOTOMETER


DOUBLE BEAM SPECTROPHOTOMETER

REFERENCES:

REFERENCES Skoog , Holler, Crouch; INSTRUMENTAL ANALYSIS , Indian Edition; Pg no: 203-213;392-396 Willard, Meritt , Dean,Settle ; INSTRUMENTAL METHODS OF ANALYSIS , seventh edition; Pg no: 118-148 A.H Beckett,J.B . Stenlake ; PRACTICAL PHARMACEUTICAL CHEMISTRY , fourth edition- part two; Pg no:

264-270;272-274 G.R. Chatwal , S.K. Anand ; INSTRUMENTAL METHODS OF CHEMICAL ANALYSIS ; Pg no: 2.167-2.172 www.google.com


Essential components of spectrophotometer are :- Source of Electro magnetic radiation Monochromator Sample compartment Detector/ transducer Recorder/ display

Block diagram of a spectrometer:

Block diagram of a spectrometer

LIGHT SOURCES:

LIGHT SOURCES Should be able to meet the following conditions: Provide sufficient radiant energy over required wavelength region. Provide stable output power during the course of measurements. Supply

continuous radiation over entire wavelength used Should be free from any fluctuation

UV Light sources:

UV Light sources HYDROGEN DISCHARGE LAMP H₂ stored under pressure. Electric discharge through lamp excite hydrogen molecules, emitting radiations. High pressure cause collisions between H₂

molecules & results in pressure broadening. Stable, robust & widely used.

DEUTERIUM DISCHARGE LAMP :

DEUTERIUM DISCHARGE LAMP Deuterium used in place of hydrogen. 3 -5 X increase in emission intensity. Expensive than hydrogen lamps.

XENON DISCHARGE LAMP :

XENON DISCHARGE LAMP Contain two tungsten electrodes separated by 8mm. Lamp filled with xenon gas stored under pressure ( 10 – 30 atm ) Low voltage application produce intense arc between

electrodes & produce UV light. High intensity of light. Limitation:- uv radiation released by lamp causes generation of ozone by ionisation of the oxygen molecule.

MERCURY ARC LAMP :

Hg (vapor form) held under high pressure. Electric discharge excites atoms. MERCURY ARC LAMP

VISIBLE LIGHT SOURCES:

VISIBLE LIGHT SOURCES TUNGSTEN FILAMENT LAM P Tungsten filament in glass envelope. Useful over range of 350 to 2000nm. Lamp life limited due to- evaporation of tungsten which darkens the inside of

the lamp reducing incident light.

TUNGSTEN HALOGEN LAMP :

TUNGSTEN HALOGEN LAMP Contain small quantity of iodine inside quartz envelope which houses tungsten filament. Iodine reacts with gaseous tungsten to form tungsten iodide. Molecules strike the

filament, tungsten is re-deposited. Quartz required for high operating temperature of the lamp.

CARBON ARC LAMP :

CARBON ARC LAMP Used when more intense source of visible light is required.

MONOCHROMATORS AND FILTERS:

MONOCHROMATORS AND FILTERS Filters Device which allows878 radiation of required wavelength to pass through Wholly or partially absorb unwanted radiation.


Absorption filters Interference filters

DIFFERENCE:

DIFFERENCE Absorption filters Work by selective absorption Made of glass Contain chemicals(dyes) that absorb all radiations except that desired to pass. Color absorbed is complement of the color of filter Interference filters Narrower bandwidth of about 15nm Consist of two parallel glass plates silvered internally Separated by thin film of dielectric material ( e.g.- cryolite ). Interference of light waves

eliminate undesired radiation

Monochromators:

Monochromators Converts polychromatic beam of light into a monochromatic beam Can isolate a selected narrow band of wavelength anywhere within a comparatively wide spectral range. Consists of:-

Entrance slit Collimator 1 Prism or grating Collimator 2 Exit slit

Types of Monochromators:

Types of Monochromators Prism monochromators : Non linear dispersion- shorter wavelength dispersed to greater degree than the longer ones. Made of glass, quartz or fused silica. Glass- visible.

Quartz, fused silica- ultraviolet region.


White light, through prism disperses into polychromatic light(rainbow.) Rotation of prism to pass required wavelength through exit slit. Effective wavelength depends on- dispersive power of wavelength

material optical angle of the prism.

Types of mounting:

Types of mounting CORNU TYPE Optical angle 60° Emerging light fall on exit slit on rotation. LITTROW TYPE Optical angle 30° One surface is aluminized. Light passes through prism & emerges on same side as

that of light source.

Diffraction gratings :

Diffraction gratings Large number of parallel lines (grooves), ruled on highly polished surface such as alumina. Done with a suitable shaped diamond tool. For UV/ Visible region about 15000- 30000

lines/square inch are drawn. Incident light diffracted over range of angles. (grooves act as scattering centre)


Construction of master grating is tedious, time consuming & expensive. Identical grooves, exactly parallel, equally spaced over length of gratings (3-10cm). Replica gratings prepared by coating original

with epoxy. Made reflective by coating surface with aluminium, gold or platinum.


Law of diffraction nλ= d (sini±sinθ)


ECHELLETE GRATING Grooved to have broad faces for reflection and narrow unused faces. Efficient diffraction of radiation due to geometry CONCAVE GRATING Grating formed on concave surface

disperses radiation and focuses it on exit slit. Eliminates need of auxillary collimating/ focusing mirrors. Cost reduction Reduction in no. of optical surface increase energy throughout monochromator .


Holographic grating Pair of identical lasers used on glass surface coated with photoresist . Interference fringes from two beams sensitize photoresist ,dissolves leaving a grooved surface. Coated with

aluminium . Greater perfection with respect to shape and dimension. Give spectra free from stray radiation & ghosts.

SAMPLE CONTAINERS:

SAMPLE CONTAINERS Cells or cuvettes used to hold the sample. Shape- rectangular or cylindrical. thickness- 1 cm Cells --- transparent in the wavelength region being measured. REGION TYPE OF

MATERIAL USED FOR CELLS OR CUVETTES VISIBLE GLASS OR PLASTIC UV REGION QUARTZ OR FUSED SILICA

Radiation Transducers/ detectors :

Radiation Transducers/ detectors Properties of radiation transducers High sensitivity High signal to noise ratio Constant response over a considerable range of wavelength Exhibit fast response time Zero output

signal in the absence of illumination

Types of radiation transducers:

Types of radiation transducers Two types :- Photon transducers/ photoelectric or quantum detectors- respond to photons Heat transducers- responds to heat Types of photon transducers -Photovoltaic cells Phototubes Photomultiplier tubes Photoconductivity transducers Silicon photodiodes Charge

transfer transducers

Barrier layer or photovoltaic cell:

Barrier layer or photovoltaic cell




Advantages Requires no power supply. Disadvantages Lack of sensitivity.

:

Photoemissive tubes

Advantages: :

Advantages: More sensitive than BLC- used in UV & Visible region Output current can be amplified- used in measuring low intensity radiations.

PHOTOMULTIPLIER TUBE :

PHOTOMULTIPLIER TUBE Successive phototubes built into one envelope. Photo cathode, series of electrodes, each at more positive potential than the one before it, an anode.

:

Disadvantages Measures low power radiations as intense light damages photo electric surface. Sensitivity limited by its dark current emission. Advantages More sensitive then phototube in UV and

visible region Ideal for measuring weak light intensity Fast response time

RECORDERS/ READ OUT SYSTEMS:

RECORDERS/ READ OUT SYSTEMS Signal from the detector is proportional to the intensity of light incident on the detector. After amplification it is displayed as percentage transmittance (%T) or as

absorbance. Common systems employed for displaying %T or absorbance are: Moving coil meter Digital display Strip chart recorder.


SINGLE BEAM SPECTROPHOTOMETER


DOUBLE BEAM SPECTROPHOTOMETER

REFERENCES:

REFERENCES Skoog , Holler, Crouch; INSTRUMENTAL ANALYSIS , Indian Edition; Pg no: 203-213;392-396 Willard, Meritt , Dean,Settle ; INSTRUMENTAL METHODS OF ANALYSIS , seventh edition; Pg no: 118-148 A.H Beckett,J.B . Stenlake ; PRACTICAL PHARMACEUTICAL CHEMISTRY , fourth edition- part two; Pg no:

264-270;272-274 G.R. Chatwal , S.K. Anand ; INSTRUMENTAL METHODS OF CHEMICAL ANALYSIS ; Pg no: 2.167-2.172 www.google.com

Applications of UV Spectroscopy:

Applications of UV Spectroscopy A seminar on Presented By Ms. Komal K. Suthar 1 st semister (2010-2011) M.Pharm. [Pharmaceutics] Roll No. 12 Guided By Mr. Shushant Thakur M.Pharm. [Pharmaceutics]

Assistant Professor Department of Pharmaceutics

Contents:

Contents Applications of UV 1.) Qualitative analysis Pharmacopoeial identification of drug Structural analysis 2.) Quantitative analysis By using beer’s law Single compound analysis Photometric titrations

Multicomponent analysis 3.) Determination of composition of complex 4.) Study of kinetics

QUALITATIVE ANALYSIS:

QUALITATIVE ANALYSIS ( a) Pharmacopoeial identification of drug : (1) By using absorbance & wavelength (2) By taking absorption ratio (3) Limit test (b)Structural analysis

2. Quantitative analysis:

2. Quantitative analysis A)By using beer’s law Using absorptivity value By using reference standard Multiple standard method B)Single compound analysis Direct analysis Using separation method After

extraction After chromatographic separation Using column chromatography Using HPLC

Slide 5:

3. Indirect analysis a)Single compound without chromophore b) Drugs with chromophoric reagent 1.For analyte which absorb weakly in UV region 2.For avoiding interference 3.Improve selectivity of assay 4. Q-techniques for chemical derivatisation C) Photometric titrations D)Multicomponent analysis Assay using

absorbance Differential spectroscopy Simultaneous equation method Absorption ratio method

Slide 6:

3. Determination of composition of complex Mole ratio method Continuous variation method ( job curve method ) 4 . Study of kinetics

(1)QUALITATIVE ANALYSIS A).Pharmacopoeial identification of drug:

(1)QUALITATIVE ANALYSIS A). Pharmacopoeial identification of drug (1)by using wavelength &absorbance e .g . morphine sulphate Take the spectrum in range of 230-360nm of 0.015 % w/v 0.1 N

HCL solution which exhibits max only at about 285nm it give absorbance approx. o.65 In alkaline pH 0.005%w/v solution of NaOH it gives 0.34 So by different wavelength we can identify different

compounds.

Slide 8:

(2)by taking absorption ratio Absorption ratio of any drug at only 2 wavelengths is constant. Aspirin at 266nm giving absorbance A1 & at 300nm it gives A2 Here absorption ratio = A1/A2 is constant For L-DOPA ratio should not be more than 0.05 p- Amino salicylic acid having absorption ratio 1.5 to 1.56

(3)Limit test:

(3)Limit test limit of light absorbing impurities in oxytetracycline Prepare 0.2%w/v solution & measure the absorbance at 430nm absorbance should not more than 0.5

Slide 10:

B. limit of p- chlorophenol in clofibrate Prepare test & standard solution of clofibrate & measure the absorbance at 455nm. Compare the absorbance of test & std. Absorbance of test must not exceed

standard.

Slide 11:

C. Salicylic Acid In Aspirin In IP & BP visual comparison method is employed in which violet colour is compared which is developed by addition of neutral FeCl 3 solution. In USP instead of visual comparison

method absorbance is measured at 305nm.

Slide 12:

D . Limit of p-amino phenol & paracetamol Sample is treated with alkaline ferricyanide reagent it produces colour with p-aminophenol & measurement is done at 710nm.

B). Structural analysis:

B). Structural analysis Chromophoric part is required for absorbance in UV. Change in chromophoric part produce predictable. Change in absorption property. E.g. Vit A exist in 2 isomeric form like vit-A 1 & A 2 Because of extra double bonds vit A2 shows bathochromic shift of 30nm. But here 25nm which is near

by thus from wavelength we can identify structure of Vit –A

(2)Quantitative Analysis :

(2)Quantitative Analysis . Single compound analysis Direct analysis Here ,the compound to be analysed does not require any chemical treatment. Compound is dissolved in suitable solvent diluted and assayed

or measured. - e.g Compound λ max Riboflavin powder 267nm Chloramphenicol capsule 278nm Chloroquine HCL injection 343nm Dexamethasone 241nm

Slide 15:

using separation method a) After extraction :- This technique applied to formation in which we have to extract that is from dosage form with the help of suitable solvent. Dilution is done with same Solvent

and measure. Absorbance drug in extracting media.

Slide 16:

Drug Extractant λ max Chlorpropramide Chloroform 232 Chloroquin phosphate tab. Chloroform 343 Chlorpheneramine Maleate syrup n- hexane 264 Chlorpromazine HCI tab Ether 277

b)After Chromatographic Separation:

b) After Chromatographic Separation Ion exchange chromatography used to separate desire component which is then measured for absorbance. E.g . Clofibrate capsules are separated by ion exchange

chromatography. It is measured by 275nm.

C)Using column chromatography:

C) Using column chromatography It is used to separate desired compound which is then analysed by spectroscopy . E.g . Aspirin tablets are subjected to column chromatography. When salicylic acid is

removed and aspirin measured at 280nm .

d)Using HPLC:

d) Using HPLC HPLC is used here for the separation of desired compound that to be estimated . E.g. Sulphamethoxazole + trimethoprim cotrimoxazole It is subjected to HPLC where both drugs are

separated. Sulfamethoxazole and trimethoprim are separately measured. It is measured at 257nm and 287nm respectively . Folic acid tablets are also analysed by this method.

Indirect Analysis:

Indirect Analysis Single component without chromophore :- Here the compound that is devoid of a chromophore is treated chemically so, that it is converted to a chromophore. e.g. phenolic compound Fecl 3 solution is added to phenolic compound to develop violet color which is then measured using an

instrument. This technique is used for large number of phenolic drug. E.g. catechol amines , paracetamol ,hormones, oestrogens , morphine and its derivatives etc.

Slide 21:

b. Drug + chromophoric reagent Drug + chromophoric reagent which give chromogen and here the absorbance of chromogen are measured in visible range.

For analyte which absorb which absorb weakly in UV region:

For analyte which absorb which absorb weakly in UV region Chemical Derivatization Indirect spectrophotometric assays are based on the conversion of the analytical by a chemical reagent to a

derivative that has different spectral properties. The majority of indirect spectrophotometric procedures

involve the conversion of the analyte to a derivative that has a longer λmax and/or a higher absorptivity. Chemical derivatisation procedures may be adopted for any of several reason.

Slide 23:

If the analyte absorbs weakly in the ultraviolet region, more sensitive method of assay is obtained by converting the substance to a derivative with a more intensely absorbing by converting chromophore. e.g. Sugars Which do not absorb significantly above 220nm can be determined. spectrophotometrically by heating with enthrone in concentrated sulphuric acid and measured the absorbance of the colored

derivatives at 625nm

e.g.:

e.g. Diazotization and Coupling of Primary aromatic Amines Condensation reactions Oxidation methods

For avoiding interference:

For avoiding interference E.g . Methyl testosterone tablet Max 240nm - drug excipients are interfering. Treat with hydrazide reagent. Oxidation of alpha ketone group of methyl testosterone with tetrazoliam

salt. Measured without interference.

Improve selectivity of assay:

Improve selectivity of assay E.g. Procaine adrenaline injection assay. Adrenaline (20 µg/ml) injection. Adrenaline measured at 270nm. Procaine at higher conc. Interference at this λ max Adrenaline reacts

with fe 2+ and gives purple colour measured by colorimetric.

Q techniques by chemical derivatization :

Q techniques by chemical derivatization Phenolic drug + FeCl 3 give violet colour. Vitamin D + antimony trichloride gives red colour and its absorbance is 500nm. By treating with primary aromatic amine + HNO

2 (HCL + NaNO 2 ) which gives diazo salt. react BMR reagent.

c) Photometric titrations:

c) Photometric titrations T itration done by photometer is called photometric titrations sample when titrated with titrant produce change in reaction in reaction mixture which is measured in terms of

absorbance change in absorbance of solution may be used to follow the change in concentration of light absorbing constituent during titration this is end point detection method

Slide 29:

principle of this technique is that formation of sample titrant complex or titration product when sample & titrant react with each other S + T = ST Where, S = sample T= Titrant

Derivative Spectroscopy:

Derivative Spectroscopy Derivative spectroscopy involves the conversion of a normal spectrum to its first, second or higher derivative spectrum. First derivative spectrum (D1) is plot of the rate of change of absorbance with wavelength against wavelength or plot of dA/ d λ The second derivative spectra (D2) is

a plot of the curvature of the first spectra against wavelength or plot of dA 2 /d λ 2 .

Slide 31:

In summery the first derivative spectrum of an absorption band is characterised by a maximum & cross over point at λ max of the absorption band. The second derivative spectra is characterised by two

satellite maxima & inverted band of which the minimum corresponds to the λ max of the fundamental band

Advantages:

Advantages Firstly an even order spectrum is of narrower spectral band width than its fundamental spectrum A derivative spectrum there fore shows better resolution of overlapping bands than the

fundamental spectrum & may permit accurate determination of λ max of individual bands Derivative spectroscopy discriminates in favour of substances of narrow spectral band widths against broad band

thus help to identify smallest interference

Differential Spectroscopy :

D ifferential Spectroscopy The selectivity and accuracy of spectrophotometric analysis of samples containing absorbing intereferents may be markedly improved by the technique of different

spectrophotometry. Different spectrophotometric assay is that measured value is the difference

absorbance (∆A ) between two equimolar solutions of the analyte in different chemical forms which exhibit different spectral characteristics.

Slide 36:

The criteria for applying difference spectrophotometry to the assay of a substance in the presence of other absorbing substance are that: (a) reproducible changes may be induced in the spectrum of the

analyte by the addition of one or more reagents. (b) the absorbance of the interfering substance is not altered by the reagents.

Slide 38:

The simplest and most commonly employed technique for altering the spectral properties of the analyte is the adjustment of the pH by means of aqueous solutions of acid, alkali or buffers. The ultra violet-visible absorption spectra of many substances containing ionisable functional groups. E.g. phenols ,

aromatic carboxylic acids and amines are dependent on the state of ionisation of the functional groups and on the PH of the solution.

Slide 39:

The absorption spectra of equimolar solutions of Phenylephrine, a phenolic sympathomimetic agent in both 0.1 M HCl (pH – 1 )and 0.1 M NaOH ( PH – 13 ). The ionisation of the phenolic group in alkaline solution generates an additional n (non bonded ) electron that interacts with the ring πelectrons to

produce a bathochromic shift of the λmax from 271nm in acidic solution to 291nm and an increase in absorbance at the λmax ( hyperchromic effect ).

Slide 40:

The different absorption spectrum is a plot of the difference in absorbance in between the solution at pH 13 and that at pH 1 against wavelength. It may be generated automatically using a double –beam

recording spectrophotometer with solution at p H 13 in the sample cell and the solution at pH 1 in the reference ( blank ) cell . At 257 and 278 nm both solutions have identical absorbance and exhibit zero

difference absorbance. Such wavelength of equal absorptivity of the two species are called isosbestic or isoabsorptive points. Above 278nm the alkaline solution absorbs more intensely than the acidic solution

and ∆A is therefore positive .

Slide 41:

Between 257 and 278nm it has a negative value. The measured value in a quantitative difference spectrophotometric assay is the ∆A at any suitable wavelength measured to the baseline , e.g. ∆ A 1 at λ 1 or amplitude between an adjacent maximum and minimum, eg ∆A 1 at λ 2 and λ 1 . At λ 1 ∆A = A alkli

– A Acid

Slide 42:

Where A alk and A acid are the individual absorbance at λ1 in 0.1M sodium hydroxide and 0.1M hydrochloric solution respectively. If the individual absorbances A alk and A acid , are proportional to the concentration of the analyte and pathlength , the ∆A also obeys the Beer – Lambert Law and a modified

equation may be derived. ∆A = ∆ abc

Slide 43:

Where ∆a is the difference absorptivity of the substance at the wavelength of measurement. If one or more other absorbing substances is present is present in the sample which at the analytical wavelength has identical absorbance in the alkaline and acidic solutions, its interference in the spectrophotometric

measurement is eliminated. ∆A = ( A alk + A x ) – ( A acid + A x ) = A alk – A acid

Determination of Composition of Complexes:

Determination of Composition of Complexes Transition metal complexes are measured by UV visible absorption spectroscopy as they form colored complexes. E.g., Fe ++ forms violet colors with Salicylic

acid. Metal (M) & ligand (L) form complex but they are of different possibilities. M + L → (M-L) 2M + L → (M-L) M + 2L → (M-L) By UV visible spectroscopy we can find out correct composition of complexes.

Fundamental Principle of Measurement:

Fundamental Principle of Measurement Complexes are colored and are easy to measure for absorbance. Color intensity is directly proportional to complex formation. Go on changing proportion of M & L &

measure absorbance & thus concentration combination having highest absorbance is the correct composition of complexes.

Mole Ratio method:

Mole Ratio method In mole ratio method concentration of either ligand or metal is kept constant. Moles of 1 component is changing & mole ratio also changes . Measure the absorbance of each mixture and

plot the graph of absorbance X v s mole ratio of Y.

Continuous variation method:

Continuous variation method It is called Job’s curve discovered by Job. Here we change proportion of both the components. X 1 +Y 1 = Z X 2 +Y 2 = Z X 3 + Y 3 = Z

Slide 48:

Proportion of both the component is changed but the sum is kept constant Measure absorbance at λ max Y is plotted as Y is changing in increasing manner. At a particular ratio there is max absorbance.

4. Study of kinetics:

4. Study of kinetics Study of reaction rate of drugs can be done using UV visible absorption spectroscopy. Absorbance of drug is measured at different time intervals & the concentration is calculated. E.g.

paracetamol is degraded to 4-amino phenol.

Slide 50:

Here we can set the instruments at 257nm & go on measuring absorbance. The rate of decrease in absorbance is equal to the rate of degradation of drugs. Also we can set the instrument at 300nm & measure the absorbance. The rate of increase in absorbance is equal to rate of degradation of drugs.

Drugs measured by UV absorption spectroscopy as per IP2007:

Drugs measured by UV absorption spectroscopy as per IP2007 SR NO DRUGS ABSORPTIVITY(a ) λ max ( nm ) 1 Chloramphenicol (anti biotic) IP 297 278 2 Caffeine (diuretic, CNS stimulant) IP 504 273 3

Atenolol tab ( β blockers) IP 53.7 275 4 Neostigmine inj (sympatholytic) IP 14.35 260 5 Promethazine tab (anti emetic) IP 910 249 6 Riboflavin ( Vit- b2) IP 328 444 7 Tinidazole ( anti amoebic) IP 356 310 8

Tamoxifen tab ( anti cancer) IP 325 275 9 Verapamil ( calcium channel blocker) IP 118 278 10 Warfarin(anti coagulant) IP 431 308

References:

References Sharma B. K.; Instrumental Method of Chemical Analysis; Krishna Prakashan media ltd; 24 th edition; 2005; S-135,164 Beckett A.H., Stenlake J.B.; Practical Pharmaceutical Chemistry; CBS publishers

and distributers; 4 th edition; 2005; 292-303 Indian Pharmacopoeia; Ministry of Health & Family welfare; Published by Indian Pharmacopoeial commission, Ghaziabad; 2007; volume I, II .

What is spectroscopy? Spectrum + Scopies “When a beam of light is allowed to pass through a prism or grating, it will dispersed into seven colors from red to violet and the set of colors or band produced is called spectrum” + Examination “Spectroscopy is the branch of the science dealt with the study of

interaction of Electro Magnetic Radiation (EMR) with matter” So the spectroscopy means examination of spectrum. From the type of radiation, which is absorbed, we can get idea about the nature (type) of

the compound and from the amount of the radiation, which is absorbed, we can get idea about the concentration (amount) of the substance. So the spectroscopy is used for qualitative and quantitative

analysis. :

What is spectroscopy? Spectrum + Scopies “When a beam of light is allowed to pass through a prism or grating, it will dispersed into seven colors from red to violet and the set of colors or band produced is

called spectrum” + Examination “Spectroscopy is the branch of the science dealt with the study of interaction of E lectro M agnetic R adiation (EMR) with matter” So the spectroscopy means examination of spectrum. From the type of radiation, which is absorbed, we can get idea about the nature (type) of

the compound and from the amount of the radiation, which is absorbed, we can get idea about the concentration (amount) of the substance. So the spectroscopy is used for qualitative and quantitative

analysis.

Slide 3:

When a beam of light is passed through a transparent cell containing a solution of an absorbing substance, reduction of the intensity of the light may occur. This is due to 1) Reflection at the inner and outer surfaces of the cell 2) Scattered by the particles present in the solution 3) Absorption of the

light by the molecules in the solution Interaction of EMR with matter

Classification of Spectroscopy: 1) Absorption Spectroscopy: the type and amount of the radiation, which is absorbed depend upon the structure of the molecules and the numbers of molecules

interacting with the radiation. The study of these dependencies is called absorption spectroscopy. (UV, IR, NMR, X-Ray, ESR) 2) Emission spectroscopy: if sufficient energy gets impinged upon a sample, the outer electrons in the species will be raised from their stable ground state to higher

energy level (unstable in nature). These excited species rapidly emits a photon and return to their ground stable energy level. The type and amount of radiation, which is emitted, is studied, this type of

spectroscopy is called emission spectroscopy. (AES, MES, Fluorimetry) 3) Scattering spectroscopy: if the incoming radiation strikes with the solid particles suspended in the solution, the light transmitted

at an angle other than 1800 from the incident light. This spectroscopy is called scattering spectroscopy. (turbidimetry, nephelometry) :

Classification of Spectroscopy: 1) Absorption Spectroscopy: the type and amount of the radiation, which is absorbed depend upon the structure of the molecules and the numbers of molecules

interacting with the radiation. The study of these dependencies is called absorption spectroscopy. (UV, IR, NMR, X-Ray, ESR) 2) Emission spectroscopy: if sufficient energy gets impinged upon a sample, the

outer electrons in the species will be raised from their stable ground state to higher energy level (unstable in nature). These excited species rapidly emits a photon and return to their ground stable

energy level. The type and amount of radiation, which is emitted, is studied, this type of spectroscopy is called emission spectroscopy. (AES, MES, Fluorimetry) 3) Scattering spectroscopy: if the incoming radiation strikes with the solid particles suspended in the solution, the light transmitted at an angle

other than 180 0 from the incident light. This spectroscopy is called scattering spectroscopy. (turbidimetry, nephelometry)

What is EMR? - EMR is a form of energy that is transmitted through space at an enormous velocity - It can travel in space with the same speed at that of light. - As the name implies an EMR is an alternating electrical and associated magnetic force field in space (It contains electrical and magnetic components) - The two components oscillate in planes perpendicular to each other and

perpendicular to the direction of propagation of the radiation. - EMR consist of a stream of discrete packets (particles) of pure energy, which is called photons or quanta. - The energy of

photon is proportional to the frequency E = hυ where E= Energy of photons, h= plank’s constant (6.624 x 10-27 erg. Sec) and υ=frequency of radiation in cycles/second :

What is EMR? - EMR is a form of energy that is transmitted through space at an enormous velocity - It can travel in space with the same speed at that of light. - As the name implies an EMR is an

alternating electrical and associated magnetic force field in space (It contains electrical and magnetic components) - The two components oscillate in planes perpendicular to each other and

perpendicular to the direction of propagation of the radiation. - EMR consist of a stream of discrete packets (particles) of pure energy, which is called photons or quanta. - The energy of photon is

proportional to the frequency E = hυ where E= Energy of photons, h= plank’s constant (6.624 x 10 -27 erg. Sec) and υ=frequency of radiation in cycles/second

- Wavelength (λ): it is the distance between two successive maxima on an electromagnetic wave. (m, cm, mm, μm, nm, and A0) - Frequency (υ): is the numbers of waves passing through a given point in

unit time. (T-1, sec-1, cycles/second, hertz, fresnel) - Wave numbers (ΰ): is the numbers of waves per centimeter in vacuum. ( cm-1) - Velocity (V): is the product of wavelength and frequency (λ X υ =V)

(cm/sec, m/sec) :

- Wavelength (λ): it is the distance between two successive maxima on an electromagnetic wave. (m, cm, mm, μm, nm, and A 0 ) - Frequency (υ): is the numbers of waves passing through a given point in

unit time. (T -1 , sec -1 , cycles/second, hertz, fresnel) - Wave numbers (ΰ): is the numbers of waves per centimeter in vacuum. ( cm -1 ) - Velocity (V): is the product of wavelength and frequency (λ X υ =V)

(cm/sec, m/sec)

Slide 7:

Classification of EMR : EMR is arbitrarily classified in to different regions according to wavelength.

Energy associated with the molecules: 1) The molecule as a whole may move this is called translation and the energy associate with this movement is called transnational energy. (Etrans) 2) The part of the molecules, that is atom or groups of atoms, may move with respect to each other. This

motion is called vibration and the associated energy is called vibrational energy. (Evib) 3) The molecule may rotate about an axis. And such rotation is characterized by the rotational energy. (Erot) 4) Besides these modes of movements, the molecule possesses an electronic configuration and the energy associated with this configuration is called electronic energy. (E ele) E total = E trans + E vib + E

rot + E ele. :

Energy associated with the molecules: 1) The molecule as a whole may move this is called translation and the energy associate with this movement is called transnational energy. (E trans ) 2) The part of the molecules, that is atom or groups of atoms, may move with respect to each other. This motion is called vibration and the associated energy is called vibrational energy. (E vib ) 3) The molecule may rotate about an axis. And such rotation is characterized by the rotational energy. (E rot ) 4) Besides

these modes of movements, the molecule possesses an electronic configuration and the energy associated with this configuration is called electronic energy. (E ele ) E total = E trans + E vib + E rot + E

ele.

Theoretical principles If a molecule is allowed to interact with the EMR of a proper frequency, the energy of the molecule is raised from one level to a higher one; we say that absorption of radiation takes place. In order for absorption to occur, the energy difference between the two energy level

must be equal to the energy of the photon absorbed E2 – E1 = hυ where E1 is energy of lower level and E2 is the energy of upper level - This energy jump from one level to another is called

transition - The graph of the light absorption against the frequency is called absorption spectra. - Visible and Ultraviolet light provides enough energy for electronic transition there for called

electronic spectra. :

Theoretical principles If a molecule is allowed to interact with the EMR of a proper frequency, the energy of the molecule is raised from one level to a higher one; we say that absorption of radiation

takes place. In order for absorption to occur, the energy difference between the two energy level must be equal to the energy of the photon absorbed E2 – E1 = hυ where E1 is energy of lower level and E2 is the energy of upper level - This energy jump from one level to another is called transition - The

graph of the light absorption against the frequency is called absorption spectra. - Visible and Ultraviolet light provides enough energy for electronic transition there for called electronic spectra.

On absorption of energy by a molecule in the ultraviolet region, changes are produced in the electronic energy of the molecule due to transitions of valence electrons in the molecule. :

On absorption of energy by a molecule in the ultraviolet region, changes are produced in the electronic energy of the molecule due to transitions of valence electrons in the molecule. E 6* Anti-bonding π*

Anti- bonding n Non- bonding π Bonding 6 Bonding

Types of transitions: 1) 6 6*: A transitions of electrons from a bonding sigma orbital to the higher energy antibonding orbitals. ( eg. Alkane). Sigma bonds are, in general, very strong, there fore they

require high energy for the transitions and this transitions requires very short wavelength (near about 150 nm) 2) n 6*: This transition involves saturated compounds with one hetero atom with unshared pair of electrons (n electrons). Corresponding band appears at 180-200 nm. 3) π π*: This transition is available in compounds with un-saturation (eg. Alkene). Corresponding band appears at 170-190 nm.

4) n π*: This type of transitions are shown by the unsaturated molecules containing one or more hetero atoms. (O, N, S):

Types of transitions: 1) 6 6*: A transitions of electrons from a bonding sigma orbital to the higher energy antibonding orbitals. ( eg. Alkane). Sigma bonds are, in general, very strong, there fore they require high energy for the transitions and this transitions requires very short wavelength (near about 150 nm) 2) n 6*: This transition involves saturated compounds with one hetero atom with unshared pair of electrons

(n electrons). Corresponding band appears at 180-200 nm. 3) π π*: This transition is available in compounds with un-saturation (eg. Alkene). Corresponding band appears at 170-190 nm. 4) n π*: This

type of transitions are shown by the unsaturated molecules containing one or more hetero atoms. (O, N, S)

Slide 12:

5) Conjugated system: In conjugated dienes, the π orbitals of the separate alkene group combine to give new orbitals i.e. the two new bonding orbitals which are designated π1 and π2 and new two anti-

bonding orbitals designated as π3* and π4*. So for the π2 π3* transition very low energy is requires corresponding to the higher wavelength.

Some important terms: 1) Chromophore: It is a group of molecules, which is responsible for the absorption of light by molecules. It is conjugated dienes. It is minimum structural requirements for the

absorption of radiation in UV range. 2) Auxochrome: It is a saturated group containing unshared electrons which when attached to a Chromophore changes both intensity as well as the wavelength of

the absorption maxima. e.g. OH, NH2, Cl etc. 3) λ-max: It is a wavelength at which there is a maximum absorption or absorption intensity. It is a physical constant and characteristic of structure

and so useful for identification of compounds. It is independent of concentration. 4) Bathochromic shift: The shifting of absorption to a longer wavelength due to substitution or solvent is called as

bathochromic shift. It is also called as Red shift. e.g., λmax of Ascorbic acid=243nm, λmax of Ascorbic acid in alkali medium=299nm.:

Some important terms: 1) Chromophore: It is a group of molecules, which is responsible for the absorption of light by molecules. It is conjugated dienes. It is minimum structural requirements for the

absorption of radiation in UV range. 2) Auxochrome: It is a saturated group containing unshared electrons which when attached to a Chromophore changes both intensity as well as the wavelength of

the absorption maxima. e.g. OH, NH2, Cl etc. 3) λ-max: It is a wavelength at which there is a maximum absorption or absorption intensity. It is a physical constant and characteristic of structure and so useful

for identification of compounds. It is independent of concentration. 4) Bathochromic shift: The shifting of absorption to a longer wavelength due to substitution or solvent is called as bathochromic shift. It is also called as Red shift. e.g., λ max of Ascorbic acid=243nm, λ max of Ascorbic acid in alkali

medium=299nm.

5) Hypsochromic shift (Blue shift): Shifting of λmax to lower value or left hand side due to substitution, solvent, pH etc is called as Hypsochromic shift. e.g. λmax of Phenol in basic

media=297nm, λmax of Phenol in acidic media=277nm. 6) Hyperchromism: Increase in absorption intensity (e) due to solvent, pH or some other factors called hyperchromic effect. 7)

Hypochromism : Decrease in absorption intensity due to substituent, solvent, pH etc. called hypochromic effect. 8) A1%1cm (A one percent one centimeter): Is the absorbance of the solution having concentration 1 gm per 100 ml of the solution. 9) Molar absorptivity (ε): Is the absorbance of the solution having concentration gm.mol.weight/1000 ml of the solution. [ε = (A1%1cm X Mol. Wt.)/10] 10) Transmittance (T): is the ratio of IT/I0 and % transmittance (%T) is given by %T=100

IT/I0 :

5) Hypsochromic shift (Blue shift): Shifting of λ max to lower value or left hand side due to substitution, solvent, pH etc is called as Hypsochromic shift. e.g. λ max of Phenol in basic media=297nm, λ max of

Phenol in acidic media=277nm. 6) Hyperchromism: Increase in absorption intensity ( e ) due to solvent, pH or some other factors called hyperchromic effect. 7) Hypochromism : Decrease in

absorption intensity due to substituent, solvent, pH etc. called hypochromic effect. 8) A 1% 1cm (A one percent one centimeter): Is the absorbance of the solution having concentration 1 gm per 100 ml of

the solution. 9) Molar absorptivity (ε): Is the absorbance of the solution having concentration

gm.mol.weight/1000 ml of the solution. [ε = (A 1% 1cm X Mol. Wt.)/10] 10) Transmittance (T): is the ratio of I T /I 0 and % transmittance (%T) is given by %T=100 I T /I 0

1) Absorbance (A): Is the degree of absorption of light by a medium through which the energy passes. It is expressed as the logarithm of the ratio of light transmitted through a pure solvent to the

intensity of light transmitted through the medium. It is the area under the curve. A= log I0/IT A= log I0 - log IT A=2- log % T Absorption Spectra The graph of the light absorption against the frequency is

called absorption spectra. It is characterized by 1) λ-max:- Position of spectra 2) Intensity of absorbance:- the amount of the radiation absorbed by the molecule. 1) Factors affecting the

position of the spectrum (λ-max) A) Structural factors i) Substitution: Placing a substituent on a Chromophore may produce change in λ-max by two mechanisms: introduction of an entirely new

transition and/or shifting the wavelength of existing transitions. E.g. each alkyl substituent produce 5 nm bathochromic shift. :

1) Absorbance (A): Is the degree of absorption of light by a medium through which the energy passes. It is expressed as the logarithm of the ratio of light transmitted through a pure solvent to the intensity of

light transmitted through the medium. It is the area under the curve. A= log I 0 /I T A= log I 0 - log I T A=2- log % T Absorption Spectra The graph of the light absorption against the frequency is called

absorption spectra. It is characterized by 1) λ-max:- Position of spectra 2) Intensity of absorbance:- the amount of the radiation absorbed by the molecule. 1) Factors affecting the position of the spectrum (λ-max) A) Structural factors i) Substitution: Placing a substituent on a Chromophore may produce

change in λ-max by two mechanisms: introduction of an entirely new transition and/or shifting the wavelength of existing transitions. E.g. each alkyl substituent produce 5 nm bathochromic shift.

ii) Solvent: the solvent effect arises because solvation is frequently different for the ground and excited states. If the ground state is solvated more strongly than the excited state, the energy

difference between the levels is increased. The increase in energy difference is reflected in a shift of the absorbance to shorter wavelengths. iii) Geometry: e.g. stilbene. Trans-stilbene absorbs at a

longer wavelength than cis-stilbene due to steric effects. Co-planarity is needed for the most effective overlap of the π-orbitals. The cis-isomer is forced in to a non planar conformation due to steric effects. The cis isomer are twisted slightly out of plane by steric interactions so that the degree of conjugation

in the π system is slightly less than the trans isomers, resulting in greater energy for the transitions. A) Non Structural factors i) PH: e.g. Phenolphthalein: in alkaline medium it is pink and in the

acidic medium it is colorless:

ii) Solvent: the solvent effect arises because solvation is frequently different for the ground and excited states. If the ground state is solvated more strongly than the excited state, the energy difference

between the levels is increased. The increase in energy difference is reflected in a shift of the absorbance to shorter wavelengths. iii) Geometry: e.g. stilbene. Trans-stilbene absorbs at a longer

wavelength than cis-stilbene due to steric effects. Co-planarity is needed for the most effective overlap

of the π-orbitals. The cis-isomer is forced in to a non planar conformation due to steric effects. The cis isomer are twisted slightly out of plane by steric interactions so that the degree of conjugation in the π

system is slightly less than the trans isomers, resulting in greater energy for the transitions. A) Non Structural factors i) PH: e.g. Phenolphthalein: in alkaline medium it is pink and in the acidic medium

it is colorless

i) Temperature: Temperature provides more energy to ground state. As a result energy required for excitation will be less, so there is bathochromic shift. 2) Factors affecting the intensity of

absorption of radiation. i) Thickness of the medium: Lambert’s law: “when a beam of monochromatic light is allowed to pass through a transparent medium, the rate of decrease of

intensity with the thickness of medium is directly proportional to the intensity of incident radiation”. It gives relationship between absorbance and the thickness of the medium. ii) Concentration of absorbing solute: Beer’s law: “when a beam of monochromatic light is allowed to pass through a

transparent medium, the rate of decrease of intensity with the concentration of absorbing solute is directly proportional to the intensity of incident radiation”. It gives relationship between absorbance

and the concentration of the medium. A = a b c (Fundamental equations of spectroscopy) :

i) Temperature: Temperature provides more energy to ground state. As a result energy required for excitation will be less, so there is bathochromic shift. 2) Factors affecting the intensity of absorption of radiation. i) Thickness of the medium: Lambert’s law: “when a beam of monochromatic light is allowed to pass through a transparent medium, the rate of decrease of intensity with the thickness of medium is directly proportional to the intensity of incident radiation”. It gives relationship between absorbance and the thickness of the medium. ii) Concentration of absorbing solute: Beer’s law:

“when a beam of monochromatic light is allowed to pass through a transparent medium, the rate of decrease of intensity with the concentration of absorbing solute is directly proportional to the intensity

of incident radiation”. It gives relationship between absorbance and the concentration of the medium. A = a b c (Fundamental equations of spectroscopy)

Deviation from the Beer’s curve. (Errors in spectrophotometric measurement) Errors may arise from instrumental of from chemical factors. Instrumental errors can arise from several sources. Noise,

fluctuation in light source. The ideal absorbance range for most measurement is in the range of 0.2 to 0.8. The calibration curve is relatively linear in this range. Other factor includes Spectral Slit Width

(SSW). As slit width is increased, the fine structure of the absorption band is lost as the incident light is no more monochromatic. Generally, fast scan rates tend to distort spectra, altering the positions of

both maxima and minima as well as diminishing peak intensities. This introduces both qualitative and quantitative errors in the measurement. :

Deviation from the Beer’s curve. (Errors in spectrophotometric measurement) Errors may arise from instrumental of from chemical factors. Instrumental errors can arise from several sources. Noise,

fluctuation in light source. The ideal absorbance range for most measurement is in the range of 0.2 to

0.8. The calibration curve is relatively linear in this range. Other factor includes Spectral Slit Width (SSW). As slit width is increased, the fine structure of the absorption band is lost as the incident light is

no more monochromatic. Generally, fast scan rates tend to distort spectra, altering the positions of both maxima and minima as well as diminishing peak intensities. This introduces both qualitative and

quantitative errors in the measurement.

A numbers of chemical factors may also produce errors in the analysis. Solute-solute interaction, e.g. aggregation, precipitation, dimerization etc. Ionization or even complexation of the analyte in solution

can also lead to apparent deviation from the Beer’s curve. Fluorescence from absorbing species in solution may also contribute to interference.:

A numbers of chemical factors may also produce errors in the analysis. Solute-solute interaction, e.g. aggregation, precipitation, dimerization etc. Ionization or even complexation of the analyte in solution

can also lead to apparent deviation from the Beer’s curve. Fluorescence from absorbing species in solution may also contribute to interference.

INSTRUMENTATION :

INSTRUMENTATION Light source Slit Monochromator Sample Holder Detector Display Light Source: (source of electromagnetic radiation): The tungsten filament lamp is a satisfactory light source for the

region 350 to 2000 nm. It consists of a tungsten filament contained in a glass envelope. The most convenient light source for UV radiation is discharge lamp. Generally deuterium discharge lamp is used.

It is consisting of deuterium-filled silica envelope. It gives radiation from 185 to 380 nm.

2) Slit: (Radiation intensity controlling device): Enough light must pass through the sample to elicit a measurable response from the detector. 3) Monochromator: (wavelength selecting device). It

converts polychromatic light in monochromatic light (light having one wavelength). a) Filters: Glass filters are pieces of colored glass, which transmit limited wavelength ranges of the spectrum. The

color is produced by incorporating oxides of such metals. :

2) Slit: (Radiation intensity controlling device): Enough light must pass through the sample to elicit a measurable response from the detector. 3) Monochromator: (wavelength selecting device). It converts

polychromatic light in monochromatic light (light having one wavelength). a) Filters: Glass filters are pieces of colored glass, which transmit limited wavelength ranges of the spectrum. The color is

produced by incorporating oxides of such metals.

b) Prisms: When a beam of light passes through a prism, it is bent or refracted. The amount of deviation is dependent on the wavelength. The prism is made up of quartz for use in the UV light,

since glass absorbs wavelengths shorter than about 330 nm. Glass prism are preferable for the visible region of the spectrum, as the dispersion is much greater than that obtained with quartz. c) Grating: Most modern UV spectrophotometer uses diffraction grating as a Monochromator. It consisting of a very large number of equispaced lines (200-2000 per mm) ruled on a glass plate. They can be used

either as transmission grating or when aluminized, as reflection grating. :

b) Prisms: When a beam of light passes through a prism, it is bent or refracted. The amount of deviation is dependent on the wavelength. The prism is made up of quartz for use in the UV light, since glass

absorbs wavelengths shorter than about 330 nm. Glass prism are preferable for the visible region of the spectrum, as the dispersion is much greater than that obtained with quartz. c) Grating: Most modern UV spectrophotometer uses diffraction grating as a Monochromator. It consisting of a very large number of

equispaced lines (200-2000 per mm) ruled on a glass plate. They can be used either as transmission grating or when aluminized, as reflection grating.

4) Sample Holder: The sample holder is known as cuvettes. Cuvettes must be transparent to the light, so the glass cells are used in the visible region and quartz or silica cells are used in the UV region. The cells used in the UV spectrophotometers are usually 1 cm in path length but cells are available from 0.1 cm to 10 cm or more. 5) Detectors (Radiation measuring device): It is also known as photocell.

They convert radiation energy in electrical energy. For the determination of substances by spectrophotometric techniques, precise determinations of the light intensities are necessary.

Photoelectric detectors are most frequently used for this purpose. They must be employed in such a way that they give a response linearly proportional to the light input and they must not suffer from

drift or fatigue.:

4) Sample Holder: The sample holder is known as cuvettes. Cuvettes must be transparent to the light, so the glass cells are used in the visible region and quartz or silica cells are used in the UV region. The cells

used in the UV spectrophotometers are usually 1 cm in path length but cells are available from 0.1 cm to 10 cm or more. 5) Detectors (Radiation measuring device): It is also known as photocell. They convert

radiation energy in electrical energy. For the determination of substances by spectrophotometric techniques, precise determinations of the light intensities are necessary. Photoelectric detectors are

most frequently used for this purpose. They must be employed in such a way that they give a response linearly proportional to the light input and they must not suffer from drift or fatigue.

a) Barrier-layer photocell: :

a) Barrier-layer photocell:

It one of the simplest detectors, which has the advantage that it requires no power supply but gives a current, which is directly proportional to the light intensity. It is consists of a metallic plate, usually

copper or iron, upon which is deposited a layer of selenium. An extremely thin transparent layer of a good conducting metal, e.g. silver, platinum or copper, is formed over the selenium to act as one

electrode, the metallic plate acting as the other. Light passes through the semitransparent silver layer causes release of an electron, which migrates, to the collector. The electron accumulating on the

collector resulting in a potential difference between the base and collector, which can be measured by a low resistance galvanometer circuit. The useful working range of selenium photocell is 380-780 nm. Their lack of sensitivity compared to phototube and photo multiplier tube, restricts their use to the

cheapest colorimeters and flame photometers. :

It one of the simplest detectors, which has the advantage that it requires no power supply but gives a current, which is directly proportional to the light intensity. It is consists of a metallic plate, usually

copper or iron, upon which is deposited a layer of selenium. An extremely thin transparent layer of a good conducting metal, e.g. silver, platinum or copper, is formed over the selenium to act as one

electrode, the metallic plate acting as the other. Light passes through the semitransparent silver layer causes release of an electron, which migrates, to the collector. The electron accumulating on the

collector resulting in a potential difference between the base and collector, which can be measured by a low resistance galvanometer circuit. The useful working range of selenium photocell is 380-780 nm. Their lack of sensitivity compared to phototube and photo multiplier tube, restricts their use to the

cheapest colorimeters and flame photometers.

It consists of an anode and a cathode sealed in an evacuated glass tube, which may have a quartz or silica window for UV measurement. :

It consists of an anode and a cathode sealed in an evacuated glass tube, which may have a quartz or silica window for UV measurement. b) Photo emissive tube:

The cathode is coated with a layer of light sensitive material that emits electrons upon absorption of photons. A power supply maintains the anode positive with respect to the cathode so that the

photoelectrons are collected at the anode. This current is directly proportional to the light intensity. Phototubes are available for use over the entire UV/visible region of the spectrum, but no single tube covers the entire range satisfactorily. Therefore many instruments with phototube detectors employ interchangeable blue and red sensitive phototube in order to provide sufficient sensitivity over the

entire spectrum. :

The cathode is coated with a layer of light sensitive material that emits electrons upon absorption of photons. A power supply maintains the anode positive with respect to the cathode so that the

photoelectrons are collected at the anode. This current is directly proportional to the light intensity. Phototubes are available for use over the entire UV/visible region of the spectrum, but no single tube

covers the entire range satisfactorily. Therefore many instruments with phototube detectors employ interchangeable blue and red sensitive phototube in order to provide sufficient sensitivity over the

entire spectrum.

c) Photo multiplier tube: :

c) Photo multiplier tube: It is very sensitive detectors with very short response times. It contains a photo cathode and a series of dynodes, which are also photosensitive.

A higher successive potential is maintained between each dynodes. A photoelectrons released from the photo cathode is accelerated toward the first dynode by their voltage difference, where it strikes to release several electrons. The secondary electrons are then accelerated toward the second dynode

where the process repeats. In this way multiplication of the electrons can be achieved. The current from phototubes and photo multiplier tubes never falls to zero. A small residual current called dark

current is produced, due to long exposure of the light. :

A higher successive potential is maintained between each dynodes. A photoelectrons released from the photo cathode is accelerated toward the first dynode by their voltage difference, where it strikes to release several electrons. The secondary electrons are then accelerated toward the second dynode

where the process repeats. In this way multiplication of the electrons can be achieved. The current from phototubes and photo multiplier tubes never falls to zero. A small residual current called dark current is

produced, due to long exposure of the light.

6) Display (Read out meter): The signal from the detector is normally proportional to the intensity of light, and after amplification may be displayed as % T or after passing through a logarithmic

conversion circuit as absorbance (Log 1/T) TYPES OF INSTRUMENTS: Instruments for measuring the absorption of light may be of the single beam or double beam type. In a single beam instrument, light

from the sources passes through a filter and then through the sample and in to the detector. The signal from the detector is proportional to the intensity of the light beam striking it. To make a

measurement of absorbance using a manually controlled single-beam instrument, the Monochromator is adjusted to the required wavelength and the appropriate lamp and photocell are

selected by means of switches. :

6) Display (Read out meter): The signal from the detector is normally proportional to the intensity of light, and after amplification may be displayed as % T or after passing through a logarithmic conversion circuit as absorbance (Log 1/T) TYPES OF INSTRUMENTS: Instruments for measuring the absorption of

light may be of the single beam or double beam type. In a single beam instrument, light from the sources passes through a filter and then through the sample and in to the detector. The signal from the

detector is proportional to the intensity of the light beam striking it. To make a measurement of

absorbance using a manually controlled single-beam instrument, the Monochromator is adjusted to the required wavelength and the appropriate lamp and photocell are selected by means of switches.

The first step is to close a shutter in the path (or adjust dark filter) and adjust 0 %T. The second step is to open the shutter and place the cell containing only the solvent in the light beam and adjust the scale on 100 % T (equivalent to 0 absorbance). The third step is to place the sample cell in the light

path and measure the intensity IT or its equivalent absorbance. In double beam spectrophotometer, the monochromatic light is split by the beam splitter in to two equal intensity light beam, which are

directed alternatively in rapid succession through a cell containing the sample and one containing the solvent only. This instrument measures the ratio of the intensity of the beam coming through the

sample and through the solvent. Changes in the intensity of the source affect both beams proportionately so the ratio of their intensities is not altered. Therefore, a high degree of stability in the light source is not required in these instruments. Difference in the lamp out put, optical system

throughput, and detector sensitivity with wavelength also affect both beams in the same way. :

The first step is to close a shutter in the path (or adjust dark filter) and adjust 0 %T. The second step is to open the shutter and place the cell containing only the solvent in the light beam and adjust the scale on

100 % T (equivalent to 0 absorbance). The third step is to place the sample cell in the light path and measure the intensity I T or its equivalent absorbance. In double beam spectrophotometer, the

monochromatic light is split by the beam splitter in to two equal intensity light beam, which are directed alternatively in rapid succession through a cell containing the sample and one containing the solvent only. This instrument measures the ratio of the intensity of the beam coming through the sample and through the solvent. Changes in the intensity of the source affect both beams proportionately so the ratio of their intensities is not altered. Therefore, a high degree of stability in the light source is not

required in these instruments. Difference in the lamp out put, optical system throughput, and detector sensitivity with wavelength also affect both beams in the same way.

APPLICATIONS: 1. Qualitative Analysis: The UV spectra of most compounds are of limited value for qualitative analysis as compared to IR and Mass spectra. Qualitative analytical use of UV spectra has

largely involved λ-max and absorptivities, occasionally includes absorption minima. In pharmacopoeias, absorption ratios have found use in identity tests, and are referred to as Q-values in

USP. 2. Quantitative Analysis: UV spectroscopy is perhaps the most widely used spectroscopic techniques for the quantitative analysis of chemical substances as pure materials and as components

of dosage forms. :

APPLICATIONS: 1. Qualitative Analysis: The UV spectra of most compounds are of limited value for qualitative analysis as compared to IR and Mass spectra. Qualitative analytical use of UV spectra has

largely involved λ-max and absorptivities, occasionally includes absorption minima. In pharmacopoeias, absorption ratios have found use in identity tests, and are referred to as Q-values in USP. 2. Quantitative

Analysis: UV spectroscopy is perhaps the most widely used spectroscopic techniques for the quantitative analysis of chemical substances as pure materials and as components of dosage forms.

A) Single component Analysis: Direct Analysis: Essentially all compounds containing conjugated double bond or aromatic rings, and many inorganic species absorb light in the UV-visible regions. In these techniques the substance to be determined is dissolved in suitable solvent and diluted to the

required concentration by appropriate dilutions and absorbance is measured. Indirect Analysis: (Analysis after addition of some reagent) indirect methods are based on the conversion of the analyte by a chemical reagent that has different spectral properties. Chemical derivatization may be adopted for any of the several reasons. 1) If the analyte absorbs weakly in the UV region. 2) The interference

form irrelevant absorption may be avoided by converting the analyte to a derivative, which absorbs in the visible region, where irrelevant absorption is negligible. 3) This technique can be used to improve

the selectivity of the assay in presence of other UV radiation absorbing substance. 4) Cost. :

A) Single component Analysis: Direct Analysis: Essentially all compounds containing conjugated double bond or aromatic rings, and many inorganic species absorb light in the UV-visible regions. In these

techniques the substance to be determined is dissolved in suitable solvent and diluted to the required concentration by appropriate dilutions and absorbance is measured. Indirect Analysis: (Analysis after addition of some reagent) indirect methods are based on the conversion of the analyte by a chemical reagent that has different spectral properties. Chemical derivatization may be adopted for any of the several reasons. 1) If the analyte absorbs weakly in the UV region. 2) The interference form irrelevant

absorption may be avoided by converting the analyte to a derivative, which absorbs in the visible region, where irrelevant absorption is negligible. 3) This technique can be used to improve the selectivity of the

assay in presence of other UV radiation absorbing substance. 4) Cost.

Methods of calculating concentration in single component analysis · By using the relationship: A = a b c · By using the formula: Cu = (Au/As) X Cs · By using the equations: Y = mX + C · By

using the Beer’s curve A) Multi component Analysis: a) Simultaneous Equations method: If a sample contains two absorbing drugs (X and Y) each of which absorbs at the λ-max of the other (λ1 and λ2), it

may be possible to determine both the drugs by the simultaneous equations method. Criteria for obtaining maximum precision, below mentioned ratio should lie out side the range 0.1-2.0 (A2/A1) /

(aX2/aX1) and (aY2/aY1) / (A2/A1) :

Methods of calculating concentration in single component analysis · By using the relationship: A = a b c · By using the formula: Cu = (Au/As) X Cs · By using the equations: Y = mX + C · By using the Beer’s curve A) Multi component Analysis: a) Simultaneous Equations method: If a sample contains two absorbing

drugs (X and Y) each of which absorbs at the λ-max of the other (λ1 and λ2), it may be possible to determine both the drugs by the simultaneous equations method. Criteria for obtaining maximum

precision, below mentioned ratio should lie out side the range 0.1-2.0 (A2/A1) / (aX2/aX1) and (aY2/aY1) / (A2/A1)

The information required is · The absorptivities of X at λ1 and λ2, aX1 and aX2 · The absorptivities of Y at λ1 and λ2, aY1 and aY2 · The absorbances of the diluted sample at λ1 and λ2, A1 and A2 Let Cx and Cy be the concentration of X and Y respectively in the sample The absorbance of the mixture is the sum of the individual absorbances of X and Y At λ1 A1 = aX1* Cx + aY1* Cy (1) At λ2 A2 = aX2* Cx + aY2* Cy (2) Multiply the equation (1) with aX2 and (2) with aX1 A1 aX2 = aX1 Cx aX2 +

aY1 Cy aX2 (3) A2 aX1 = aX2 Cx aX1+ aY2 Cy aX1 (4) :

The information required is · The absorptivities of X at λ1 and λ2, aX1 and aX2 · The absorptivities of Y at λ1 and λ2, aY1 and aY2 · The absorbances of the diluted sample at λ1 and λ2, A1 and A2 Let Cx and Cy be the concentration of X and Y respectively in the sample The absorbance of the mixture is the sum of the

individual absorbances of X and Y At λ1 A1 = aX1* Cx + aY1* Cy (1) At λ2 A2 = aX2* Cx + aY2* Cy (2) Multiply the equation (1) with aX2 and (2) with aX1 A1 aX2 = aX1 Cx aX2 + aY1 Cy aX2 (3) A2 aX1 = aX2

Cx aX1+ aY2 Cy aX1 (4)

A1 aX2 - A2 aX1 = aY1 Cy aX2 - aY2 Cy aX1 A1 aX2 - A2 aX1 = Cy (aY1 aX2 - aY2 aX1) Cy = (A1 aX2 - A2 aX1) / (aY1 aX2 - aY2 aX1) (5) Same way we can derive Cx = (A2 aY1 – A1 aY2) / (aY1 aX2 - aY2 aX1) (6) Equations 5 and 6 are known as simultaneous equations and by solving these simultaneous equations

we can determine the concentration of X and Y in the sample. :

A1 aX2 - A2 aX1 = aY1 Cy aX2 - aY2 Cy aX1 A1 aX2 - A2 aX1 = Cy (aY1 aX2 - aY2 aX1) Cy = (A1 aX2 - A2 aX1) / (aY1 aX2 - aY2 aX1) (5) Same way we can derive Cx = (A2 aY1 – A1 aY2) / (aY1 aX2 - aY2 aX1) (6) Equations 5 and 6 are known as simultaneous equations and by solving these simultaneous equations

we can determine the concentration of X and Y in the sample.

b) Q-Absorbance ratio method The absorbance ratio method is a modification of the simultaneous equations procedure. It depends on the property that, for a substance, which obeys Beer’s law at all

wavelength, the ratio of absorbances at any two wavelengths is a constant value independent of concentration or path length. In the quantitative assay of two components in admixture by the

absorbance ratio method, absorbances are measured at two wavelengths, one being the λ-max of one of the components (λ2) and other being a wavelength of equal absorptivity of two components (λ1),

i.e. an iso-absorptive point. At λ1 A1 = aX1* Cx + aY1* Cy (1) At λ2 A2 = aX2* Cx + aY2* Cy (2) :

b) Q-Absorbance ratio method The absorbance ratio method is a modification of the simultaneous equations procedure. It depends on the property that, for a substance, which obeys Beer’s law at all

wavelength, the ratio of absorbances at any two wavelengths is a constant value independent of concentration or path length. In the quantitative assay of two components in admixture by the

absorbance ratio method, absorbances are measured at two wavelengths, one being the λ-max of one

of the components (λ2) and other being a wavelength of equal absorptivity of two components (λ1), i.e. an iso-absorptive point. At λ1 A1 = aX1* Cx + aY1* Cy (1) At λ2 A2 = aX2* Cx + aY2* Cy (2)

Now divide (2) with (1) A2/A1 = (aX2* Cx + aY2* Cy) (aX1* Cx + aY1* Cy) Divide each term with (Cx + Cy) A2/A1 = (aX2* Cx + aY2* Cy) / (Cx + Cy) (Cx + Cy) (aX1* Cx + aY1* Cy) / (Cx + Cy) Put Fx = Cx / (Cx + Cy) and Fy = Cy / (Cx + Cy) A2/A1 = [aX2 Fx + aY2 Fy] / [aX1 Fx + aY1Fy] Where Fx is the fraction of X

and Fy is the fraction of Y i.e. Fy = 1-Fx There fore A2/A1 = [aX2 Fx + aY2 (1-Fx)] / [aX1 Fx + aY1(1-Fx)] = [aX2 Fx + aY2 – aY2Fx] / [aX1 Fx + aY1 – aY1Fx] :

Now divide (2) with (1) A2/A1 = (aX2* Cx + aY2* Cy) (aX1* Cx + aY1* Cy) Divide each term with (Cx + Cy) A2/A1 = (aX2* Cx + aY2* Cy) / (Cx + Cy) (Cx + Cy) (aX1* Cx + aY1* Cy) / (Cx + Cy) Put Fx = Cx / (Cx + Cy)

and Fy = Cy / (Cx + Cy) A2/A1 = [aX2 Fx + aY2 Fy] / [aX1 Fx + aY1Fy] Where Fx is the fraction of X and Fy is the fraction of Y i.e. Fy = 1-Fx There fore A2/A1 = [aX2 Fx + aY2 (1-Fx)] / [aX1 Fx + aY1(1-Fx)] = [aX2 Fx +

aY2 – aY2Fx] / [aX1 Fx + aY1 – aY1Fx]

At iso-absorptive point aX1 = aY1 and Cx = Cy There fore A2/A1 = [aX2 Fx + aY2 – aY2Fx] / aX1 = (aX2 Fx/ aX1) + (aY2/ aX1) –( aY2Fx/ aX1) Let Qx = aX2/aX1 , Qy = aY2/aY1 and absorption ratio Qm =

A2/A1 Qm = Fx Qx + Qy - Fx Qy = Fx (Qx-Qy) + Qy Fx = (Qm – Qy) / (Qx – Qy) (3) From the equations (1) A1 = aX1 (Cx + Cy) there fore Cx + Cy = A1 / aX1 There fore Cx = (A1/aX1) – Cy (4) From the equation (3) Cx / (Cx + Cy) = (Qm – Qy) / (Qx – Qy) There fore Cx / (A1 / aX1) = (Qm – Qy) / (Qx – Qy) There fore Cx

= [(Qm – Qy) / (Qx – Qy)] X (A1 / aX1) (5) :

At iso-absorptive point aX1 = aY1 and Cx = Cy There fore A2/A1 = [aX2 Fx + aY2 – aY2Fx] / aX1 = (aX2 Fx/ aX1) + (aY2/ aX1) –( aY2Fx/ aX1) Let Qx = aX2/aX1 , Qy = aY2/aY1 and absorption ratio Qm = A2/A1 Qm = Fx Qx + Qy - Fx Qy = Fx (Qx-Qy) + Qy Fx = (Qm – Qy) / (Qx – Qy) (3) From the equations (1) A1 = aX1 (Cx + Cy) there fore Cx + Cy = A1 / aX1 There fore Cx = (A1/aX1) – Cy (4) From the equation (3) Cx / (Cx + Cy) = (Qm – Qy) / (Qx – Qy) There fore Cx / (A1 / aX1) = (Qm – Qy) / (Qx – Qy) There fore Cx = [(Qm – Qy) / (Qx

– Qy)] X (A1 / aX1) (5)

a) Derivative spectroscopy :

a) Derivative spectroscopy

Derivative spectroscopy involves the conversion of a normal spectra to its first, second or higher derivative spectra. The normal spectrum is known as fundamental, zero order or D0 spectra. The first

derivative spectrum (D1) is a plot of the rate of change of absorbance with wavelength against wavelength, i.e. plot of ΔA/Δλ vs. λ. The second derivative spectrum is a plot of Δ2A/ Δλ2 vs. λ. For the

quantitative estimation of binary mixtures by the derivative spectroscopy, first of all we have to find out the Zero Crossing Points (ZCP) for both the components (A and B). Now select ZCP for A and B so that at that particular ZCP other component shows remarkable absorbance. Now prepare calibration

curve of A at the ZCP of B and of B at the ZCP of A. Find out the unknown concentration using calibration curves. :

Derivative spectroscopy involves the conversion of a normal spectra to its first, second or higher derivative spectra. The normal spectrum is known as fundamental, zero order or D 0 spectra. The first

derivative spectrum (D 1 ) is a plot of the rate of change of absorbance with wavelength against wavelength, i.e. plot of ΔA/Δλ vs. λ. The second derivative spectrum is a plot of Δ 2 A/ Δλ 2 vs. λ. For the quantitative estimation of binary mixtures by the derivative spectroscopy, first of all we have to find out the Zero Crossing Points (ZCP) for both the components (A and B). Now select ZCP for A and B so that at that particular ZCP other component shows remarkable absorbance. Now prepare calibration curve of A

at the ZCP of B and of B at the ZCP of A. Find out the unknown concentration using calibration curves.

1) Determination of Dissociation constant of an indicators Indicators give different color at different pH. Methyl red is red in color in acidic medium and is yellow in alkaline medium because in acidic

medium it remains as HMR (Unionized form) and in alkaline medium as MR- (Ionized form). HMR MR- + H+ Ka = [(MR-) (H+)] / (HMR) There fore Pka = pH – Log [(MR-) (H+)] 2) Determination of

composition of Complex. M + L = Complex There is two methods for the determination of composition of complex first is Mole ratio method. In this technique concentration of on of the components of the

complex is kept constant and other is increased and the absorbance of the resulting solution is measured. Now from the plot of absorbance Vs concentration. Another method is Job’s curve method

(Continuous variation method). 5) As a detector in HPLC:

1) Determination of Dissociation constant of an indicators Indicators give different color at different pH. Methyl red is red in color in acidic medium and is yellow in alkaline medium because in acidic

medium it remains as HMR (Unionized form) and in alkaline medium as MR - (Ionized form). HMR MR - + H + Ka = [(MR - ) (H + )] / (HMR) There fore Pka = pH – Log [(MR - ) (H + )] 2) Determination of

composition of Complex. M + L = Complex There is two methods for the determination of composition of complex first is Mole ratio method. In this technique concentration of on of the components of the

complex is kept constant and other is increased and the absorbance of the resulting solution is measured. Now from the plot of absorbance Vs concentration. Another method is Job’s curve method

(Continuous variation method). 5) As a detector in HPLC

References: 1) Beckett, A.H. and Stenlake, J.B., In; Practical Pharmaceutical Chemistry, 4th Edn., Part One, CBS Publishers, New Delhi, 2000. 2) Schirmer, R.E., In; Modern Methods of

Pharmaceutical Analysis, 2nd Edn., Volume-I, CRC Press, Florida, 2000. 3) Christian, G.D., In; Analytical Chemistry, 6th Edn., John Wiley and Sons, Inc., Singapore, 2004. 4) Ohannesian, L. and Streeter, A.J., In; Handbook of Pharmaceutical Analysis, Marcel Dekker, Inc., New York, 2002. 5)

Connors, K.A., In; A Text Book of Pharmaceutical Analysis, 3rd Edn., A Wiley-Interscience Publications, New York, 1982. 6) Kalsi, P.S., In; Spectroscopy of Organic Compounds, 6th Edn., New Age

International Publishers, new Delhi, 2004. :

References: 1) Beckett, A.H. and Stenlake, J.B., In; Practical Pharmaceutical Chemistry, 4 th Edn., Part One, CBS Publishers, New Delhi, 2000. 2) Schirmer, R.E., In; Modern Methods of Pharmaceutical

Analysis, 2 nd Edn., Volume-I, CRC Press, Florida, 2000. 3) Christian, G.D., In; Analytical Chemistry, 6 th Edn., John Wiley and Sons, Inc., Singapore, 2004. 4) Ohannesian, L. and Streeter, A.J., In; Handbook of

Pharmaceutical Analysis, Marcel Dekker, Inc., New York, 2002. 5) Connors, K.A., In; A Text Book of Pharmaceutical Analysis, 3 rd Edn., A Wiley-Interscience Publications, New York, 1982. 6) Kalsi, P.S., In; Spectroscopy of Organic Compounds, 6 th Edn., New Age International Publishers, new Delhi, 2004.


2 UV Spectroscopy Introduction UV radiation and Electronic Excitations The difference in energy between molecular bonding, non-bonding and anti-bonding orbitals ranges from 125-650 kJ/mole This energy corresponds to EM radiation in the ultraviolet (UV) region, 100-350 nm, and visible (VIS) regions 350-700 nm of the spectrum For comparison, recall the EM spectrum: Using IR we observed vibrational

transitions with energies of 8-40 kJ/mol at wavelengths of 2500-15,000 nm For purposes of our discussion, we will refer to UV and VIS spectroscopy as UV UV X-rays IR g -rays Radio Microwave Visible


3 UV Spectroscopy Introduction The Spectroscopic Process In UV spectroscopy, the sample is irradiated with the broad spectrum of the UV radiation If a particular electronic transition matches the energy of a

certain band of UV, it will be absorbed The remaining UV light passes through the sample and is observed From this residual radiation a spectrum is obtained with “gaps” at these discrete energies –

this is called an absorption spectrum


4 UV Spectroscopy Introduction Observed electronic transitions The lowest energy transition (and most often obs. by UV) is typically that of an electron in the Highest Occupied Molecular Orbital (HOMO) to the Lowest Unoccupied Molecular Orbital (LUMO) For any bond (pair of electrons) in a molecule, the

molecular orbitals are a mixture of the two contributing atomic orbitals; for every bonding orbital “created” from this mixing ( s , p ), there is a corresponding anti-bonding orbital of symmetrically higher

energy ( s * , p * ) The lowest energy occupied orbitals are typically the s; likewise, the corresponding

anti-bonding s * orbital is of the highest energy p -orbitals are of somewhat higher energy, and their complementary anti-bonding orbital somewhat lower in energy than s *. Unshared pairs lie at the energy of the original atomic orbital, most often this energy is higher than p or s (since no bond is

formed, there is no benefit in energy)


5 UV Spectroscopy Introduction Observed electronic transitions Here is a graphical representation Energy s* p s p* n Atomic orbital Atomic orbital Molecular orbitals Occupied levels Unoccupied levels


6 UV Spectroscopy Introduction Observed electronic transitions From the molecular orbital diagram, there are several possible electronic transitions that can occur, each of a different relative energy:

Energy s* p s p* n s s p n n s * p * p * s * p * alkanes carbonyls unsaturated cmpds. O, N, S, halogens carbonyls


7 UV Spectroscopy Introduction Observed electronic transitions Although the UV spectrum extends below 100 nm (high energy), oxygen in the atmosphere is not transparent below 200 nm Special

equipment to study vacuum or far UV is required Routine organic UV spectra are typically collected from 200-700 nm This limits the transitions that can be observed: s s p n n s * p * p * s * p * alkanes carbonyls

unsaturated cmpds. O, N, S, halogens carbonyls 150 nm 170 nm 180 nm √ - if conjugated! 190 nm 300 nm √


8 UV Spectroscopy Introduction Selection Rules Not all transitions that are possible are observed For an electron to transition, certain quantum mechanical constraints apply – these are called “ selection rules

” For example, an electron cannot change its spin quantum number during a transition – these are “ forbidden ” Other examples include: the number of electrons that can be excited at one time symmetry properties of the molecule symmetry of the electronic states To further complicate matters, “forbidden”

transitions are sometimes observed (albeit at low intensity) due to other factors


9 UV Spectroscopy Introduction Band Structure Unlike IR (or later NMR), where there may be upwards of 5 or more resolvable peaks from which to elucidate structural information, UV tends to give wide,

overlapping bands It would seem that since the electronic energy levels of a pure sample of molecules would be quantized, fine, discrete bands would be observed – for atomic spectra, this is the case In

molecules, when a bulk sample of molecules is observed, not all bonds (read – pairs of electrons) are in the same vibrational or rotational energy states This effect will impact the wavelength at which a

transition is observed – very similar to the effect of H-bonding on the O-H vibrational energy levels in neat samples


10 UV Spectroscopy Introduction Band Structure When these energy levels are superimposed, the effect can be readily explained – any transition has the possibility of being observed Energy E 0 E 1


11 UV Spectroscopy Instrumentation and Spectra Instrumentation The construction of a traditional UV-VIS spectrometer is very similar to an IR, as similar functions – sample handling, irradiation, detection

and output are required Here is a simple schematic that covers most modern UV spectrometers: sample reference detector I 0 I 0 I 0 I log( I 0 / I ) = A 200 700 l , nm monochromator/ beam splitter optics UV-VIS

sources


12 UV Spectroscopy Instrumentation and Spectra Instrumentation Two sources are required to scan the entire UV-VIS band: Deuterium lamp – covers the UV – 200-330 Tungsten lamp – covers 330-700 As with the dispersive IR, the lamps illuminate the entire band of UV or visible light; the monochromator (grating

or prism) gradually changes the small bands of radiation sent to the beam splitter The beam splitter sends a separate band to a cell containing the sample solution and a reference solution The detector

measures the difference between the transmitted light through the sample ( I ) vs. the incident light ( I 0 ) and sends this information to the recorder


13 UV Spectroscopy Instrumentation and Spectra Instrumentation As with dispersive IR, time is required to cover the entire UV-VIS band due to the mechanism of changing wavelengths A recent improvement is the diode-array spectrophotometer - here a prism (dispersion device) breaks apart the full spectrum

transmitted through the sample Each individual band of UV is detected by a individual diodes on a

silicon wafer simultaneously – the obvious limitation is the size of the diode, so some loss of resolution over traditional instruments is observed sample Polychromator – entrance slit and dispersion device UV-

VIS sources Diode array


14 UV Spectroscopy Instrumentation and Spectra Instrumentation – Sample Handling Virtually all UV spectra are recorded solution-phase Cells can be made of plastic, glass or quartz Only quartz is transparent in the full 200-700 nm range; plastic and glass are only suitable for visible spectra

Concentration (we will cover shortly) is empirically determined A typical sample cell (commonly called a cuvet ):


15 UV Spectroscopy Instrumentation and Spectra Instrumentation – Sample Handling Solvents must be transparent in the region to be observed; the wavelength where a solvent is no longer transparent is referred to as the cutoff Since spectra are only obtained up to 200 nm, solvents typically only need to lack conjugated p systems or carbonyls Common solvents and cutoffs: acetonitrile 190 chloroform 240 cyclohexane 195 1,4-dioxane 215 95% ethanol 205 n -hexane 201 methanol 205 isooctane 195 water

190


16 UV Spectroscopy Instrumentation and Spectra Instrumentation – Sample Handling Additionally solvents must preserve the fine structure (where it is actually observed in UV!) where possible H-

bonding further complicates the effect of vibrational and rotational energy levels on electronic transitions, dipole-dipole interacts less so The more non-polar the solvent, the better (this is not always

possible)


17 UV Spectroscopy Instrumentation and Spectra The Spectrum The x-axis of the spectrum is in wavelength; 200-350 nm for UV, 200-700 for UV-VIS determinations Due to the lack of any fine

structure, spectra are rarely shown in their raw form, rather, the peak maxima are simply reported as a numerical list of “lamba max” values or l max l max = 206 nm 252 317 376


18 UV Spectroscopy Instrumentation and Spectra The Spectrum The y-axis of the spectrum is in absorbance, A From the spectrometers point of view, absorbance is the inverse of transmittance: A = log 10 ( I 0 / I ) From an experimental point of view, three other considerations must be made: a longer path

length, l through the sample will cause more UV light to be absorbed – linear effect the greater the concentration, c of the sample, the more UV light will be absorbed – linear effect some electronic

transitions are more effective at the absorption of photon than others – molar absorptivity, e this may vary by orders of magnitude…


19 UV Spectroscopy Instrumentation and Spectra The Spectrum These effects are combined into the Beer-Lambert Law: A = e c l for most UV spectrometers, l would remain constant (standard cells are

typically 1 cm in path length) concentration is typically varied depending on the strength of absorption observed or expected – typically dilute – sub .001 M molar absorptivities vary by orders of magnitude:

values of 10 4 -10 6 10 4 -10 6 are termed high intensity absorptions values of 10 3 -10 4 are termed low intensity absorptions values of 0 to 10 3 are the absorptions of forbidden transitions A is unitless, so the units for e are cm -1 · M -1 and are rarely expressed Since path length and concentration effects can be

easily factored out, absorbance simply becomes proportional to e , and the y-axis is expressed as e directly or as the logarithm of e


20 UV Spectroscopy Instrumentation and Spectra Practical application of UV spectroscopy UV was the first organic spectral method, however, it is rarely used as a primary method for structure determination It is most useful in combination with NMR and IR data to elucidate unique electronic features that may be ambiguous in those methods It can be used to assay (via l max and molar absorptivity) the proper

irradiation wavelengths for photochemical experiments, or the design of UV resistant paints and coatings The most ubiquitous use of UV is as a detection device for HPLC; since UV is utilized for solution phase samples vs. a reference solvent this is easily incorporated into LC design UV is to HPLC what mass

spectrometry (MS) will be to GC


21 UV Spectroscopy Chromophores Definition Remember the electrons present in organic molecules are involved in covalent bonds or lone pairs of electrons on atoms such as O or N Since similar functional groups will have electrons capable of discrete classes of transitions, the characteristic energy of these

energies is more representative of the functional group than the electrons themselves A functional group capable of having characteristic electronic transitions is called a chromophore ( color loving )

Structural or electronic changes in the chromophore can be quantified and used to predict shifts in the observed electronic transitions


22 UV Spectroscopy Chromophores Organic Chromophores Alkanes – only posses s -bonds and no lone pairs of electrons, so only the high energy s s * transition is observed in the far UV This transition is

destructive to the molecule, causing cleavage of the s -bond s* s


23 UV Spectroscopy Chromophores Organic Chromophores Alcohols, ethers, amines and sulfur compounds – in the cases of simple, aliphatic examples of these compounds the n s * is the most often

observed transition; like the alkane s s * it is most often at shorter l than 200 nm Note how this transition occurs from the HOMO to the LUMO s* CN s CN n N sp 3


24 UV Spectroscopy Chromophores Organic Chromophores Alkenes and Alkynes – in the case of isolated examples of these compounds the p p * is observed at 175 and 170 nm, respectively Even though this

transition is of lower energy than s s *, it is still in the far UV – however, the transition energy is sensitive to substitution p* p


25 UV Spectroscopy Chromophores Organic Chromophores Carbonyls – unsaturated systems incorporating N or O can undergo n p * transitions (~285 nm) in addition to p p * Despite the fact this

transition is forbidden by the selection rules ( e = 15), it is the most often observed and studied transition for carbonyls This transition is also sensitive to substituents on the carbonyl Similar to alkenes

and alkynes, non-substituted carbonyls undergo the p p * transition in the vacuum UV (188 nm, e = 900); sensitive to substitution effects


26 UV Spectroscopy Chromophores Organic Chromophores Carbonyls – n p * transitions (~285 nm); p p * (188 nm) p p* n s CO transitions omitted for clarity It has been determined from spectral studies,

that carbonyl oxygen more approximates sp rather than sp 2 !


27 UV Spectroscopy Chromophores Substituent Effects General – from our brief study of these general chromophores, only the weak n p * transition occurs in the routinely observed UV The attachment of substituent groups (other than H) can shift the energy of the transition Substituents that increase the

intensity and often wavelength of an absorption are called auxochromes Common auxochromes include alkyl, hydroxyl, alkoxy and amino groups and the halogens


28 UV Spectroscopy Chromophores Substituent Effects General – Substituents may have any of four effects on a chromophore Bathochromic shift (red shift) – a shift to longer l ; lower energy Hypsochromic

shift (blue shift) – shift to shorter l ; higher energy Hyperchromic effect – an increase in intensity Hypochromic effect – a decrease in intensity 200 nm 700 nm e Hypochromic Hypsochromic

Hyperchromic Bathochromic


29 UV Spectroscopy Chromophores Substituent Effects Conjugation – most efficient means of bringing about a bathochromic and hyperchromic shift of an unsaturated chromophore: l max nm e 175 15,000

217 21,000 258 35,000 n p * 280 27 p p * 213 7,100 465 125,000 n p * 280 12 p p * 189 900


30 UV Spectroscopy Chromophores Substituent Effects Conjugation – Alkenes The observed shifts from conjugation imply that an increase in conjugation decreases the energy required for electronic excitation

From molecular orbital (MO) theory two atomic p orbitals, f 1 and f 2 from two sp 2 hybrid carbons combine to form two MOs Y 1 and Y 2 * in ethylene Y 2 * p Y 1 f 1 f 2


31 UV Spectroscopy Chromophores Substituent Effects Conjugation – Alkenes When we consider butadiene, we are now mixing 4 p orbitals giving 4 MOs of an energetically symmetrical distribution compared to ethylene Y 2 * p Y 1 Y 1 Y 2 Y 3 * Y 4 * D E for the HOMO LUMO transition is reduced


32 UV Spectroscopy Chromophores Substituent Effects Conjugation – Alkenes Extending this effect out to longer conjugated systems the energy gap becomes progressively smaller: Energy ethylene butadiene

hexatriene octatetraene Lower energy = Longer wavelengths


33 UV Spectroscopy Chromophores Substituent Effects Conjugation – Alkenes Similarly, the lone pairs of electrons on N, O, S, X can extend conjugated systems – auxochromes Here we create 3 MOs – this

interaction is not as strong as that of a conjugated p -system Y 2 p Y 1 p * n A Y 3 * Energy


34 UV Spectroscopy Chromophores Substituent Effects Conjugation – Alkenes Methyl groups also cause a bathochromic shift, even though they are devoid of p - or n -electrons This effect is thought to be

through what is termed “hyperconjugation” or sigma bond resonance


35 UV Spectroscopy Next time – We will find that the effect of substituent groups can be reliably quantified from empirical observation of known conjugated structures and applied to new systems This

quantification is referred to as the Woodward-Fieser Rules which we will apply to three specific chromophores: Conjugated dienes Conjugated dienones Aromatic systems


36 UV Spectroscopy Structure Determination Dienes General Features For acyclic butadiene, two conformers are possible – s-cis and s-trans The s-cis conformer is at an overall higher potential energy than the s-trans ; therefore the HOMO electrons of the conjugated system have less of a jump to the

LUMO – lower energy, longer wavelength s - trans s - cis


37 UV Spectroscopy Structure Determination Dienes General Features Two possible p p * transitions can occur for butadiene Y 2 Y 3 * and Y 2 Y 4 * The Y 2 Y 4 * transition is not typically observed: The energy of this transition places it outside the region typically observed – 175 nm For the more favorable

s-trans conformation, this transition is forbidden The Y 2 Y 3 * transition is observed as an intense absorption s - trans s - cis 175 nm –forb. 217 nm 253 nm 175 nm Y 4 * Y 2 Y 1 Y 3 *


38 UV Spectroscopy Structure Determination Dienes General Features The Y 2 Y 3 * transition is observed as an intense absorption ( e = 20,000+) based at 217 nm within the observed region of the UV

While this band is insensitive to solvent (as would be expected) it is subject to the bathochromic and hyperchromic effects of alkyl substituents as well as further conjugation Consider: l max = 217 253 220

227 227 256 263 nm


39 UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules Woodward and the Fiesers performed extensive studies of terpene and steroidal alkenes and noted similar substituents and

structural features would predictably lead to an empirical prediction of the wavelength for the lowest energy p p * electronic transition This work was distilled by Scott in 1964 into an extensive treatise on

the Woodward-Fieser rules in combination with comprehensive tables and examples – (A.I. Scott, Interpretation of the Ultraviolet Spectra of Natural Products , Pergamon, NY, 1964) A more modern

interpretation was compiled by Rao in 1975 – (C.N.R. Rao, Ultraviolet and Visible Spectroscopy , 3 rd Ed., Butterworths, London, 1975)


40 UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules - Dienes The rules begin with a base value for l max of the chromophore being observed: acyclic butadiene = 217 nm The

incremental contribution of substituents is added to this base value from the group tables: Group Increment Extended conjugation +30 Each exo-cyclic C=C +5 Alkyl +5 -OCOCH 3 +0 -OR +6 -SR +30 -Cl, -Br

+5 -NR 2 +60


41 UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules - Dienes For example: Isoprene - acyclic butadiene = 217 nm one alkyl subs. + 5 nm 222 nm Experimental value 220 nm

Allylidenecyclohexane - acyclic butadiene = 217 nm one exocyclic C=C + 5 nm 2 alkyl subs. +10 nm 232 nm Experimental value 237 nm


42 UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes There are two major types of cyclic dienes, with two different base values Heteroannular (transoid): Homoannular (cisoid): e = 5,000 – 15,000 e = 12,000-28,000 base l max = 214 base l max = 253 The increment table is the same as for acyclic butadienes with a couple additions: Group Increment Additional homoannular

+39 Where both types of diene are present, the one with the longer l becomes the base


43 UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes In the pre-NMR era of organic spectral determination, the power of the method for discerning isomers is readily

apparent Consider abietic vs. levopimaric acid: levopimaric acid abietic acid


44 UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes For example: 1,2,3,7,8,8a-hexahydro-8a-methylnaphthalene heteroannular diene = 214 nm 3 alkyl subs. (3 x

5) +15 nm 1 exo C=C + 5 nm 234 nm Experimental value 235 nm


45 UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes heteroannular diene = 214 nm 4 alkyl subs. (4 x 5) +20 nm 1 exo C=C + 5 nm 239 nm homoannular diene

= 253 nm 4 alkyl subs. (4 x 5) +20 nm 1 exo C=C + 5 nm 278 nm


46 UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes Be careful with your assignments – three common errors: This compound has three exocyclic double bonds; the

indicated bond is exocyclic to two rings This is not a heteroannular diene; you would use the base value for an acyclic diene Likewise, this is not a homooannular diene; you would use the base value for an

acyclic diene


47 UV Spectroscopy Structure Determination Enones General Features Carbonyls, as we have discussed have two primary electronic transitions: p p* n Remember, the p p * transition is allowed and gives a

high e , but lies outside the routine range of UV observation The n p * transition is forbidden and gives a very low e, but can routinely be observed


48 UV Spectroscopy Structure Determination Enones General Features For auxochromic substitution on the carbonyl, pronounced hypsochromic shifts are observed for the n p * transition ( l max ) : This is explained by the inductive withdrawal of electrons by O, N or halogen from the carbonyl carbon – this

causes the n -electrons on the carbonyl oxygen to be held more firmly It is important to note this is different from the auxochromic effect on p p * which extends conjugation and causes a bathochromic

shift In most cases, this bathochromic shift is not enough to bring the p p * transition into the observed range 293 nm 279 235 214 204 204


49 UV Spectroscopy Structure Determination Enones General Features Conversely, if the C=O system is conjugated both the n p * and p p * bands are bathochromically shifted Here, several effects must be noted: the effect is more pronounced for p p * if the conjugated chain is long enough, the much higher intensity p p * band will overlap and drown out the n p * band the shift of the n p * transition is not

as predictable For these reasons, empirical Woodward-Fieser rules for conjugated enones are for the higher intensity, allowed p p * transition


50 UV Spectroscopy Structure Determination Enones General Features These effects are apparent from the MO diagram for a conjugated enone: p Y 1 Y 2 Y 3 * Y 4 * p* n p p* n


51 UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Group Increment 6-membered ring or acyclic enone Base 215 nm 5-membered ring parent enone Base 202 nm Acyclic dienone Base 245 nm Double bond extending conjugation 30 Alkyl group or ring residue a, b, g and

higher 10, 12, 18 -OH a, b, g and higher 35, 30, 18 -OR a, b, g, d 35, 30, 17, 31 -O(C=O)R a, b, d 6 -Cl a, b 15, 12 -Br a, b 25, 30 -NR 2 b 95 Exocyclic double bond 5 Homocyclic diene component 39


52 UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Aldehydes, esters and carboxylic acids have different base values than ketones Unsaturated system Base Value Aldehyde

208 With a or b alkyl groups 220 With a,b or b,b alkyl groups 230 With a,b,b alkyl groups 242 Acid or ester With a or b alkyl groups 208 With a,b or b,b alkyl groups 217 Group value – exocyclic a,b double

bond +5 Group value – endocyclic a,b bond in 5 or 7 membered ring +5


53 UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Unlike conjugated alkenes, solvent does have an effect on l max These effects are also described by the



54 UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Some examples – keep in mind these are more complex than dienes cyclic enone = 215 nm 2 x b - alkyl subs. (2 x 12) +24 nm 239 nm Experimental value 238 nm cyclic enone = 215 nm extended conj. +30 nm b -ring residue

+12 nm d -ring residue +18 nm exocyclic double bond + 5 nm 280 nm Experimental 280 nm


55 UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Take home problem – can these two isomers be discerned by UV-spec Eremophilone allo- Eremophilone Problem

Set 1: (text) – 1,2,3a,b,c,d,e,f,j, 4, 5, 6 (1 st , 2 nd and 5 th pairs), 8a, b, c Problem Set 2: outside problems/key -Tuesday


56 UV Spectroscopy Structure Determination Aromatic Compounds General Features Although aromatic rings are among the most widely studied and observed chromophores, the absorptions that arise from the various electronic transitions are complex On first inspection, benzene has six p -MOs, 3 filled p , 3

unfilled p * p 4 * p 5 * p 6 * p 2 p 1 p 3


57 UV Spectroscopy Structure Determination Aromatic Compounds General Features One would expect there to be four possible HOMO-LUMO p p * transitions at observable wavelengths (conjugation) Due to symmetry concerns and selection rules, the actual transition energy states of benzene are illustrated at the right: p 4 * p 5 * p 6 * p 2 p 1 p 3 A 1 g B 2 u B 1 u E 1 u 260 nm (forbidden) 200 nm (forbidden)

180 nm (allowed)


58 UV Spectroscopy Structure Determination Aromatic Compounds General Features The allowed transition ( e = 47,000) is not in the routine range of UV obs. at 180 nm, and is referred to as the primary

band The forbidden transition ( e = 7400) is observed if substituent effects shift it into the obs. region; this is referred to as the second primary band At 260 nm is another forbidden transition ( e = 230),

referred to as the secondary band. This transition is fleetingly allowed due to the disruption of symmetry by the vibrational energy states, the overlap of which is observed in what is called fine structure


59 UV Spectroscopy Structure Determination Aromatic Compounds General Features Substitution, auxochromic, conjugation and solvent effects can cause shifts in wavelength and intensity of aromatic systems similar to dienes and enones However, these shifts are difficult to predict – the formulation of

empirical rules is for the most part is not efficient (there are more exceptions than rules) There are some general qualitative observations that can be made by classifying substituent groups --


60 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Substituents with Unshared Electrons If the group attached to the ring bears n electrons, they can induce a shift in the

primary and secondary absorption bands Non-bonding electrons extend the p -system through resonance – lowering the energy of transition p p * More available n -pairs of electrons give greater

shifts


61 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Substituents with Unshared Electrons The presence of n -electrons gives the possibility of n p * transitions If this occurs, the electron now removed from G, becomes an extra electron in the anti-bonding p * orbital of the ring

This state is referred to as a charge-transfer excited state


62 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Substituents with Unshared Electrons pH can change the nature of the substituent group deprotonation of oxygen gives

more available n -pairs, lowering transition energy protonation of nitrogen eliminates the n -pair, raising transition energy Primary Secondary Substituent l max e l max e -H 203.5 7,400 254 204 -OH 211 6,200 270 1,450 -O - 235 9,400 287 2,600 -NH 2 230 8,600 280 1,430 -NH 3 + 203 7,500 254 169 -C(O)OH 230

11,600 273 970 -C(O)O - 224 8,700 268 560


63 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Substituents Capable of p -conjugation When the substituent is a p -chromophore, it can interact with the benzene p -system With benzoic acids, this causes an appreciable shift in the primary and secondary bands For the

benzoate ion, the effect of extra n -electrons from the anion reduces the effect slightly Primary Secondary Substituent l max e l max e -C(O)OH 230 11,600 273 970 -C(O)O - 224 8,700 268 560


64 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Electron-donating and electron-withdrawing effects No matter what electronic influence a group exerts, the

presence shifts the primary absorption band to longer l Electron-withdrawing groups exert no influence on the position of the secondary absorption band Electron-donating groups increase the l and e of the

secondary absorption band


65 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Electron-donating and electron-withdrawing effects Primary Secondary Substituent l max e l max e -H 203.5 7,400 254 204 -CH 3 207 7,000 261 225 -Cl 210 7,400 264 190 -Br 210 7,900 261 192 -OH 211 6,200 270 1,450 -

OCH 3 217 6,400 269 1,480 -NH 2 230 8,600 280 1,430 -CN 224 13,000 271 1,000 C(O)OH 230 11,600 273 970 -C(O)H 250 11,400 -C(O)CH 3 224 9,800 -NO 2 269 7,800 Electron donating Electron

withdrawing


66 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Di-substituted and multiple group effects With di-substituted aromatics, it is necessary to consider both groups If both groups are electron donating or withdrawing, the effect is similar to the effect of the stronger of the two

groups as if it were a mono -substituted ring If one group is electron withdrawing and one group electron donating and they are para - to one another, the magnitude of the shift is greater than the sum

of both the group effects Consider p -nitroaniline:


67 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Di-substituted and multiple group effects If the two electonically dissimilar groups are ortho- or meta- to one another, the effect is usually the sum of the two individual effects ( meta - no resonance; ortho -steric hind.) For

the case of substituted benzoyl derivatives, an empirical correlation of structure with observed l max has been developed This is slightly less accurate than the Woodward-Fieser rules, but can usually predict

within an error of 5 nm


68 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Di-substituted and multiple group effects Substituent increment G o m p Alkyl or ring residue 3 3 10 -O-Alkyl, -OH, -O-

Ring 7 7 25 -O - 11 20 78 -Cl 0 0 10 -Br 2 2 15 -NH 2 13 13 58 -NHC(O)CH 3 20 20 45 -NHCH 3 73 -N(CH 3 ) 2 20 20 85 Parent Chromophore l max R = alkyl or ring residue 246 R = H 250 R = OH or O-Alkyl 230


69 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Polynuclear aromatics When the number of fused aromatic rings increases, the l for the primary and secondary

bands also increase For heteroaromatic systems spectra become complex with the addition of the n p * transition and ring size effects and are unique to each case


70 UV Spectroscopy Visible Spectroscopy Color General The portion of the EM spectrum from 400-800 is observable to humans- we (and some other mammals) have the adaptation of seeing color at the

expense of greater detail 400 500 600 800 700 l , nm Violet 400-420 Indigo 420-440 Blue 440-490 Green 490-570 Yellow 570-585 Orange 585-620 Red 620-780


71 UV Spectroscopy Visible Spectroscopy Color General When white (continuum of l ) light passes through, or is reflected by a surface, those ls that are absorbed are removed from the transmitted or reflected light respectively What is “seen” is the complimentary colors (those that are not absorbed)

This is the origin of the “color wheel”


72 UV Spectroscopy Visible Spectroscopy Color General Organic compounds that are “colored” are typically those with extensively conjugated systems (typically more than five) Consider b -carotene l max

is at 455 – in the far blue region of the spectrum – this is absorbed The remaining light has the complementary color of orange


73 UV Spectroscopy Visible Spectroscopy Color General Likewise: l max for lycopene is at 474 – in the near blue region of the spectrum – this is absorbed, the compliment is now red l max for indigo is at 602

– in the orange region of the spectrum – this is absorbed, the compliment is now indigo!


74 UV Spectroscopy Visible Spectroscopy Color General One of the most common class of colored organic molecules are the azo dyes: From our discussion of di-subsituted aromatic chromophores, the

effect of opposite groups is greater than the sum of the individual effects – more so on this heavily conjugated system Coincidentally, it is necessary for these to be opposite for the original synthetic

preparation!


75 UV Spectroscopy Visible Spectroscopy Color General These materials are some of the more familiar colors of our “environment”


76 The colors of M&M’s Bright Blue Common Food Uses Beverages, dairy products, powders, jellies, confections, condiments, icing. Royal Blue Common Food Uses Baked goods, cereals, snack foods, ice-

cream, confections, cherries. Orange-red Common Food Uses Gelatins, puddings, dairy products, confections, beverages, condiments. Lemon-yellow Common Food Uses Custards, beverages, ice-cream,

confections, preserves, cereals. Orange Common Food Uses Cereals, baked goods, snack foods, ice-cream, beverages, dessert powders, confections


77 UV Spectroscopy Visible Spectroscopy Color General In the biological sciences these compounds are used as dyes to selectively stain different tissues or cell structures Biebrich Scarlet - Used with picric

acid/aniline blue for staining collagen, recticulum, muscle, and plasma. Luna's method for erythrocytes & eosinophil granules. Guard's method for sex chromatin and nuclear chromatin.


78 UV Spectroscopy Visible Spectroscopy Color General In the chemical sciences these are the acid-base indicators used for the various pH ranges: Remember the effects of pH on aromatic substituents

Absorption and Intensity Shifts:-:

3 Red shift :- λmax shifted towards longer wavelength Bathochromic shift. λ max shifted towards longer wavelength due to i) Presence of an auxochrome. ii) By change of solvent. Absorption and Intensity

Shifts :- 200 nm 800 nm e Hypochromic Hypsochromic Hyperchromic Bathochromic NKV


4 Benzen Aniline λ max -255nm λ max- 280 Here the NH 2 group is acts as a Auxochrome : Anilinium λ max 254nm But in anilinium there is no lone pair on nitrogen atom . No shairing take place. NKV


5 Blue shifts: - λmax shifted towards shorter wavelength or hypsochromic shift or effect. due to i) removal of conjugation ii) by changing polarity of solvent. E.g. The aniline experienced a blue shift by

removal of conjugation in acidic medium. Aniline λmax= 280nm Anilinium cation λmax = 253nm .. NKV


6 Hyperchromic effect:- increases in the intensity of absorption maximum. i.e. Є max is increased. The introduction of auxochrome usually increases intensity of absorption Pyridine 2 methyl pyridine λmax=

257nm λmax = 262nm εmax = 2750 εmax = 3650 NKV


7 Hypochromic effect :- decreases in the intensity of absorption maximum . i.e. Є max is decreased. 200 nm 800 nm e Hypochromic Hypsochromic Hyperchromic Bathochromic NKV Ex. Biphenyl 2methyle

biphenyl Є max=19000 Є max= 10250 Distortion take place

Woodward-Fieser Rules:

8 Woodward- Fieser Rules NKV Woodward-Fieser rule is used to calculate the position of λ max for given structure ….. …..By relating the position of λ max with the position and degree of chromophores. #

various types of double bond in a conjgugation describes below… Homoannular dienes Heteroannular dienes Endocyclic double bond Exocyclic double bond.

1) Homoannular dienes :

9 1) Homoannular dienes NKV It is cyclic dienes having conjugated double bond in a same ring. It is also called as homo dienes. Ex..

2) Heteroannular dienes :

10 2) Heteroannular dienes NKV It is cyclic dienese in which double bond in conjugation are present in different ring Ex..

3) Endocyclic double bond:

11 3) Endocyclic double bond NKV Double bond present in a ring Ex..

4) Exocyclic double bond. :

12 4) Exocyclic double bond. NKV It is double bond in which one of the double bonded atom is a part of a ring system Ex…


13 NKV Endocyclic double bond Exocyclic double bond Ring A has one endocyclic and one exocyclic double bonds . While ring B has one endocyclic double bond only.

Woodward-Fieser Rules for Dienes , Trines ,polyenes etc. :

14 Woodward- Fieser Rules for Dienes , Trines , polyenes etc. NKV The constitutes the basic values or parent value . The constribution made by various alkyl substituents, ring residues, double bond

extending conjugation and polar groups such as –Cl,-Br, -OR ,… ………are added to the basic value to obtain λ max for particular compound.

Woodward-Fieser Rules for Dienes:

15 Parent values: Heteroannular conjugated dienes ( transoid ) λ = 214 nm Acyclic transoid λ =217nm Homoannular conjugated dienes ( cisoid ) λ = 253 nm Increments for: Double bond extending

conjugation 30 nm Alkyl substituent or ring residue 5 nm Exocyclic double bond 5 nm Auxochrome - OR 6 nm -SR 30 nm - Cl , -Br 5 nm -NR 2 60 nm Woodward-Fieser Rules for Dienes NKV


16 NKV trans cis


17 NKV


18 nkv Woodward-Fieser Rules - Dienes For example:1 Isoprene – transoid acyclic butadiene = 217nm one alkyl subs. + 5 nm Calculated value 222nm Experimental value 220 nm


19 Woodward- Fieser Rules for Dienes NKV Ex..2 Solution: A given compound is homoannular dienes which is having two alkyl substituents and two ring residue Parent value………………………………..253nm 2

Alkyl substituents ……………………….. 10nm 2 Ring residues …………………………… 10nm Calculated value………………………… 273nm Observed value…………………………… 265nm 1,4-dimethylcyclohex-13 diene


20 Woodward- Fieser Rules for Dienes NKV Ex..3 Solution: The given compound is hetero annular dienes . It is having 4 ring residue as shown bellow. Parent value……………………………………..215nm 4 ring

residue …………………………………… 20nm Calculated value……………………………….. 235nm Observed value………………………………… 236nm


21 21 Woodward-Fieser Rules – Cyclic Dienes For example:4 1,2,3,7,8,8a-hexahydro-8a-methylnaphthalene heteroannular diene = 214 nm 3 alkyl subs. (3 x 5) +15 nm 1 exo C=C + 5 nm

Calculated value 234 nm Experimental value 235 nm NKV


22 Ex..5 Solution Basic value…………….. 217nm 2 alkyl subt. …………… 10nm 2 ring residue………….. 10nm 1 exocyclic double bond … 5nm Calculated value…………… 242nm Observed value…………….. 242nm NKV


23 Woodward-Fieser Rules for Dienes Example…6 Acyclic transoid 217 nm Alkyl group or ring residue 3x5 = 15 nm Calculated 232 nm Observed 234 nm NKV


24 NKV Cisoid : 253 nm Alkyl groups or ring residues: 2 x 5 = 10 nm Calculated: 263 nm Observed: 256 nm Example…7 Woodward-Fieser Rules for Dienes


25 Woodward-Fieser Rules for Dienes Example..8 Transoid 214 nm Alkyl group or ring residue 3x5 = 15 nm Exocyclic double bond ( * ) 5 nm Calculated 234 nm Observed 235 nm NKV *


26 NKV Cisoid: 253 nm Alkyl substituent ring residues: 3 x 5 = 5 nm 15 nm Exocyclic double bond( * ): 5 nm Calculated: 278 nm Observed: 275 nm Example…9 Woodward-Fieser Rules for Dienes *


27 Base value 214nm Alkyl substitution 5*5 25nm Exocyclic double bond 5*3 15nm Extra conjugation 30nm Calculated 284nm Observed 283nm Woodward-Fieser Rules for Dienes Example…10


28 Base value 214nm Alkyl substitution 5*4 20nm Exocyclic double bond 5 5nm Calculated 239nm Observed 238nm Woodward-Fieser Rules for Dienes Abetic acid Example…11


29 Base value 214nm Alkyl substitution 5*5 25nm Exocyclic double bond 5*2 10nm Calculated 278nm Observed 281nm Woodward-Fieser Rules for Dienes Example…12 Neoabetic acid


30 30 Woodward-Fieser Rules – Cyclic Dienes Be careful with your assignments – two common errors: This is not a heteroannular diene; you would use the base value for an acyclic diene Likewise, this is not

a homooannular diene; you would use the base value for an acyclic diene λ max =232nm NKV

Woodward’s Rules for Conjugated Carbonyl Compounds:

31 Woodward’s Rules for Conjugated Carbonyl Compounds NKV Woodward fieser rule for calculating λ max for α – β unsaturated carbonyl compound modified by Scott.


32 Woodward’s Rules for Conjugated Carbonyl Compounds Base values: X = R ( ketone ) α β unsaturated acyclic …. Six- membered ring or acyclic parent enone λ =215nm Five- membered ring parent enone λ

=202nm X = H (aldehyde) λ =208nm X = OH (alcohol), OR (ester) λ =195nm NKV


33 Increments for: Double bond extending conjugation 30 Exocyclic double bond 5 Endocyclic double bond in a 5- or 7-membered ring for X = OH, OR 5 Homocyclic diene component 39 Alkyl substituent or

ring residue α 10 β 12 γ or higher 18 Woodward’s Rules for Conjugated Carbonyl Compounds NKV


34 Polar groupings: - α β γ δ -OH 35 30 - 50 -OC(O)CH3 6 6 6 6 -OCH 3 35 30 17 31 -Cl 15 12 12 12 -Br 25 30 25 25 -NR 2 - 95 - - Woodward’s Rules for Conjugated Carbonyl Compounds NKV Auxochrome


35 35 Group Increment 6-membered ring or acyclic enone Base 215 nm 5-membered ring parent enone Base 202 nm Acyclic dienone Base 245 nm Double bond extending conjugation 30 Alkyl group or ring

residue a, b, g and higher 10, 12, 18 -OH a, b, g and higher 35, 30, 18 -OR a, b, g, d 35, 30, 17, 31 -O(C=O)R a, b, d 6 -Cl a, b 15, 12 -Br a, b 25, 30 -NR 2 b 95 Exocyclic double bond 5 Homocyclic diene

component 39 NKV

Solvent shifts for various solvents: :

36 Solvent shifts for various solvents : NKV Solvent λ max shift (nm) water + 8 chloroform - 1 ether - 7 cyclohexane - 11 dioxane - 5 hexane - 11 In the compounds the actual spectra obtained are affected

considerably by the nature of the solvent employed. Hence a solvent correction is applied to calculate value for that particular solvent


37 Woodward’s Rules for Conjugated Carbonyl Compounds NKV β α Ex..1 Solution: The given compound is an α - β unsaturated acyclic ketone which is having alkyl substitution to α - position. Parent

value………………………………….. 215nm 1 alkyl substituent at α - position……………... 10nm Calculated value……………………………… 225nm Observed value………………………………. 220nm


38 Woodward’s Rules for Conjugated Carbonyl Compounds NKV Ex…2 The given compound has been α - β -unsaturated six membered ring structure ketone . It is having one ring residue at α - position and two

at β - position and has double bonded exocyclic to two rings.


39 NKV β α Parent value……………………………….. 215nm 1 ring residue at α -position ………………... 5nm 2 ring residue at β -position ………………. 24nm Double bond exocyclic to two rings………. 10nm Calculated

value…………………………… 254nm Observed value……………………………. 256nm


40 Acyclic enone: 215 nm α -Alkyl groups or ring residues: 10 nm β -Alkyl groups or ring residues: 2 x 12 = 24 nm Calculated: 249 nm Observed: 249 nm Woodward’s Rules for Conjugated Carbonyl Compounds

NKV Ex…3


41 Five-membered ring parent enone: 202 nm β -Alkyl groups or ring residues: 2 x 12 = 24 nm Exocyclic double bond: 5 nm Calculated: 231 nm Observed: 226 nm Woodward’s Rules for Conjugated Carbonyl

Compounds NKV Ex…4 β α


42 Five-membered ring parent enone: 202 nm α -Br: 25 nm β -Alkyl groups or ring residues:2 x 12 = 24 nm Exocyclic double bond: 5 nm Calculated: 256 nm Observed: 251 nm Woodward’s Rules for

Conjugated Carbonyl Compounds NKV Ex…5 α β


43 Woodward’s Rules for Conjugated Carbonyl Compounds NKV Ester: 195 nm β -Alkyl groups or ring residues: 12 nm Endocyclic double bond in 7-membered ring ( *) 5 nm Calculated: 212 nm Observed:

218 nm Ex…6 α β *


44 Woodward’s Rules for Conjugated Carbonyl Compounds NKV Aldehyde : 208 nm α -Alkyl groups or ring residues: 10 nm β -Alkyl groups or ring residues: 2 x 12 = 24 nm Calculated: 242 nm Observed: 242

nm Ex…7 α β


45 Basic value 215nm β alkul substitution 2*12 24nm Exocycliclated double bond 5nm Calculated 244nm Observed 241nm Ex…8


46 Basic value 215nm β alkul substitution 2*12 24nm Exocycliclated double bond 5nm Calculated 244nm Observed 241nm Ex…9


47 Basic value 215nm β alkyl substitution 1*12 12nm σ alkyl substitution 1*18 18nm Exocycliclated double bond 1*5 5nm Extra conjugation 1*30 30nm Calculated 280nm Observed 277nm Ex…10

Instrumentation:

48 Instrumentation NKV

SPECTROPHOTOMETER:

49 A spectrophotometer is a device which detects the percentage transmittance of light radiation when light of certain intensity & frequency range is passed through the sample. Thus the instrument compares

the intensity of the transmitted light with that of the incident light. SPECTROPHOTOMETER NKV Transmittance, T = P / P0 % Transmittance, %T = 100 T


50 These are expensive and more sophisticated and are designed to read % transmittance or absorbance, Record the absorption spectrum using plotter or recorder . These are of double beam type

where we can use sample and reference solution at a time. Wavelength of accuracy is normally +/- 0.1nm . These instruments are hence more accurate & reliable than the other types. NKV

Charecterestics of spectophotometer

Instrumentation:

51 Instruments for measuring the absorption of U.V. or visible radiation are made up of the following components; Sources (UV and visible ), Wavelength selector ( monochromators ), Detector, Recording

system, Sample containers, Matched cell, Solvent. Instrumentation NKV

1) Sources of Light:

52 A) Hydrogen Discharge Lamp B) Deuterium Discharge Lamp C) Incandescent filament lamps D) Tungsten filament lamp E) Xenon Arc discharge lamp, F) Mercury Vapor Arc 1 ) Sources of Light NKV

#Requirements - stable -constant intensity -Sufficient radiant energy #Basic


53 A) HYDROGEN DISCHARGE LAMP: In these lamps hydrogen gas is stored under relatively high pressure. when an electric discharge is passed through the lamp, excited hydrogen molecules will be

produced which emit UV radiations. These cover the range of 3500-1200 A. NKV


54 3-5 times more intensity than other type. Measurement about 350 nm & near IR to 2.5um are usually made It gives continuous spectrum over the range Filament are usually coiled NKV


55 C)TUNGSTEN LAMP: it is similar in its functioning to an electric bulb. The tungsten filament is heated electrically to white heat. To maintain the constant intensity ,the electrical current to the lamp must be

carefully controlled. The lamps are generally stable & easy to use. NKV

D) XENON DISCHARGE LAMP:

56 In these ,xenon gas is stored under pressure in the range of 10-30 atmospheres. The xenon lamp possesses two tungsten electrodes separated by about 8mm. When an intense arc is formed between

two tungsten electrodes by applying a low voltage, the UV light is produced. The intensity of UV radiation produced by xenon lamp is much greater than that of hydrogen lamp. D) XENON DISCHARGE

LAMP NKV

E) Murcury Arc:

57 E) Murcury Arc If the mercury discharge lamp is enclosed in a glass tube, it is used to provide visible radiation. If the mercury discharge is fused in a silica envelope, it is used to provide U.V-radiation. It

emits radiation in the range 350nm-800nm. NKV

2)MONOCHROMATORS:

58 PRISM - Refractive , Reflective GRATING – Transmission Grating Diffraction Grating 2 )MONOCHROMATORS NKV

FILTERS:

59 Absorption Filter Interference Filter FILTERS NKV Is a device , which allows light of required wavelength to pass through, but absorbs other wavelength wholly or partially Thus suitable filter can

select desired wavelength band. Types of filters

Absorption Filter :

60 Absorption Filter Theory of complimentary colors. Color of the solution Complimentary colored filter Transmitted wavelength in nm Violet Yellowish Green 400-435 Blue Yellow 435-480 Greenish Blue

orange 480-490 Bluish Green Red 490-500 Green Purple 500-560 Yellowish Green Violet 560-580 Yellow Blue 580-595 Orange Greenish Blue 595-610 Red Bluish Green 610-750 NKV


61 Selection of absorption filter is done according to the following procedure: Write the color VIBGYOR in clockwise or anticlockwise manner, omitting Indigo. If solution to be analyzed is BLUE in color a filter

having a complimentary color ORANGE is used in the analysis. NKV


62 The color in the glass filters are produced by incorporating metal oxides like (V, Cr, Mn, Fe, Ni, Co, Cu etc.). Gelatin filter is prepared by adding organic pigments; Some natural organic pigments NKV


63 NKV Some natural organic pigments






66 Merits: Simple in construction, Cheaper, Selection of filter is easy. Demerits: Less accurate since there is a deviation of ±30nm, Intensity of radiation decreases due to absorption by filters. NKV

Interference Filter:

67 Interference Filter Interference filters are constructed by using two parallel glass plates which are silver coated internally. They are separated by a thin film of transparent dielectric spacer film made up of Calcium Fluoride (CaF 2 ) or Magnesium Fluoride (MgF 2 ) or Silicon oxide (Sio). NKV Works on the interference phenomenon, causes rejection of unwanted wavelength by selective reflection. These

filters have a band pass of 10-15nm with peak transmittance of 40-60%.


68 Merits: Inexpensive compared to prisms and gratings. More accurate compared to absorption filters. Demerits: One filter is used to provide only one specific wavelength. Therefore to cover the entire

region of uv-visible region a number of filters are required NKV

Wavelength selector (monochromator) :

69 Constructions: All monochromators contain the following component parts; An entrance slit. A collimating lens. A dispersing device (usually a prism or a grating). A focusing lens. An exit slit.

Wavelength selector (monochromator ) NKV

Monochromator:

70 Working:- Polychromatic radiation (radiation of more than one wavelength) enters the monochromator through the entrance slit. Monochromator NKV NKV

A dispersing device :

71 Rotating prism : made of glass, quartz or fused silica. Glass prims - used in visible range. Quartz and Fused silica prism – used in UV range. A dispersing device NKV


72 NKV


73 As a resolution of ±0.1nm can be achieved by using a grating, they are commonly used in a spectrometer. They are made up of glass , quartz or aluminum Gratings: NKV

They are of two types:

74 They are of two types a) Diffraction Gratings b) Transmission Gratings a ) Diffraction Gratings: NKV


75 The mechanism is that diffraction produces reinforcement. The rays which are incident upon the grating get reinforced with the reflected rays and hence the resulting radiation has the wavelength

which is governed by the equation: mλ = b (Sin i ± Sin r) λ = wavelength of radiation produced b = grating spacing i = angle of incidence r = angle of reflection m = order 0, 1, 2, 3 etc. NKV


76 Czerney-Turner grating monochromator NKV

b)Transmission Gratings: :

77 b )Transmission Gratings: It is similar to diffraction grating but refraction takes place instead of reflection. NKV λ = d Sin θ m where… λ = wavelength produced d = 1/lines per cm m = order 0, 1, 2, 3

etc. θ = angle of deflection Merits: Produces constant dispersion over the entire UV-visible region. Demerits: Difficult in construction.


78 Comparison Prism Grating Made of Glass-: Visible Quartz/fused silica-: UV Alkali halide:-IR Grooved on highly polished surface like alumina. Working Principle Angle of Incident Law of diffraction mλ= d

(sini±sinθ) Merits/demerits Prisms give non-liner dispersion hence no overlap of spectral order Grating gives liner dispersion hence overlap of spectral order. NKV


79 Merits/ demerits Prisms are subjected to etching from atmospheric moisture. It can’t be used over considerable wavelength ranges. Prisms are not sturdy long lasting. Expensive Moisture resistant It can

be used over considerable wavelength ranges. Grating are sturdy and long lasting. Economical. NKV Comparison Prism Grating

3)Detectors:

80 3)Detectors It is a component that converts EMR into an electron flow and subsequently into a current flow or voltage. NKV The types of detectors used in UV-visible spectroscopy are: a) Photo voltaic cell or barrier layer cell b) Photo tubes or photo emissive cells c) Photo multiplier tubes d) Silicon diode.

a).Photo voltaic cells or barrier layer cells: :

81 a). Photo voltaic cells or barrier layer cells: NKV

b).Photo tubes or Photo emissive cells: :

82 b). Photo tubes or Photo emissive cells: NKV

c)Photo multiplier tubes: :

83 c )Photo multiplier tubes: These are the most sensitive of all the detectors, expensive and used in sophisticated instruments. The principle involves multiplication of photoelectrons by secondary

emission of electrons. This is achieved by using a photo cathode and a series of anodes (dynodes), up to 10 dynodes are used. NKV

Each dynode is maintained at 75-100 volts higher than the preceding one. .At each stage the electron emission is multiplied by a factor 4 or 5 due to secondary emission of electrons. :

84 Each dynode is maintained at 75-100 volts higher than the preceding one. .At each stage the electron emission is multiplied by a factor 4 or 5 due to secondary emission of electrons. Cross section of a

photomultiplier tube NKV


85 NKV

D. Thermal detectors : :

86 D. Thermal detectors : sensitive to IR ( λ >750 nm) Thermocouple s - junction thermometer Bolometers - resistance thermometer Pyroelectric devices - piezoelectric effect NKV

4) Sample cell:

87 4) Sample cell Sample cell or cuvettes are used to hold a sample solution. For visible radiation the sample cells are made up of glass and for the UV the cells are made up of quartz, since glass absorbs UV radiations. The path length of sample cell ranges from 1cm to 10cm. Sample cells may be rectangular or cylindrical in shape. The material of the sample cell should not absorb at the wavelength observed NKV

5) Recording system:

88 5) Recording system The signals from the detector finally received by the recording system The recording is done by recorder pen In this DC current signals are produce that are amplified by DC amplifier & read on analog meter, recorders, digital voltmeter, display of computer system NKV

6) Matched cells:

89 6) Matched cells When double beam instrumentation is used two cell are needed one for the reference and one for the sample It normal for absorption and can lead to analytical error For most

accurate work matched cells are used “ these are cells in which absorption of each one is equal to or very nearly equal to absorption of other those within very similar absorptivities are put together and

designed matched cell .” NKV

7) Solvents:

90 7) Solvents Solvents must be transparent in the region to be observed; the wavelength where a solvent is no longer transparent is referred to as the cutoff Common solvents and cutoffs: acetonitrile

…………………… 190 chloroform……………………. . 240 cyclohexane …………… ... 195 1,4-dioxane………. ……….. 215 95% ethanol…….. ………… 205 n-hexane ………………… 201 methanol …………………….. 205 isooctane

……………………… 195 water …………………….. 190 NKV

Power supply:

91 Power supply A power supply serves a triple function It decreases line voltage to the instrument operating level with a transformer It converts AC to DC with a rectifier if direct current is required by Instrument . It smooth out in line voltage in order to deliver a constant voltage to source lamp and

instrument. NKV

U.V. Spectroscopic Instruments:

92 U.V. Spectroscopic Instruments NKV

Types of U.V. Spectroscopic Instruments :

93 Types of U.V. Spectroscopic Instruments A) Single beam spectrophotometer B) Double beam spectrophotometer NKV

A) Single-beam Spectrophotometer :

94 A) Single-beam Spectrophotometer The measurement of % transmittance with a manual single beam instrument involves three steps: 0% transmittance adjustment 100% transmittance adjustment

Determination of % of sample NKV

Disadvantages:

95 Disadvantages NKV It measures total amount of light reaching the detector rather the percentage absorbed Light may lost at reflecting surface or may be absorbed by solvent used to dissolve the sample.

Response of detector varies significantly with wavelength of the reading falling on it . So this problems of instrument variation can be largely overcome by using double beam system


96 reference 200 700 l , nm monochromator/ beam splitter optics UV-VIS sources sample Detector I 0 I 0 I 0 I log(I 0 /I) = A Double-Beam Spectrometer NKV

Double beam spectrophotometer:

97 The radiation from the source is allowed to pass via a mirror system to the monochromator unit. The radiation coming out of the monochromator through the exit slit is received by rotating sector which divides the beam into two beams, one passing through the reference & the other through the sample

cell. After passing through the sample & reference cells, the light beams are focused onto the detector. Double beam spectrophotometer NKV


98 The output of the detector is connected to a phase sensitive amplifier which responds to any change in transmission through sample & reference. The phase sensitive amplifier transmits the signals to the

recorder which is followed by the movement of the pen on chart. NKV

Advantages of double beam Spectrophotometer:

99 Advantages of double beam Spectrophotometer 1)It is not necessary to continuously replace the blank with the sample or adjust to zero for each sample as in single beam instruments. 2)The ratio of the

intensities of the sample and reference beams is constantly obtained. 3)Any error due to variation in

intensity of the source or fluctuation in the detector is minimized. 4)Rapid scanning over a wide λ region. NKV

COMPARISON::

100 COMPARISON: SL. NO SINGLE BEAM INSTRUMENT DOUBL BEAM INSTRUMENT 1. 2. Calibration should be done with blank every time, before measuring the absorbance or transmittance of sample

Radiant energy intensity changes with fluctuation of voltage. Calibration is done only in the beginning It permits a large degree of inherent compensation for fluctuations in the intensity of the radiant energy.


101 3 4 5 6 In single beam it’s not possible to compare blank and sample together. It measure the total amount of transmitted light reaching the detector In single beam radiant energy wavelength has to be

adjusted every time Working on single beam is tedious and time consuming. In double beam it’s possible to do direct one step comparison of sample in one path with a standard in the other path. It measures the percentage of light absorbed by the sample In this scanning can be done over a wide wavelength

region Working on double beam is fast and non tedious.

Reference: -:

102 Willard Merritt and Dean“Instrumental Method of Analysis” 7 th edition. Pavia “Introduction to spectoscopy” 3 rd edition William Kemp, “Organic Spectroscopy” 3rd edition . Sharma Y.R., “Elementary

Organic Spectroscopy” 3rd edition. Chatwal and Anand, “Instrumental Method of Chemical Analysis”, 13th edition. Spectroscopy of organic compounds by p. S.Kalsi Internet source.

( www.chemistry.ccsu.edu/glagovich/ teaching/316/uvvis/uvvis.html. www.shu.ac.uk/schools/sci/ chem/tutorials/molspec/lumin3.html ) Reference: - NKV

thaNK you !!!:

103 thaNK you !!! All the Best for exam NKV


104 Basic value 215nm β alkul substitution 2*12 24nm Exocycliclated double bond 5nm Calculated 244nm Observed 241nm


105


106


107 Base value 214nm Alkyl substitution 5*3 15nm Exocyclic double bond 5nm Homoannular 39nm Calculated 273nm Observed 275nm Woodward-Fieser Rules for Dienes


108 Base value 214nm Alkyl substitution 5*4 20nm Exocyclic double bond 5*2 5nm Homoannular 39nm Calculated 278nm Observed 281nm Woodward-Fieser Rules for Dienes


109 Base value 214nm Alkyl substitution 5*5 25nm Homoannular 39nm Calculated 268nm Observed 272nm Woodward-Fieser Rules for Dienes

:

ULTRA-VIOLET SPECTROSCOPY

Slide 3:

Spectroscopy Spectroscopy is the tool for study of atomic & molecular structure. It deals with interaction of electronic radiation with matter involving the measurement & interpretation of the

extension of absorption or emission of electromagnetic radiation by molecule. Most important consequence of such interaction is the energy is absorbed or emitted by the matter in discrete amount

called as quanta.

Slide 4:

Terminology Wavelength (λ) :- distance between two successive maxima of one electromagnetic wave. express in Angstron units or nm Frequency (ν) :- Number of wavelength passing through a given point in Unit time. Hertz or cycles per second Wave number (ΰ) :- Number of waves per centimeter in vacuum. ΰ

=(1/λ) Reciprocal of wavelength, express as per nm. λ=(c/v) Relation between frequency, velocity & wave number v = c ΰ

Slide 5:

UV radiation The wavelength range of uv radiation starts at blue end of visible light(4000A) & ends at 2000A. It is sub-divided into two spectral region- Near UV region b\w 2000 A- 4000 A. Far or vacuum UV

region below 2000 A. UV-spectroscopy involved with electronic excitation & have sufficient energy to excite valance electrons in Many atoms or mole. Commercial uv equipment operates b\w 200 and 800

nm.

Slide 6:

Origin & Theory Ultra violet absorption spectra arises from transition of electron with in molecule from lower to higher energy level and emission spectra Aries from reverse type of transition . For radiation to

cause electronic excitation, it must be in the uv region of electromagnetic spectrum This energy difference given by E= hν Etotal = Eelectro + Evib + Erota Eelectronic > Evibrational >Erotational

Slide 7:

But actually energy difference in energy between ground & excited states of electrons E= E1 – E0 E1 – E0 = hv

Slide 8:

Three types of electrons are involved in organic molecule σ-electrons- Involved in the saturated bonds. Such as found in the carbon, hydrogen in the paraffin. Energy required to excite electron in σ bonds is

more than the produced by the UV light. So compounds containing σ bonds do not absorb uv radiation.

b) π- electrons- Involved in unsaturated hydrocarbon. Present in triens & aromatic compounds. n electrons – these does not involved in the bonding b/w atoms in molecules. e.g.. Organic compounds

containing nitrogen, oxygen and halogen

Slide 10:

Type of transition in organic mole. σ to σ* n to π* n to σ* π to π * Energy required for various transitions are in the order σ-σ*> n-σ* > π-π*> n- π* Thus, n-π* transition required less energy than a π-π* or σ-σ*

transition. n to π * transition : shown by unsaturated mole. which contain atom such as oxygen, nitrogen and sulphur.

Slide 11:

They exhibit a weak band in their spectrum. σ to σ* transition : These type transition occur in compounds in which all electrons are involved in single bonds and no lone pair of electrons. large energy

required for these transition so absorption band occur in far region. commercial spectrophotometer generally do not operate at wavelength less than 180-200 nm so these transition cannot normally

observed. e.g.. Saturated hydrocarbons.

Slide 12:

n to σ* transition saturated compounds with lone pair of electrons undergoes this type transition less energy required for these type transition. absorption band occurs in longer wavelength in near uv

region. π to π * transition occurs in mole. having π electron system. These transitions fall in an experimentally convenient region of the spectrum (200 - 700 nm). These transitions need an

unsaturated group in the molecule to provide the p electrons.

Slide 13:

Chromophores Earlier the term was denoted to any functional group which presence gives colour to the compound. e.g.. Nitro group. But now the term is used for any group which exhibits absorption of

electromagnetic radiation in the visible or uv region. It may or may not impart colour to the compound. Mainly two types are known : 1. In which the group is having π electrons undergo π to π * transition.

e.g.. Ethylene and acetylenes. 2. Chromophores having both n and π electrons undergoes π to π * and n to π * transition.eg. Azo , nitro compound.

UV / visible Spectroscopy :

UV / visible Spectroscopy 1. Bathochromic shift lower energy, longer wavelength CONJUGATION. 2. Hypsochromic shift higher energy, shorter wavelength. 3. Hyperchromic effect increase in intensity 4.

Hypochromic effect decrease in intensity

Slide 16:

The first criatian for solvent A good solvent should not absorb ultraviolet radiation in the same region as the substance whose spectrum is being determined. Usually solvents which do not contain conjugated systems are most suitable for this purpose, although they vary as to the shortest wavelength at which

they remain transparent to ultraviolet radiation. The solvents most commonly used are water, 95% ethanol, and n-hexane.

Slide 17:

Property of solvent The ability of a solvent to influence the wavelength of ultraviolet light which will be absorbed. Polar solvents may not form hydrogen bonds as readily with excited states as with ground

states of polar solvents. Transitions of the type are shifted to shorter wavelengths by polar solvents. On the other hand, in some cases the excited states may form n→π stronger hydrogen bonds than the

corresponding ground state. In such cases, a polar solvent would shift an absorption to longer wavelength, since the energy of the electronic transition would be decreased. Transitions of the type are

shifted to longer wavelengths by polar solvents.

Choice of Solvents :

Choice of Solvents Some solvent used in uv Solvent min. wavelength for 1cm cell, nm Acetonitrile 190 Water 191 Cyclohexane 195 Hexane 201 Methanol 203 Ethanol 204 Ether 215 Methylene dichloride 220

Chloroform 237 Carbon tetrachloride 257

Slide 19:

CELLS UV Spectrophotometer Quartz (crystalline silica) Visible Spectrophotometer Glass IR Spectrophotometer NaCl

LAYOUT DIAGRAM OF U.V.SPECTROSCOPY :

LAYOUT DIAGRAM OF U.V.SPECTROSCOPY

Slide 21:

Instrumentation Components of spectrophotometer. .light Source , Monochromator, Sample compartment, Detector Recorder Light source tungsten-halogen lamp Hydrogen discharge lamp

Deuterium lamp Xenon discharge lamp Mercury arc VISIBLE SPECTROPHOTOMETER tungsten lamp

Slide 22:

Monochromator It used to disperse the radiation according to wavelength. Filters – Glass filters- Made from pieces of colored glass which transmit limited wavelength range of spectrum. Color produced by

incorporation of oxide of vanadium, chromium, iron, nickel, copper. Wide band width 150nm

Slide 23:

Prisms- Prism bends the monochromatic light. Amount of deviation depends on wavelength. Quartz prism used in UV-region. Glass prism used in visible region spectrum. Function – They produce non

linear dispersion. Grating– Large number of equispaced lines on a glass blank coated with aluminum film. Blaze angle Normal surface vector Normal to groove face .

Slide 24:

Detectors Three common types of detectors are used Barrier layer cell Photo cell detector Photomultiplier , Photo voltaic cells barrier layer cells It consist of flat Cu or Fe electrode on which

semiconductor such as selenium is deposited. on the selenium a thin layer of silver or gold is sputtered over the surface.

Slide 25:

Photocell detector: It consist of high sensitive cathode in the form of a half cylinder of metal which is evacuated. Anode also present which fixed along the axis of the tube Photocell is more sensitive than

photovoltaic cell. + - light Fig.- photocell detector Photomultiplier tube It is generally used as detector in UV- spectrophotometer It is the combination of photodiode & electron multiplier. It consist of

evacuated tube contains photocathode. 9-16 electrodes known as dynodes.

Slide 26:

Recorder Recorder Signal from detector received by the recording system The recording done by recorder pan.

Slide 27:

Description of UV- spectrophotometer Description of UV- spectrophotometer Single beam spectrophotometer Double beam spectrophotometer Advantage of double beam spectrophotometer:- It is not necessary to continually replace the blank with the sample or to adjust the auto zero. The ratio

of the powers of the sample & reference is constantly obtained. It has rapid scanning over the wide wavelength region because of the above two factors.

Slide 28:

Lamberts & Beer’s law “when a beam of light is allowed to pass through a transparent medium, rate of decrease of intensity with the thickness of medium is directly proportional to the intensity of light” - dI/ dt αI - dI/ dt = kI……..eqn..A) Where I denotes the intensity of incident light of λ wavelength, t denotes

the thickness of medium and k denotes the proportionally factor. On integrating eqn A and putting I = I₀ when t =0 we get Log I₀ / It = k t It = I₀ e-k t where I₀ denotes intensity of incident light, It denotes

intensity of transmitted light.

Slide 29:

Beer’s law Beer’s observed that a similar relationship holds between transmittance and concentration i.e. the intensity of a beam of monochromatic light decreases exponentially with the increase in

concentration of the e absorbing substance arithmetically It=Iο e¯k’c =Iο 10¯°˙4343 k’c=Iο10¯K’c k’ and K’ are constant and c is the concentration of the absorbing substance It/Iο 10¯act log Iο/It=act( eqn 1) eqn

1 is termed as beer –lembert law

Slide 30:

a is replaced by the ε and termed as molar absorption coefficient . Limits to beer law Chemical Deviations absorbing undergo association, dissociation or reaction with the solvent Instrumental

Deviations non-monochromatic radiation stray light

Limits to Beer’s Law Chemical Deviations :

Limits to Beer’s Law Chemical Deviations high concentration－particles too close Average distance between ions and molecules are diminished to the point. Affect the charge distribution and extent of

absorption. Cause deviations from linear relationship.

Limits to Beer’s Law Chemical Deviations :

Limits to Beer’s Law Chemical Deviations chemical interactions－monomer-dimer equilibria, metal complexation equilibria, acid/base equilibria and solvent-analyte association equilibria The extent of

such departure can be predicted from molar absorptivities and equilibrium constant.

Slide 33:

Woodward- Fieser Rules The Woodward- Fieser Rules for Dienes conjugated dienes π→ π* transition ε=20,000 to 26,000 λ =217 to 245nm Butadiene and many simple conjugated dienes exist in a planar

trans conformation. Generally, alkyl substitution produces bathochromic shifts and hyperchromic effects. With certain patterns of alkyl substitution, the wavelength

Slide 34:

The central bond is a part of the ring system, the diene chromospheres is usually held rigidly in either the s trans or the s cis conformation Homoannular Diene Heteroannular Diene (s-cis) (s-trans) less

intense ε= 5 000 –15000 more intense, ε= 12,000 λ longer (273nm) 28,000 λ shorter (234nm) The actual rules for predicting the absorption of open chain and six membered ring diene were first made by

Woodward in 1941.

Slide 35:

Since that time they have been modified by Fieser and Scott as a result of experience with a very large number of dienes and trienes. Rules for diene and triene absorption Value assigned to parent

heteroannular or open diene 214 nm Value assigned to parent homoannular diene 253 nm Increment for (a) each alkyl substituent or ring residue 5 nm (b) the exocyclic double bond 5 nm (c) a double-bond

extension 30 nm

Slide 36:

(d) auxochrome ―OCOCH3 0 nm ―OR 6 nm ―SR 30 nm ―Cl, ―Br 5 nm ―NR2 60 nm λcalc Total carbonyl compounds; enones Substitution by an auxochrome with a lone pair of electrons, such as –

NR2, – OH, –OR, –NH2, or –X, as in amides, acids, esters, or acid chlorides, gives a pronounced hypsochromic effect on the n → π* transition and lesser bathochromic effect on the π → π* transition.

(resonance interaction)

Slide 37:

The Hypsocromic Effect of Lone Pair Auxochromes on the n→π* Transition of a Carbonyl Group λmax εmax Solvent COCH3 H 293 nm 12 Hexane COCH3 CH3 279 15 Hexane CH3 CO Cl 235 53 Hexane CH3

CONH2 214 - Water effect. 46

Slide 38:

COCH3 OCH2CH3 204 60 Water This shift is due primarily to the inductive effect of the oxygen, nitrogen, or halogen atoms. They withdraw electrons from the carbonyl carbon, causing the lone pair of electrons

on oxygen to be held more firmly than would be the case in the absence of an inductive Effect. Woodward’s Rules for Enones Conjugation of a double bond with a carbonyl group leads to absorption

(ε = 8,000 to 20,000), π→π*, at 220 ~ 250 nm, predictable n→π*, at 310 ~ 330 nm, much less intense (ε= 50 to 100), not predictable

Slide 39:

Applications of UV uv was the first organic spectral method, however, it is rarely used as a primary method for structure determinationIt is most useful in combination with NMR and IR data to elucidate unique electronic features that may be ambiguous in those methodsIt can be used to assay (via lmax

and molar absorptivity) the proper irradiation wavelengths for photochemical experiments, or the design of UV resistant paints and coatings

Slide 40:

Quantitative determination of solutions of transition metal ions Quantitative determination of highly conjugated organic compounds As a detector for HPLC Identification of inorganic and organic species

Widely used method Magnitude of molar absorptivities Absorbing species methods

Slide 41:

Detection of geometrical isomers. Detection of functional groups. Detection of impurities. Molecular weight determination. Dissociation constant for acids and base. chemical kinetics. Charge transfer

transition. tautomeric equilibrium. structure of choral.

Slide 1:

1 CHMBD 449 – Organic Spectral Analysis Fall 2005 Chapter 7: UV Spectroscopy UV & electronic transitions Usable ranges & observations Selection rules Band Structure Instrumentation & Spectra

Beer-Lambert Law Application of UV-spec

Slide 2:

2 UV Spectroscopy Introduction UV radiation and Electronic Excitations The difference in energy between molecular bonding, non-bonding and anti-bonding orbitals ranges from 125-650 kJ/mole This energy corresponds to EM radiation in the ultraviolet (UV) region, 100-350 nm, and visible (VIS) regions 350-700 nm of the spectrum For comparison, recall the EM spectrum: Using IR we observed vibrational

transitions with energies of 8-40 kJ/mol at wavelengths of 2500-15,000 nm For purposes of our discussion, we will refer to UV and VIS spectroscopy as UV UV X-rays IR g -rays Radio Microwave Visible

Slide 3:

3 UV Spectroscopy Introduction The Spectroscopic Process In UV spectroscopy, the sample is irradiated with the broad spectrum of the UV radiation If a particular electronic transition matches the energy of a

certain band of UV, it will be absorbed The remaining UV light passes through the sample and is observed From this residual radiation a spectrum is obtained with “gaps” at these discrete energies –

this is called an absorption spectrum

Slide 4:

4 UV Spectroscopy Introduction Observed electronic transitions The lowest energy transition (and most often obs. by UV) is typically that of an electron in the Highest Occupied Molecular Orbital (HOMO) to the Lowest Unoccupied Molecular Orbital (LUMO) For any bond (pair of electrons) in a molecule, the

molecular orbitals are a mixture of the two contributing atomic orbitals; for every bonding orbital “created” from this mixing ( s , p ), there is a corresponding anti-bonding orbital of symmetrically higher

energy ( s * , p * ) The lowest energy occupied orbitals are typically the s; likewise, the corresponding anti-bonding s * orbital is of the highest energy p -orbitals are of somewhat higher energy, and their

complementary anti-bonding orbital somewhat lower in energy than s *. Unshared pairs lie at the energy of the original atomic orbital, most often this energy is higher than p or s (since no bond is

formed, there is no benefit in energy)

Slide 5:

5 UV Spectroscopy Introduction Observed electronic transitions Here is a graphical representation Energy s* p s p* n Atomic orbital Atomic orbital Molecular orbitals Occupied levels Unoccupied levels

Slide 6:

6 UV Spectroscopy Introduction Observed electronic transitions From the molecular orbital diagram, there are several possible electronic transitions that can occur, each of a different relative energy:

Energy s* p s p* n s s p n n s * p * p * s * p * alkanes carbonyls unsaturated cmpds. O, N, S, halogens carbonyls

Slide 7:

7 UV Spectroscopy Introduction Observed electronic transitions Although the UV spectrum extends below 100 nm (high energy), oxygen in the atmosphere is not transparent below 200 nm Special

equipment to study vacuum or far UV is required Routine organic UV spectra are typically collected from 200-700 nm This limits the transitions that can be observed: s s p n n s * p * p * s * p * alkanes carbonyls

unsaturated cmpds. O, N, S, halogens carbonyls 150 nm 170 nm 180 nm √ - if conjugated! 190 nm 300 nm √

Slide 8:

8 UV Spectroscopy Introduction Selection Rules Not all transitions that are possible are observed For an electron to transition, certain quantum mechanical constraints apply – these are called “ selection rules

” For example, an electron cannot change its spin quantum number during a transition – these are “ forbidden ” Other examples include: the number of electrons that can be excited at one time symmetry properties of the molecule symmetry of the electronic states To further complicate matters, “forbidden”

transitions are sometimes observed (albeit at low intensity) due to other factors

Slide 9:

9 UV Spectroscopy Introduction Band Structure Unlike IR (or later NMR), where there may be upwards of 5 or more resolvable peaks from which to elucidate structural information, UV tends to give wide,

overlapping bands It would seem that since the electronic energy levels of a pure sample of molecules would be quantized, fine, discrete bands would be observed – for atomic spectra, this is the case In

molecules, when a bulk sample of molecules is observed, not all bonds (read – pairs of electrons) are in the same vibrational or rotational energy states This effect will impact the wavelength at which a

transition is observed – very similar to the effect of H-bonding on the O-H vibrational energy levels in neat samples

Slide 10:

10 UV Spectroscopy Introduction Band Structure When these energy levels are superimposed, the effect can be readily explained – any transition has the possibility of being observed Energy E 0 E 1

Slide 11:

11 UV Spectroscopy Instrumentation and Spectra Instrumentation The construction of a traditional UV-VIS spectrometer is very similar to an IR, as similar functions – sample handling, irradiation, detection

and output are required Here is a simple schematic that covers most modern UV spectrometers: sample reference detector I 0 I 0 I 0 I log( I 0 / I ) = A 200 700 l , nm monochromator/ beam splitter optics UV-VIS

sources

Slide 12:

12 UV Spectroscopy Instrumentation and Spectra Instrumentation Two sources are required to scan the entire UV-VIS band: Deuterium lamp – covers the UV – 200-330 Tungsten lamp – covers 330-700 As with the dispersive IR, the lamps illuminate the entire band of UV or visible light; the monochromator (grating

or prism) gradually changes the small bands of radiation sent to the beam splitter The beam splitter sends a separate band to a cell containing the sample solution and a reference solution The detector

measures the difference between the transmitted light through the sample ( I ) vs. the incident light ( I 0 ) and sends this information to the recorder

Slide 13:

13 UV Spectroscopy Instrumentation and Spectra Instrumentation As with dispersive IR, time is required to cover the entire UV-VIS band due to the mechanism of changing wavelengths A recent improvement is the diode-array spectrophotometer - here a prism (dispersion device) breaks apart the full spectrum

transmitted through the sample Each individual band of UV is detected by a individual diodes on a

silicon wafer simultaneously – the obvious limitation is the size of the diode, so some loss of resolution over traditional instruments is observed sample Polychromator – entrance slit and dispersion device UV-

VIS sources Diode array

Slide 14:

14 UV Spectroscopy Instrumentation and Spectra Instrumentation – Sample Handling Virtually all UV spectra are recorded solution-phase Cells can be made of plastic, glass or quartz Only quartz is transparent in the full 200-700 nm range; plastic and glass are only suitable for visible spectra

Concentration (we will cover shortly) is empirically determined A typical sample cell (commonly called a cuvet ):

Slide 15:

15 UV Spectroscopy Instrumentation and Spectra Instrumentation – Sample Handling Solvents must be transparent in the region to be observed; the wavelength where a solvent is no longer transparent is referred to as the cutoff Since spectra are only obtained up to 200 nm, solvents typically only need to lack conjugated p systems or carbonyls Common solvents and cutoffs: acetonitrile 190 chloroform 240 cyclohexane 195 1,4-dioxane 215 95% ethanol 205 n -hexane 201 methanol 205 isooctane 195 water

190

Slide 16:

16 UV Spectroscopy Instrumentation and Spectra Instrumentation – Sample Handling Additionally solvents must preserve the fine structure (where it is actually observed in UV!) where possible H-

bonding further complicates the effect of vibrational and rotational energy levels on electronic transitions, dipole-dipole interacts less so The more non-polar the solvent, the better (this is not always

possible)

Slide 17:

17 UV Spectroscopy Instrumentation and Spectra The Spectrum The x-axis of the spectrum is in wavelength; 200-350 nm for UV, 200-700 for UV-VIS determinations Due to the lack of any fine

structure, spectra are rarely shown in their raw form, rather, the peak maxima are simply reported as a numerical list of “lamba max” values or l max l max = 206 nm 252 317 376

Slide 18:

18 UV Spectroscopy Instrumentation and Spectra The Spectrum The y-axis of the spectrum is in absorbance, A From the spectrometers point of view, absorbance is the inverse of transmittance: A = log 10 ( I 0 / I ) From an experimental point of view, three other considerations must be made: a longer path

length, l through the sample will cause more UV light to be absorbed – linear effect the greater the concentration, c of the sample, the more UV light will be absorbed – linear effect some electronic

transitions are more effective at the absorption of photon than others – molar absorptivity, e this may vary by orders of magnitude…

Slide 19:

19 UV Spectroscopy Instrumentation and Spectra The Spectrum These effects are combined into the Beer-Lambert Law: A = e c l for most UV spectrometers, l would remain constant (standard cells are

typically 1 cm in path length) concentration is typically varied depending on the strength of absorption observed or expected – typically dilute – sub .001 M molar absorptivities vary by orders of magnitude:

values of 10 4 -10 6 10 4 -10 6 are termed high intensity absorptions values of 10 3 -10 4 are termed low intensity absorptions values of 0 to 10 3 are the absorptions of forbidden transitions A is unitless, so the units for e are cm -1 · M -1 and are rarely expressed Since path length and concentration effects can be

easily factored out, absorbance simply becomes proportional to e , and the y-axis is expressed as e directly or as the logarithm of e

Slide 20:

20 UV Spectroscopy Instrumentation and Spectra Practical application of UV spectroscopy UV was the first organic spectral method, however, it is rarely used as a primary method for structure determination It is most useful in combination with NMR and IR data to elucidate unique electronic features that may be ambiguous in those methods It can be used to assay (via l max and molar absorptivity) the proper

irradiation wavelengths for photochemical experiments, or the design of UV resistant paints and coatings The most ubiquitous use of UV is as a detection device for HPLC; since UV is utilized for solution phase samples vs. a reference solvent this is easily incorporated into LC design UV is to HPLC what mass

spectrometry (MS) will be to GC

Slide 21:

21 UV Spectroscopy Chromophores Definition Remember the electrons present in organic molecules are involved in covalent bonds or lone pairs of electrons on atoms such as O or N Since similar functional groups will have electrons capable of discrete classes of transitions, the characteristic energy of these

energies is more representative of the functional group than the electrons themselves A functional group capable of having characteristic electronic transitions is called a chromophore ( color loving )

Structural or electronic changes in the chromophore can be quantified and used to predict shifts in the observed electronic transitions

Slide 22:

22 UV Spectroscopy Chromophores Organic Chromophores Alkanes – only posses s -bonds and no lone pairs of electrons, so only the high energy s s * transition is observed in the far UV This transition is

destructive to the molecule, causing cleavage of the s -bond s* s

Slide 23:

23 UV Spectroscopy Chromophores Organic Chromophores Alcohols, ethers, amines and sulfur compounds – in the cases of simple, aliphatic examples of these compounds the n s * is the most often

observed transition; like the alkane s s * it is most often at shorter l than 200 nm Note how this transition occurs from the HOMO to the LUMO s* CN s CN n N sp 3

Slide 24:

24 UV Spectroscopy Chromophores Organic Chromophores Alkenes and Alkynes – in the case of isolated examples of these compounds the p p * is observed at 175 and 170 nm, respectively Even though this

transition is of lower energy than s s *, it is still in the far UV – however, the transition energy is sensitive to substitution p* p

Slide 25:

25 UV Spectroscopy Chromophores Organic Chromophores Carbonyls – unsaturated systems incorporating N or O can undergo n p * transitions (~285 nm) in addition to p p * Despite the fact this

transition is forbidden by the selection rules ( e = 15), it is the most often observed and studied transition for carbonyls This transition is also sensitive to substituents on the carbonyl Similar to alkenes

and alkynes, non-substituted carbonyls undergo the p p * transition in the vacuum UV (188 nm, e = 900); sensitive to substitution effects

Slide 26:

26 UV Spectroscopy Chromophores Organic Chromophores Carbonyls – n p * transitions (~285 nm); p p * (188 nm) p p* n s CO transitions omitted for clarity It has been determined from spectral studies,

that carbonyl oxygen more approximates sp rather than sp 2 !

Slide 27:

27 UV Spectroscopy Chromophores Substituent Effects General – from our brief study of these general chromophores, only the weak n p * transition occurs in the routinely observed UV The attachment of substituent groups (other than H) can shift the energy of the transition Substituents that increase the

intensity and often wavelength of an absorption are called auxochromes Common auxochromes include alkyl, hydroxyl, alkoxy and amino groups and the halogens

Slide 28:

28 UV Spectroscopy Chromophores Substituent Effects General – Substituents may have any of four effects on a chromophore Bathochromic shift (red shift) – a shift to longer l ; lower energy Hypsochromic

shift (blue shift) – shift to shorter l ; higher energy Hyperchromic effect – an increase in intensity Hypochromic effect – a decrease in intensity 200 nm 700 nm e Hypochromic Hypsochromic

Hyperchromic Bathochromic

Slide 29:

29 UV Spectroscopy Chromophores Substituent Effects Conjugation – most efficient means of bringing about a bathochromic and hyperchromic shift of an unsaturated chromophore: l max nm e 175 15,000

217 21,000 258 35,000 n p * 280 27 p p * 213 7,100 465 125,000 n p * 280 12 p p * 189 900

Slide 30:

30 UV Spectroscopy Chromophores Substituent Effects Conjugation – Alkenes The observed shifts from conjugation imply that an increase in conjugation decreases the energy required for electronic excitation

From molecular orbital (MO) theory two atomic p orbitals, f 1 and f 2 from two sp 2 hybrid carbons combine to form two MOs Y 1 and Y 2 * in ethylene Y 2 * p Y 1 f 1 f 2

Slide 31:

31 UV Spectroscopy Chromophores Substituent Effects Conjugation – Alkenes When we consider butadiene, we are now mixing 4 p orbitals giving 4 MOs of an energetically symmetrical distribution compared to ethylene Y 2 * p Y 1 Y 1 Y 2 Y 3 * Y 4 * D E for the HOMO LUMO transition is reduced

Slide 32:

32 UV Spectroscopy Chromophores Substituent Effects Conjugation – Alkenes Extending this effect out to longer conjugated systems the energy gap becomes progressively smaller: Energy ethylene butadiene

hexatriene octatetraene Lower energy = Longer wavelengths

Slide 33:

33 UV Spectroscopy Chromophores Substituent Effects Conjugation – Alkenes Similarly, the lone pairs of electrons on N, O, S, X can extend conjugated systems – auxochromes Here we create 3 MOs – this

interaction is not as strong as that of a conjugated p -system Y 2 p Y 1 p * n A Y 3 * Energy

Slide 34:

34 UV Spectroscopy Chromophores Substituent Effects Conjugation – Alkenes Methyl groups also cause a bathochromic shift, even though they are devoid of p - or n -electrons This effect is thought to be

through what is termed “hyperconjugation” or sigma bond resonance

Slide 35:

35 UV Spectroscopy Next time – We will find that the effect of substituent groups can be reliably quantified from empirical observation of known conjugated structures and applied to new systems This

quantification is referred to as the Woodward-Fieser Rules which we will apply to three specific chromophores: Conjugated dienes Conjugated dienones Aromatic systems

Slide 36:

36 UV Spectroscopy Structure Determination Dienes General Features For acyclic butadiene, two conformers are possible – s-cis and s-trans The s-cis conformer is at an overall higher potential energy than the s-trans ; therefore the HOMO electrons of the conjugated system have less of a jump to the

LUMO – lower energy, longer wavelength s - trans s - cis

Slide 37:

37 UV Spectroscopy Structure Determination Dienes General Features Two possible p p * transitions can occur for butadiene Y 2 Y 3 * and Y 2 Y 4 * The Y 2 Y 4 * transition is not typically observed: The energy of this transition places it outside the region typically observed – 175 nm For the more favorable

s-trans conformation, this transition is forbidden The Y 2 Y 3 * transition is observed as an intense absorption s - trans s - cis 175 nm –forb. 217 nm 253 nm 175 nm Y 4 * Y 2 Y 1 Y 3 *

Slide 38:

38 UV Spectroscopy Structure Determination Dienes General Features The Y 2 Y 3 * transition is observed as an intense absorption ( e = 20,000+) based at 217 nm within the observed region of the UV

While this band is insensitive to solvent (as would be expected) it is subject to the bathochromic and hyperchromic effects of alkyl substituents as well as further conjugation Consider: l max = 217 253 220

227 227 256 263 nm

Slide 39:

39 UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules Woodward and the Fiesers performed extensive studies of terpene and steroidal alkenes and noted similar substituents and

structural features would predictably lead to an empirical prediction of the wavelength for the lowest energy p p * electronic transition This work was distilled by Scott in 1964 into an extensive treatise on

the Woodward-Fieser rules in combination with comprehensive tables and examples – (A.I. Scott, Interpretation of the Ultraviolet Spectra of Natural Products , Pergamon, NY, 1964) A more modern

interpretation was compiled by Rao in 1975 – (C.N.R. Rao, Ultraviolet and Visible Spectroscopy , 3 rd Ed., Butterworths, London, 1975)

Slide 40:

40 UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules - Dienes The rules begin with a base value for l max of the chromophore being observed: acyclic butadiene = 217 nm The

incremental contribution of substituents is added to this base value from the group tables: Group Increment Extended conjugation +30 Each exo-cyclic C=C +5 Alkyl +5 -OCOCH 3 +0 -OR +6 -SR +30 -Cl, -Br

+5 -NR 2 +60

Slide 41:

41 UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules - Dienes For example: Isoprene - acyclic butadiene = 217 nm one alkyl subs. + 5 nm 222 nm Experimental value 220 nm

Allylidenecyclohexane - acyclic butadiene = 217 nm one exocyclic C=C + 5 nm 2 alkyl subs. +10 nm 232 nm Experimental value 237 nm

Slide 42:

42 UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes There are two major types of cyclic dienes, with two different base values Heteroannular (transoid): Homoannular (cisoid): e = 5,000 – 15,000 e = 12,000-28,000 base l max = 214 base l max = 253 The increment table is the same as for acyclic butadienes with a couple additions: Group Increment Additional homoannular

+39 Where both types of diene are present, the one with the longer l becomes the base

Slide 43:

43 UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes In the pre-NMR era of organic spectral determination, the power of the method for discerning isomers is readily

apparent Consider abietic vs. levopimaric acid: levopimaric acid abietic acid

Slide 44:

44 UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes For example: 1,2,3,7,8,8a-hexahydro-8a-methylnaphthalene heteroannular diene = 214 nm 3 alkyl subs. (3 x

5) +15 nm 1 exo C=C + 5 nm 234 nm Experimental value 235 nm

Slide 45:

45 UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes heteroannular diene = 214 nm 4 alkyl subs. (4 x 5) +20 nm 1 exo C=C + 5 nm 239 nm homoannular diene

= 253 nm 4 alkyl subs. (4 x 5) +20 nm 1 exo C=C + 5 nm 278 nm

Slide 46:

46 UV Spectroscopy Structure Determination Dienes Woodward-Fieser Rules – Cyclic Dienes Be careful with your assignments – three common errors: This compound has three exocyclic double bonds; the

indicated bond is exocyclic to two rings This is not a heteroannular diene; you would use the base value for an acyclic diene Likewise, this is not a homooannular diene; you would use the base value for an

acyclic diene

Slide 47:

47 UV Spectroscopy Structure Determination Enones General Features Carbonyls, as we have discussed have two primary electronic transitions: p p* n Remember, the p p * transition is allowed and gives a

high e , but lies outside the routine range of UV observation The n p * transition is forbidden and gives a very low e, but can routinely be observed

Slide 48:

48 UV Spectroscopy Structure Determination Enones General Features For auxochromic substitution on the carbonyl, pronounced hypsochromic shifts are observed for the n p * transition ( l max ) : This is explained by the inductive withdrawal of electrons by O, N or halogen from the carbonyl carbon – this

causes the n -electrons on the carbonyl oxygen to be held more firmly It is important to note this is different from the auxochromic effect on p p * which extends conjugation and causes a bathochromic

shift In most cases, this bathochromic shift is not enough to bring the p p * transition into the observed range 293 nm 279 235 214 204 204

Slide 49:

49 UV Spectroscopy Structure Determination Enones General Features Conversely, if the C=O system is conjugated both the n p * and p p * bands are bathochromically shifted Here, several effects must be noted: the effect is more pronounced for p p * if the conjugated chain is long enough, the much higher intensity p p * band will overlap and drown out the n p * band the shift of the n p * transition is not

as predictable For these reasons, empirical Woodward-Fieser rules for conjugated enones are for the higher intensity, allowed p p * transition

Slide 50:

50 UV Spectroscopy Structure Determination Enones General Features These effects are apparent from the MO diagram for a conjugated enone: p Y 1 Y 2 Y 3 * Y 4 * p* n p p* n

Slide 51:

51 UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Group Increment 6-membered ring or acyclic enone Base 215 nm 5-membered ring parent enone Base 202 nm Acyclic dienone Base 245 nm Double bond extending conjugation 30 Alkyl group or ring residue a, b, g and

higher 10, 12, 18 -OH a, b, g and higher 35, 30, 18 -OR a, b, g, d 35, 30, 17, 31 -O(C=O)R a, b, d 6 -Cl a, b 15, 12 -Br a, b 25, 30 -NR 2 b 95 Exocyclic double bond 5 Homocyclic diene component 39

Slide 52:

52 UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Aldehydes, esters and carboxylic acids have different base values than ketones Unsaturated system Base Value Aldehyde

208 With a or b alkyl groups 220 With a,b or b,b alkyl groups 230 With a,b,b alkyl groups 242 Acid or ester With a or b alkyl groups 208 With a,b or b,b alkyl groups 217 Group value – exocyclic a,b double

bond +5 Group value – endocyclic a,b bond in 5 or 7 membered ring +5

Slide 53:

53 UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Unlike conjugated alkenes, solvent does have an effect on l max These effects are also described by the


Slide 54:

54 UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Some examples – keep in mind these are more complex than dienes cyclic enone = 215 nm 2 x b - alkyl subs. (2 x 12) +24 nm 239 nm Experimental value 238 nm cyclic enone = 215 nm extended conj. +30 nm b -ring residue

+12 nm d -ring residue +18 nm exocyclic double bond + 5 nm 280 nm Experimental 280 nm

Slide 55:

55 UV Spectroscopy Structure Determination Enones Woodward-Fieser Rules - Enones Take home problem – can these two isomers be discerned by UV-spec Eremophilone allo- Eremophilone Problem

Set 1: (text) – 1,2,3a,b,c,d,e,f,j, 4, 5, 6 (1 st , 2 nd and 5 th pairs), 8a, b, c Problem Set 2: outside problems/key -Tuesday

Slide 56:

56 UV Spectroscopy Structure Determination Aromatic Compounds General Features Although aromatic rings are among the most widely studied and observed chromophores, the absorptions that arise from the various electronic transitions are complex On first inspection, benzene has six p -MOs, 3 filled p , 3

unfilled p * p 4 * p 5 * p 6 * p 2 p 1 p 3

Slide 57:

57 UV Spectroscopy Structure Determination Aromatic Compounds General Features One would expect there to be four possible HOMO-LUMO p p * transitions at observable wavelengths (conjugation) Due to symmetry concerns and selection rules, the actual transition energy states of benzene are illustrated at the right: p 4 * p 5 * p 6 * p 2 p 1 p 3 A 1 g B 2 u B 1 u E 1 u 260 nm (forbidden) 200 nm (forbidden)

180 nm (allowed)

Slide 58:

58 UV Spectroscopy Structure Determination Aromatic Compounds General Features The allowed transition ( e = 47,000) is not in the routine range of UV obs. at 180 nm, and is referred to as the primary

band The forbidden transition ( e = 7400) is observed if substituent effects shift it into the obs. region; this is referred to as the second primary band At 260 nm is another forbidden transition ( e = 230),

referred to as the secondary band. This transition is fleetingly allowed due to the disruption of symmetry by the vibrational energy states, the overlap of which is observed in what is called fine structure

Slide 59:

59 UV Spectroscopy Structure Determination Aromatic Compounds General Features Substitution, auxochromic, conjugation and solvent effects can cause shifts in wavelength and intensity of aromatic systems similar to dienes and enones However, these shifts are difficult to predict – the formulation of

empirical rules is for the most part is not efficient (there are more exceptions than rules) There are some general qualitative observations that can be made by classifying substituent groups --

Slide 60:

60 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Substituents with Unshared Electrons If the group attached to the ring bears n electrons, they can induce a shift in the

primary and secondary absorption bands Non-bonding electrons extend the p -system through resonance – lowering the energy of transition p p * More available n -pairs of electrons give greater

shifts

Slide 61:

61 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Substituents with Unshared Electrons The presence of n -electrons gives the possibility of n p * transitions If this occurs, the electron now removed from G, becomes an extra electron in the anti-bonding p * orbital of the ring

This state is referred to as a charge-transfer excited state

Slide 62:

62 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Substituents with Unshared Electrons pH can change the nature of the substituent group deprotonation of oxygen gives

more available n -pairs, lowering transition energy protonation of nitrogen eliminates the n -pair, raising transition energy Primary Secondary Substituent l max e l max e -H 203.5 7,400 254 204 -OH 211 6,200 270 1,450 -O - 235 9,400 287 2,600 -NH 2 230 8,600 280 1,430 -NH 3 + 203 7,500 254 169 -C(O)OH 230

11,600 273 970 -C(O)O - 224 8,700 268 560

Slide 63:

63 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Substituents Capable of p -conjugation When the substituent is a p -chromophore, it can interact with the benzene p -system With benzoic acids, this causes an appreciable shift in the primary and secondary bands For the

benzoate ion, the effect of extra n -electrons from the anion reduces the effect slightly Primary Secondary Substituent l max e l max e -C(O)OH 230 11,600 273 970 -C(O)O - 224 8,700 268 560

Slide 64:

64 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Electron-donating and electron-withdrawing effects No matter what electronic influence a group exerts, the

presence shifts the primary absorption band to longer l Electron-withdrawing groups exert no influence on the position of the secondary absorption band Electron-donating groups increase the l and e of the

secondary absorption band

Slide 65:

65 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Electron-donating and electron-withdrawing effects Primary Secondary Substituent l max e l max e -H 203.5 7,400 254 204 -CH 3 207 7,000 261 225 -Cl 210 7,400 264 190 -Br 210 7,900 261 192 -OH 211 6,200 270 1,450 -

OCH 3 217 6,400 269 1,480 -NH 2 230 8,600 280 1,430 -CN 224 13,000 271 1,000 C(O)OH 230 11,600 273 970 -C(O)H 250 11,400 -C(O)CH 3 224 9,800 -NO 2 269 7,800 Electron donating Electron

withdrawing

Slide 66:

66 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Di-substituted and multiple group effects With di-substituted aromatics, it is necessary to consider both groups If both groups are electron donating or withdrawing, the effect is similar to the effect of the stronger of the two

groups as if it were a mono -substituted ring If one group is electron withdrawing and one group electron donating and they are para - to one another, the magnitude of the shift is greater than the sum

of both the group effects Consider p -nitroaniline:

Slide 67:

67 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Di-substituted and multiple group effects If the two electonically dissimilar groups are ortho- or meta- to one another, the effect is usually the sum of the two individual effects ( meta - no resonance; ortho -steric hind.) For

the case of substituted benzoyl derivatives, an empirical correlation of structure with observed l max has been developed This is slightly less accurate than the Woodward-Fieser rules, but can usually predict

within an error of 5 nm

Slide 68:

68 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Di-substituted and multiple group effects Substituent increment G o m p Alkyl or ring residue 3 3 10 -O-Alkyl, -OH, -O-

Ring 7 7 25 -O - 11 20 78 -Cl 0 0 10 -Br 2 2 15 -NH 2 13 13 58 -NHC(O)CH 3 20 20 45 -NHCH 3 73 -N(CH 3 ) 2 20 20 85 Parent Chromophore l max R = alkyl or ring residue 246 R = H 250 R = OH or O-Alkyl 230

Slide 69:

69 UV Spectroscopy Structure Determination Aromatic Compounds Substituent Effects Polynuclear aromatics When the number of fused aromatic rings increases, the l for the primary and secondary

bands also increase For heteroaromatic systems spectra become complex with the addition of the n p * transition and ring size effects and are unique to each case

Slide 70:

70 UV Spectroscopy Visible Spectroscopy Color General The portion of the EM spectrum from 400-800 is observable to humans- we (and some other mammals) have the adaptation of seeing color at the

expense of greater detail 400 500 600 800 700 l , nm Violet 400-420 Indigo 420-440 Blue 440-490 Green 490-570 Yellow 570-585 Orange 585-620 Red 620-780

Slide 71:

71 UV Spectroscopy Visible Spectroscopy Color General When white (continuum of l ) light passes through, or is reflected by a surface, those ls that are absorbed are removed from the transmitted or reflected light respectively What is “seen” is the complimentary colors (those that are not absorbed)

This is the origin of the “color wheel”

Slide 72:

72 UV Spectroscopy Visible Spectroscopy Color General Organic compounds that are “colored” are typically those with extensively conjugated systems (typically more than five) Consider b -carotene l max

is at 455 – in the far blue region of the spectrum – this is absorbed The remaining light has the complementary color of orange

Slide 73:

73 UV Spectroscopy Visible Spectroscopy Color General Likewise: l max for lycopene is at 474 – in the near blue region of the spectrum – this is absorbed, the compliment is now red l max for indigo is at 602

– in the orange region of the spectrum – this is absorbed, the compliment is now indigo!

Slide 74:

74 UV Spectroscopy Visible Spectroscopy Color General One of the most common class of colored organic molecules are the azo dyes: From our discussion of di-subsituted aromatic chromophores, the

effect of opposite groups is greater than the sum of the individual effects – more so on this heavily conjugated system Coincidentally, it is necessary for these to be opposite for the original synthetic

preparation!

Slide 75:

75 UV Spectroscopy Visible Spectroscopy Color General These materials are some of the more familiar colors of our “environment”

Slide 76:

76 The colors of M&M’s Bright Blue Common Food Uses Beverages, dairy products, powders, jellies, confections, condiments, icing. Royal Blue Common Food Uses Baked goods, cereals, snack foods, ice-

cream, confections, cherries. Orange-red Common Food Uses Gelatins, puddings, dairy products, confections, beverages, condiments. Lemon-yellow Common Food Uses Custards, beverages, ice-cream,

confections, preserves, cereals. Orange Common Food Uses Cereals, baked goods, snack foods, ice-cream, beverages, dessert powders, confections

Slide 77:

77 UV Spectroscopy Visible Spectroscopy Color General In the biological sciences these compounds are used as dyes to selectively stain different tissues or cell structures Biebrich Scarlet - Used with picric

acid/aniline blue for staining collagen, recticulum, muscle, and plasma. Luna's method for erythrocytes & eosinophil granules. Guard's method for sex chromatin and nuclear chromatin.

Slide 78:

78 UV Spectroscopy Visible Spectroscopy Color General In the chemical sciences these are the acid-base indicators used for the various pH ranges: Remember the effects of pH on aromatic substituents

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Presentation Transcript

UV-Visible spectroscopy :

1 UV-Visible spectroscopy U.A. Deokate Copyright © by U. A. Deokate, all rights reserved.

EMR & Electromagnetic spectrum :

Deokate U.A. 2 09/20/2006 8:58 AM EMR & Electromagnetic spectrum The radiations which travels with speed are called Electro magnetic radiations. It vibrates perpendicular to the direction of propagation with a wave motion. It is broken in to several regions called as Electro Magnetic Spectrum Different

regions of the electromagnetic spectrum provide different kinds of information as a result of interactions. Light may be considered to have both wave-like and particle-like characteristics.

Electromagnetic Spectrum :

Deokate U.A. 3 09/20/2006 8:58 AM Electromagnetic Spectrum

UV-Visible spectrum :

Deokate U.A. 4 09/20/2006 8:58 AM UV-Visible spectrum The part of the electromagnetic radiation spectrum that you are most familiar with is "visible light" but this is just a small portion of all the

possible types Violet : 400 - 420 nm Indigo : 420 - 440 nm Blue : 440 - 490 nm Green : 490 - 570 nm Yellow : 570 - 585 nm Orange : 585 - 620 nm Red : 620 - 780 nm

Ultraviolet and visible spectroscopy :

Deokate U.A. 5 09/20/2006 8:58 AM Ultraviolet and visible spectroscopy It is used to measure the multiple bonds or atomic conjugation within the molecule. The UV-Visible region is subdivided as below Vacuum UV: 100-200 nm Near UV: 200 to 400 nm Visible region: 400 to 750 nm Vacuum UV is so named

because molecule of air absorb radiation in these region. The radiation is assessable only in special vacuum equipments.

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Basics of UV Light Absorption :

Deokate U.A. 6 09/20/2006 8:58 AM Basics of UV Light Absorption Ultraviolet/visible spectroscopy involves the absorption of ultraviolet/visible light by a molecule causing the promotion of an electron from a ground electronic state to an excited electronic state Absorption of this relatively high-energy light causes electronic excitation. The easily accessible part of this region (wavelengths of 200 to 800

nm) shows absorption only if conjugated pi-electron systems are present.

Electronic spectroscopy :

Deokate U.A. 7 09/20/2006 8:58 AM Electronic spectroscopy The visible region of the spectrum comprises photon energies of 36 to 72 kcal/mole, and the near ultraviolet region, out to 200 nm,

extends this energy range to 143 kcal/mole. Ultraviolet radiation having wavelengths less than 200 nm is difficult to handle, and is seldom used as a routine tool for structural analysis The energies are sufficient

to promote or excite a molecular electron to a higher energy orbital. The absorption spectroscopy carried out in this region is sometimes called "electronic spectroscopy".

Electronic transactions involved :

Deokate U.A. 8 09/20/2006 8:58 AM Electronic transactions involved

Electronic spectroscopy :

Deokate U.A. 9 09/20/2006 8:58 AM Electronic spectroscopy Out of the six transitions outlined, only the two lowest energy ones (left-most, colored blue) are achieved by the energies available in the 200 to

800 nm spectrum. As a rule, energetically favored electron promotion will be from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO), and the resulting species

is called an excited state

Types of Transitions :

Deokate U.A. 10 09/20/2006 8:58 AM Types of Transitions There are several types of electronic transitions available to a molecule including: σ to σ * (alkanes) σ to π * (carbonyl compounds) π to π *

(alkenes, carbonyl compounds, alkynes, azo compounds) ή to σ * (oxygen, nitrogen, sulfur, and halogen compounds) ή to π * (carbonyl compounds)

Electronic transactions involved :

Deokate U.A. 11 09/20/2006 8:58 AM Electronic transactions involved

σ σ * Transitions :

Deokate U.A. 12 09/20/2006 8:58 AM σ σ * Transitions An electron in a bonding σ orbital is excited to the corresponding antibonding orbital. The energy required is large. For example, alkanes as methane (which has only C-H bonds, and can only undergo σ σ * transitions) shows an absorbance maximum at

125 nm. Absorption maxima due to σ σ * transitions are not seen in typical UV-Visible spectra (200 - 700 nm). σ bonds are very strong and requires higher energy of vacuum UV.

ή σ * Transitions :

Deokate U.A. 13 09/20/2006 8:58 AM ή σ * Transitions Saturated compounds containing atoms with lone pairs (non-bonding electrons) are capable of ή σ * as transitions. These transitions usually need less energy than σ σ * transitions. They can be initiated by light whose wavelength is in the range 150 - 250

nm. The number of organic functional groups with n σ * peaks in the UV region is small. These transitions are involved in saturated compound with one hetero atom with unshared pair of electron i.e.

saturated halides, ethers, aldehide, ketones, amines etc. These transitions are sensitive to hydrogen bonding eg alcohol and ethers which absorbs at wavelength shorter than 185 nm therefore used as a

solvent in UV

π π * Transitions :

Deokate U.A. 14 09/20/2006 8:58 AM π π * Transitions For molecules that possess π bonding as in alkenes, alkynes, aromatics, acyl compounds or nitriles, energy that is available can promote electrons

from a π Bonding molecular orbital to a π Antibonding molecular orbital (π * ). This is called a π π * transition. The absorption peaks for these transitions fall in an experimentally convenient region of the spectrum (200 - 700 nm). These transitions need an unsaturated group in the molecule to provide the π

electrons.

ή π * Transitions :

Deokate U.A. 15 09/20/2006 8:58 AM ή π * Transitions Even lone pairs that exist on Oxygen atoms and Nitrogen atoms may be promoted from their non-bonding molecular orbital to a π antibonding

molecular orbital within the molecule. This is called an ή π * transition and requires less energy (longer

wavelength) compared to a ή π * transition within the same chromaphore. These are available in compounds with unsaturated centers. eg. Alkenes. They requires lowest energy as compare to others

Terminology Chromophore: :

Deokate U.A. 16 09/20/2006 8:58 AM Terminology Chromophore: Chromophore: A covalently unsaturated group responsible for electronic absorption. Or Any group of atoms that absorbs light

whether or not a color is thereby produced. Eg c=c, c=o, No2 etc A compound containing chromophore is called chromogen. There are two types of chromophore Independent chromophore: single

chromophore is sufficient to import color to the compound eg. Azo group Dependent chromophore: When more then one chromophore is required to produce color. Eg acetone having 1 kentone group is

colorless where as diacetyl having two kentone group is yellow

Auxochrome: :

Deokate U.A. 17 09/20/2006 8:58 AM Auxochrome: Auxochrome: A saturated group with non bonding electron when attached to chromophore alters both wavelengths as well as intensity of absorption. Eg

OH, NH2, NHR etc. OR A group which extends the conjugation of a chromophore by sharing of nonbonding electrons. Bathochromic group: The group which deepen the colour of chromophore is

called bathochromic group. Eg. Primary, secondary and tertiary amino groups

Terminology :

Deokate U.A. 18 09/20/2006 8:58 AM Terminology Bathochromic shift: (Red shift) shift of lambda max to longer side or less energy is called bathochromic shift or read shift. This is due to substitution or solvent

effect. Hypsochromic shift: (Blue shift) shift of lambda max to shorter side and higher energy is called hypsochromic or blue shift. Eg solvent effect. Hyperchromic effect: an increase in absorption intensity

Hypochromic effect: an decrease in absorption intensity

Substituent Effects :

Deokate U.A. 19 09/20/2006 8:58 AM Substituent Effects General – Substituents may have any of four effects on a chromophore Bathochromic shift (red shift) – a shift to longer l; lower energy Hypsochromic

shift (blue shift) – shift to shorter l; higher energy Hyperchromic effect – an increase in intensity Hypochromic effect 200 nm 700nm – a decrease in intensity Hypochromic Hypsochromic Hyperchromic

Bathochromic e

Instrumentation: :

Deokate U.A. 20 09/20/2006 8:58 AM Instrumentation: GENERAL INSTRUMENTATION

Instrumentation: :

Deokate U.A. 21 09/20/2006 8:58 AM Instrumentation: A beam of light from a visible and/or UV light source (colored red) is separated into its component wavelengths by a prism or diffraction grating. Each

monochromatic (single wavelength) beam in turn is split into two equal intensity beams by a half-mirrored device. One beam, the sample beam (colored magenta), passes through a small transparent container (cuvette) containing a solution of the compound being studied in a transparent solvent. The

other beam, the reference (colored blue), passes through an identical cuvette containing only the solvent. The intensities of these light beams are then measured by electronic detectors and compared. The intensity of the reference beam, which should have suffered little or no light absorption, is defined

as I0. The intensity of the sample beam is defined as I. Over a short period of time, the spectrometer automatically scans all the component wavelengths in the manner described. The ultraviolet (UV) region

scanned is normally from 200 to 400 nm, and the visible portion is from 400 to 800 nm.

Solvent effect: :

Deokate U.A. 22 09/20/2006 8:58 AM Solvent effect: Different compounds may have very different absorption maxima and absorbances. Intensely absorbing compounds must be examined in dilute

solution, so that significant light energy is received by the detector, and this requires the use of completely transparent (non-absorbing) solvents. The most commonly used solvents are water, ethanol,

hexane and cyclohexane. Solvents having double or triple bonds, or heavy atoms (e.g. S, Br & I) are generally avoided. Because the absorbance of a sample will be proportional to its molar concentration in

the sample cuvette, a corrected absorption value known as the molar absorptivity is used when comparing the spectra of different compounds. This is defined as: Molar Absorptivity,ε = A/bc ( where A= absorbance, c = sample concentration in moles/literb = length of light path through the cuvette in

cm.)

Choice of Solvent :

Deokate U.A. 23 09/20/2006 8:58 AM Choice of Solvent

Absorption Intensity :

Deokate U.A. 24 09/20/2006 8:58 AM Absorption Intensity Molar absoptivities may be very large for strongly absorbing chromophores (>10,000) and very small if absorption is weak (10 to 100). The magnitude of ε reflects both the size of the chromophore and the probability that light of a given

wavelength will be absorbed when it strikes the chromophore. A general equation stating this relationship may be written as follows: ε = 0.87 • 1020 Ρ • a (where Ρ is the transition probability ( 0 to 1

) & a is the chromophore area in cm2 )

UV-Visible spectrum :

Deokate U.A. 25 09/20/2006 8:58 AM UV-Visible spectrum A spectrum is a plot of the intensity of energy detected versus the wavelength (or mass or momentum or frequency, etc.) of the energy.

Applications of UV/Visible Spectrophotometry :

Deokate U.A. 26 09/20/2006 8:58 AM Applications of UV/Visible Spectrophotometry UV spectra and Visible spectra can be used to identify an unknown compound by a comparative analysis. One can compare the UV or Visible spectra of the unknown with the spectra of known suspects. Those that

match are evidence that they could be one and the same. However using a match on UV or Visible is not conclusive. UV and Visble spectra can also be used to determine the concentration of a chromaphore compound in a mixture using the Beer Lambert Law. Usually 3-5 standard sample of the chromaphore

are prepared, and the absorbance of each standard is measured. The Absorbance can be plotted against the concentration and a standard curve can be generated. By measuring the absorbance of the unknown mixture, one can locate the absorbance on the "y" axis of the Standard curve, draw a perpindicular over till it intersects the standard curve and then a perpindicular down until it intersects the "x" axis. At the

point of intersection on the "x" axis, the concentration of the chromaphore can be determined. One can also use a proportional method where the Absorbance of a known concentration sample is measured

and the Absorbance of the unknown can be measured. Then at lamda max:

Woodward Fisher rule :

Deokate U.A. 27 09/20/2006 8:58 AM Woodward Fisher rule Woodward fisher rule for calculation of lambda max position

NMR


PRINCIPLE,INSTRUMENTATION AND APPLICATIONS OF NMR SPECTROSCOPY BY SUJITH THOKALA 1


Introduction: NMR Spectroscopy is the study of spin changes at the nuclear level when a radiofrequency energy is absorbed in the presence of magnetic field More important to the organic chemist than

infrared spectroscopy Gives information about the number of magnetically distinct atoms of the type being studied Unlike other spectroscopic techniques nuclei of atoms rather than other electros are

involved in the absorption process The combination of IR and NMR data is often sufficient to determine completely the structure of an unknown sample Nuclei with odd mass number only give NMR spectra 2


Consider a spinning top, in which the spinning axis of the top moves slowly around the vertical. This is precessional motion and the top is said to be pressing around the vertical axis of earth’s gravitational field. Precessional Motion: Likewise the proton(tiny magnet) precesses about the axis of the external

magnetic field in the same manner as above Precessional Frequency: The precessional frequency of the spinning magnet(nucleus) may be defined as equal to the frequency of electromagnetic radiations in

megacycles per second necessary to induce a transition from one spin state to another 3


Principle When energy in the form of radiofrequency is applied and when Applied frequency = Precessional frequency absorption of energy occurs and a NMR signal is recorded ** Without application

of magnetic field, there is no two spin states and there is only one average spin ** Hence radio frequency radiation cannot be absorbed ** Therefore, application of magnetic field and radio frequency

is necessary to cause a NMR spectra Note: 4


Excitation Process in NMR Spectroscopy 5


The nuclear magnetic resonance process; absorption occurs when ν = ω ω =60MHz ν =60MHz h ν B o 6


Relaxation Process: Involve some non-radiative transitions by which a nucleus in an upper transition state returns to the lower spin state Three kinds of relaxation processes are: i ) Spin-spin Relaxation: It

involves the transfer of energy from one nucleus to the other There is no net loss of energy 7


ii) Spin-lattice Relaxation (Longitudinal relaxation): It involves the transfer of energy from the nucleus in its higher energy state to the molecular lattice As the additional transitional, vibrational and rotational

energy The total energy of the system remains same This mechanism is not effective in solids 8


Only for nuclei with I > 1/2 There must be a permanent (or transient) electric field gradient across the nucleus (i.e., QR is much less effective in molecules in which the nucleus is at a center of tetrahedral or

octahedral symmetry) For nuclei with a large electric quadrupole moment, this mechanism is so effective Characteristic features: iii) Quadrupole Relaxation: 9


Instrumentation 10


11


Sample Holders: Holder should be chemically inert, durable and transparent to rf radiation Generally glass tubes are employed 8.5cm long and approximately 0.3cm in diameter Permanent Magnet: The factors which are important in the design of the magnets for NMR spectroscopy - Homogeneity or

uniformity of the field - The constancy of the field strength - Maximum obtainable strength of the field Sweeping coils: For the nucleus to resonate, the precession frequency of the nucleus must equal the

frequency of the applied RF radiation So by keeping the RF frequency constant and changing the B o by using a pair of Helmholtz coils this can be achieved This is called sweeping the field 12


Radio Frequency Generator : Radio frequency oscillator is used to generate RF radiation The coil of oscillator is wound around the sample container The oscillator coil is wound perpendicular to the

applied magnetic field 13


Radio Frequency Receiver: Absorption signal Dispersion signal Detector should be capable of separating absorption signal from dispersion signal. There are two methods of detection i ) By using a radio

frequency bridge ii) By using a separate receiver coil Absorption and dispersion signals differ in phase by 90 o Phase sensitive detector helps the operator to select the phase of the signal to be detected 14


Readout System: The absorption signal received from radio frequency receiver is extremely weak Therefore, it requires considerable amplification before it is fed to a chart recorder Chart recorder 15


Block Diagram of Fourier Transform NMR Spectrometer 16


Applications of NMR Spectroscopy The NMR spectroscopy is widely used for the detailed investigation of an unknown compound Identification of structural isomers Detection of hydrogen bonding Detection of aromaticity Distinction between Cis-Trans isomers and conformers Detection of electronegative atom or

group Detection of some double bond character due to resonance Importance in quantitative analysis 17


Other applications: To study interactions between different molecules To study molecular motions in liquids and polymers For determining the three dimensional structure of proteins and other

macromolecules Solid state NMR is used in material science Also used in medical diagnosis (MRI) Oil and natural gas exploration and recovery in petroleum industry 18


Limitations of NMR Spectroscopy Various limitations of NMR spectroscopy are as follows: One of the serious problem with NMR is its lack of sensitivity If two atoms resonate at similar resonance

frequencies results in overlap of spectra While characterizing, the organic compounds, no information about molecular weight is obtained by NMR In most of the cases, only liquids can be studied by NMR

spectroscopy 19


INTERPRETATION OF NMR SPECTRA 20


NMR Problem-1 21


Solve-1 2-Butanone 22


NMR Problem-2 23


4-Heptanone Solve-2 24


NMR Problem-3 25


Solve-3 4-Methylbenzaldehyde 26


NMR Problem-4 27


4-Propylbutanoate Solve-4 28


CONCLUSION NMR spectroscopy has been advancing over the decades with the development of more and more powerful magnets. Although the method has all the earmarks of a very competent

quantitative analytical tool, its quantitative potential remains largely untapped due to the cost of such instruments. 29


Donald L.Pavia, Gary M.Lampman, George S.Krig , Introduction to Spectroscopy , 3 rd edition,105-176, Harcourt college publishers(2007) Gurudeep R. Chatwal,Sham k. Anand, Instrumental Methods of

Chemical Analysis ,5 th Edition,2.185-2.234,Himalaya Publishing House(2009) Y.R.Sharma, Elementary Organic Spectroscopy ,4 th Edition,181-255, S.Chand & Company Ltd(2010) David G.Watson,

Pharmaceutical Analysis , 2 nd Edition, 163-185, Elsevier(2005) http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/nmr/nmr1.htm

http://www.nanonet.go.jp/english/info/nanoproject/kitaguchi.html http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/nmr1.htm

http://chem.ch.huji.ac.il/nmr/whatisnmr/whatisnmr.html http://www.spsj.or.jp/c5/kobunshi/kobu2009/hottopics0907.html REFERENCES 30

Chapter 13Nuclear Magnetic

Resonance Spectroscopy:

Chapter 13 Nuclear Magnetic Resonance Spectroscopy Jo Blackburn Richland College, Dallas, TX Dallas County Community College District ã 2003, Prentice Hall Organic Chemistry, 5th Edition L. G. Wade, Jr.

Introduction:

Introduction NMR is the most powerful tool available for organic structure determination. It is used to study a wide variety of nuclei: 1H 13C 15N 19F 31P =>

Nuclear Spin:

Nuclear Spin A nucleus with an odd atomic number or an odd mass number has a nuclear spin. The spinning charged nucleus generates a magnetic field.

External Magnetic Field:

External Magnetic Field When placed in an external field, spinning protons act like bar magnets. =>

Two Energy States:

Two Energy States The magnetic fields of the spinning nuclei will align either with the external field, or against the field. A photon with the right amount of energy can be absorbed and cause the spinning

proton to flip. =>

E and Magnet Strength:

E and Magnet Strength Energy difference is proportional to the magnetic field strength. E = h = h B0 2 Gyromagnetic ratio, , is a constant for each nucleus (26,753 s-1gauss-1 for H). In a 14,092 gauss

field, a 60 MHz photon is required to flip a proton. Low energy, radio frequency. =>

Magnetic Shielding:

Magnetic Shielding If all protons absorbed the same amount of energy in a given magnetic field, not much information could be obtained. But protons are surrounded by electrons that shield them from

the external field. Circulating electrons create an induced magnetic field that opposes the external magnetic field. =>

Shielded Protons:

Shielded Protons Magnetic field strength must be increased for a shielded proton to flip at the same frequency.

Protons in a Molecule:

Protons in a Molecule Depending on their chemical environment, protons in a molecule are shielded by different amounts.

NMR Signals:

NMR Signals The number of signals shows how many different kinds of protons are present. The location of the signals shows how shielded or deshielded the proton is. The intensity of the signal shows the number of protons of that type. Signal splitting shows the number of protons on adjacent atoms. =>

The NMR Spectrometer:

The NMR Spectrometer =>

The NMR Graph:

The NMR Graph =>

Tetramethylsilane:

Tetramethylsilane TMS is added to the sample. Since silicon is less electronegative than carbon, TMS protons are highly shielded. Signal defined as zero. Organic protons absorb downfield (to the left) of the

TMS signal. =>

Chemical Shift:

Chemical Shift Measured in parts per million. Ratio of shift downfield from TMS (Hz) to total spectrometer frequency (Hz). Same value for 60, 100, or 300 MHz machine. Called the delta scale. =>

Delta Scale:

Delta Scale =>

Location of Signals:

Location of Signals More electronegative atoms deshield more and give larger shift values. Effect decreases with distance. Additional electronegative atoms cause increase in chemical shift. =>

Typical Values:

Typical Values =>

Aromatic Protons, 7-8:

Aromatic Protons, 7-8 =>

Vinyl Protons, 5-6:

Vinyl Protons, 5-6 =>

Acetylenic Protons, 2.5:

Acetylenic Protons, 2.5 =>

Aldehyde Proton, 9-10:

Aldehyde Proton, 9-10 => Electronegative oxygen atom

O-H and N-H Signals:

O-H and N-H Signals Chemical shift depends on concentration. Hydrogen bonding in concentrated solutions deshield the protons, so signal is around 3.5 for N-H and 4.5 for O-H. Proton exchanges

between the molecules broaden the peak. =>

Carboxylic Acid Proton, 10+:

Carboxylic Acid Proton, 10+ =>

Number of Signals:

Number of Signals Equivalent hydrogens have the same chemical shift. =>

Intensity of Signals:

Intensity of Signals The area under each peak is proportional to the number of protons. Shown by integral trace.

How Many Hydrogens?:

How Many Hydrogens? When the molecular formula is known, each integral rise can be assigned to a particular number of hydrogens.

Spin-Spin Splitting:

Spin-Spin Splitting Nonequivalent protons on adjacent carbons have magnetic fields that may align with or oppose the external field. This magnetic coupling causes the proton to absorb slightly downfield

when the external field is reinforced and slightly upfield when the external field is opposed. All possibilities exist, so signal is split. =>

1,1,2-Tribromoethane:

1,1,2-Tribromoethane Nonequivalent protons on adjacent carbons. =>

Doublet: 1 Adjacent Proton:

Doublet: 1 Adjacent Proton =>

Triplet: 2 Adjacent Protons:

Triplet: 2 Adjacent Protons =>

The N + 1 Rule:

The N + 1 Rule If a signal is split by N equivalent protons, it is split into N + 1 peaks. =>

Range of Magnetic Coupling:

Range of Magnetic Coupling Equivalent protons do not split each other. Protons bonded to the same carbon will split each other only if they are not equivalent. Protons on adjacent carbons normally will

couple. Protons separated by four or more bonds will not couple. =>

Splitting for Ethyl Groups:

Splitting for Ethyl Groups =>

Splitting for Isopropyl Groups:

Splitting for Isopropyl Groups =>

Coupling Constants:

Coupling Constants Distance between the peaks of multiplet Measured in Hz Not dependent on strength of the external field Multiplets with the same coupling constants may come from adjacent groups of

protons that split each other. =>

Values for Coupling Constants:

Values for Coupling Constants =>

Complex Splitting:

Complex Splitting Signals may be split by adjacent protons, different from each other, with different coupling constants. Example: Ha of styrene which is split by an adjacent H trans to it (J = 17 Hz) and an

adjacent H cis to it (J = 11 Hz). =>

Splitting Tree:

Splitting Tree

Spectrum for Styrene:

Spectrum for Styrene =>

Stereochemical Nonequivalence:

Stereochemical Nonequivalence Usually, two protons on the same C are equivalent and do not split each other. If the replacement of each of the protons of a -CH2 group with an imaginary “Z” gives

stereoisomers, then the protons are non-equivalent and will split each other. =>

Some Nonequivalent Protons:

Some Nonequivalent Protons

Time Dependence:

Time Dependence Molecules are tumbling relative to the magnetic field, so NMR is an averaged spectrum of all the orientations. Axial and equatorial protons on cyclohexane interconvert so rapidly

that they give a single signal. Proton transfers for OH and NH may occur so quickly that the proton is not split by adjacent protons in the molecule. =>

Hydroxyl Proton:

Hydroxyl Proton Ultrapure samples of ethanol show splitting. Ethanol with a small amount of acidic or basic impurities will not show splitting.

N-H Proton:

N-H Proton Moderate rate of exchange. Peak may be broad.

Identifying the O-H or N-H Peak:

Identifying the O-H or N-H Peak Chemical shift will depend on concentration and solvent. To verify that a particular peak is due to O-H or N-H, shake the sample with D2O Deuterium will exchange with the O-H

or N-H protons. On a second NMR spectrum the peak will be absent, or much less intense. =>

Carbon-13:

Carbon-13 12C has no magnetic spin. 13C has a magnetic spin, but is only 1% of the carbon in a sample. The gyromagnetic ratio of 13C is one-fourth of that of 1H. Signals are weak, getting lost in noise.

Hundreds of spectra are taken, averaged. =>

Fourier Transform NMR:

Fourier Transform NMR Nuclei in a magnetic field are given a radio-frequency pulse close to their resonance frequency. The nuclei absorb energy and precess (spin) like little tops. A complex signal is produced, then decays as the nuclei lose energy. Free induction decay is converted to spectrum. =>

Hydrogen and Carbon Chemical Shifts:

Hydrogen and Carbon Chemical Shifts

Combined 13C and 1H Spectra:

Combined 13C and 1H Spectra =>

Differences in 13C Technique:

Differences in 13C Technique Resonance frequency is ~ one-fourth, 15.1 MHz instead of 60 MHz. Peak areas are not proportional to number of carbons. Carbon atoms with more hydrogens absorb more

strongly. =>

Spin-Spin Splitting:

Spin-Spin Splitting It is unlikely that a 13C would be adjacent to another 13C, so splitting by carbon is negligible. 13C will magnetically couple with attached protons and adjacent protons. These complex

splitting patterns are difficult to interpret. =>

Proton Spin Decoupling:

Proton Spin Decoupling To simplify the spectrum, protons are continuously irradiated with “noise,” so they are rapidly flipping. The carbon nuclei see an average of all the possible proton spin states. Thus,

each different kind of carbon gives a single, unsplit peak. =>

Off-Resonance Decoupling:

Off-Resonance Decoupling 13C nuclei are split only by the protons attached directly to them. The N + 1 rule applies: a carbon with N number of protons gives a signal with N + 1 peaks. =>

Interpreting 13C NMR:

Interpreting 13C NMR The number of different signals indicates the number of different kinds of carbon. The location (chemical shift) indicates the type of functional group. The peak area indicates the numbers

of carbons (if integrated). The splitting pattern of off-resonance decoupled spectrum indicates the number of protons attached to the carbon. =>

Two 13C NMR Spectra:

Two 13C NMR Spectra =>

MRI:

MRI Magnetic resonance imaging, noninvasive “Nuclear” is omitted because of public’s fear that it would be radioactive. Only protons in one plane can be in resonance at one time. Computer puts

together “slices” to get 3D. Tumors readily detected. =>

Nuclear Magnetic Resonance Spectroscopy (NMR):

1 Nuclear Magnetic Resonance Spectroscopy (NMR) Presented By :- Mr.Jayprakash S.Nogaja M Pharmacy-IIyr (Pharmaceutics) [email protected]


2 ( NMR ) H-NMR / Proton NMR “ NMR is the branch of spectroscopy in which radiofrequency waves induces transitions between magnetic energy levels of nuclei of a molecule .The energy levels are

created by keeping the nuclei in a magnetic field .” Measures Magnetic properties of nuclei. Nuclear Magnetic Resonance Spectroscopy


3 Protons of different chemical environment produces their own unique pick. hence different types of protons in am molecule produce different picks NMR Spectroscopy is based up on the measurement of absorption of electromagnetic waves by the spinning nuclei in the radiofrequency region of range 4 to

900 MHZ (V)


4 What is meaning of Different Chemical & Magnetic Environment ?


5 Eg – in Aliphatic compounds


6


7 What is meaning of Different Chemical & Magnetic Environment : 5 ring Protons 6 ring Protons 5 ring Protons 3 methy Protons 3 methyl Protons 5 u 3u Reff.std. 6 u Reff.std. Eg – in Aromatic compounds


8 What is Spinning of protons ? This behaves as Induced Field magnet Nucleus (1 proton,0 neutron ) Positive Center Electron Negative Center The nucleus of hydrogen atom (proton) behaves as a tiny

Spinning bar magnet .


9 What is The need of Magnetic Field ? Aligned /Parallal /attration energy /low energy (E1)/ +1/2 / α Opposed /Antiparallal /Repulsion Energy/ High Energy (E2) /-1/2 / β Ans :To create magnetic energy levels in nuclei of a molecule The transition from one energy state to another is called as Flipping of

proton occurs due to absorption of EMR


10 Induced Magnetic Field in proton-


11 Now this Aligned or Opposed precessing protons will absorb energy from the radiofrequency sourse only if its precessing frequency is equals to ferquency of beam.when this occures,the nucleus and the

radiofrequency beam are said to be in resonance ,hence the term NMR


12 Radiation source /Energy source : The wave lengths and energy required in NMR spectroscopy are far different from that of UV visible & IR . In NMR radiofrequency waves are used which are long

wavelength and therefore have less energy associated with them .These do not affect the nuclei in strong magnetic field . Radiofrequency region of range 4 to 900 MHZ


13 Concepts of 1)Precessional Motion.2)Precessional Frequency .3)Gyroscopic Motion. Aligned Opposed Δ E low energy (E1 ) High Energy (E2) h γ Consider the behavior of spinning top & its spinning motion The

top performs warts like motion in which it spins around its vertical axis under the influence of earts gravitational field.This type motion is called as Precessional Motion the top is said to be precessing

around vertical axis and at what rate/frequency/revolutions per second is called as Precessional Frequency


14 The Precession arises from the interaction of spin with earth’s gravity acting vertically downwards is called as Gyroscopic motion. Only Spinning top will precess ,a static top will merely fall over Each type of magnetic nuclei have their different characteristic Precessional Frequency( V ) , So at Specific frequency

of RF Radiation only specific proton absorb radiation.


15 According to Larmor Precession Theory … “The Precessional Frequency( V ) is Directly proportional to Strength of external magnetic field( H 0) .” ω α γ H 0 H 0 = ω ω = Angular precessional Vlocity γ =

Gyromagnetic ratio = 2 πμ / h I μ = magnetic moment of the spinning bar magnet I = Spin quantum number of spinning bar magnet. h = Planck’s constant


16 According to Fundamental equation of NMR γ H 0 = 2 π V V = Frequency of EMR which is equal to Precessional frequency at resonance “The precessional frequency may be defined as the number of revolutions per second mad by the magnetic moment vector of nucleus around the external field H

0 .Alternately is equal to the frequency of EMR in megacycles per second necessary to induce a transition from one spinning state to another”


17 All nuclei carry charge ,so they will possess spin angular momentum .But only those nuclei having Spin Quantum number (I) greater than 0 (I > 0 )will possess angular momentum along the axis of

rotation. So the atomic nuclei with I > 0 show the NMR Phenomenon The spine quantum is depend on Atomic Mass number (A) & Atomic Number (Z) of atom. Atomic Mass number (A) Atomic Number (Z) Spin Quantum number (I) Odd Odd / Even ½,3/2,5/2 … Even Even 0 Even Odd 1,2,3… So, C-13,F-19, P-

31,N-14, N-15,O-17 NMR are available NMR is applicable to any nucleus possessing spin .

Instrumentation :

18 Instrumentation When NMR active nuclei (such as 1H or 13C) placed in a magnetic field,it absorb radiation at a frequency characteristic of the isotope . ie.the resonant frequency. It is complex collection

of electronic equipment most of power is converted in to heat and vary little converted in to signal To maintain temperature N 2 (-96 º ) gas is used .


19 1)Sample holder : Generally made up of Glass tube of about 8.5 Cm long & 0.3 Cm diameter. Sample is in Solution form 2 to 10 % diluted solution. Ideal Properties of Solvents : Solvent should not contain H/Proton Devoid of hydrogen atom Non polar Chemically Inert , no reactive with sample Available in

pure form. Dissolve sufficient quantity of solute (up to 10 %) Example :CCl 4 ,CS 2 , CDCl 3 ,F3C-COOH,DMSO,D 2 O Ideal Properties of Sample Holder : Transparent to RF radiation. Chemically inert

Durable


20 2) Permanent magnet / External magnet : The permanent magnet or electro magnet can be used for providing stable & homogenous field . The energy of the absorption and the intensity of the signal are proportional to the strength of the magnetic field so field should be strong & controllable / modulable.

Generally Magnet size is 15”in diameter produce 23,500 gause for 100MHz


21 3)Sweep Generator / Sweep Coil : Sweep coil is used for producing & controlling uniform magnetic field. This can be done by 2 ways : 1 ) Field sweep method – If the applied magnetic field is kept constant and RF radiations are changing ,so that it become to resonance frequency 2 ) Frequency Sweep Method

– if the frequency of RF radiation is maintained constant & applied magnetic field is changing to bring resonance. Generally the field sweep method is more in use because it easy to modulate frequency of

RF radiation than the magnetic field of large stable magnetic field.


22 Sweep coil is the pair of coils fixed in the pole faces of the stable magnet the magnetic field produced by the coil is added to field which is in the direction of main field. Magnetic field of coil con be varied by

varying current flowing through it. 4 )RF Generator /RF Oscillator – The RF Oscillator coil is installed perpendicular to magnetic field and transmit RF waves


23 5 ) RF Receiver /Detector – A few turns of wire wound tightly around the sample tube perpendicular to both magnetic field and RF Oscillator. When the precessional frequency is matched with the RF of RF

Oscillator the nuclei induce electro magnetic field (emf) in the detector coil This signals are amplified and send to recorder . 6 ) Amplifier & Record & Readout System – The absorption signal received from

RF is extremely weak so amplification before readout is necessary.


24 Reference :- 1 ) Organic Spectroscopy By-WILLIAM KEMP ,3 rd Edition page no :100 to 109. 2 ) Instrumental methods of chemical analysis By- Gurdeep R. Chartwal & Sham K. Anand ,Himalaya

Publishing House,2009. Page no-2.185-2.23 3) Pharmceutical analysis ,Vol-2, Instrumental method,7 th edition,topic no25. By Dr.K.R.Mahadic,Dr.H.N.More,Dr.A.V.Kasture,Dr.S.G.Wadodkar, Nirali

publication.Page no :-222-233. 4 )Organic Spectroscopy By Y.R.Sharma.Page no-132-165

NUCLEAR MAGNETIC RESONANCE spectroscopy:

NUCLEAR MAGNETIC RESONANCE spectroscopy BY SNEHA THAKUR

CONTENTS:

CONTENTS Introduction Principle Relaxation process Instrumentation Chemical shift C13 NMR 2D and COSY NMR Applications Recent advances Scope

What is NMR spectroscopy? :

What is NMR spectroscopy? Study of spin changes at the nuclear level when a radiofrequency energy is absorbed in the presence of magnetic field. Nuclei with odd mass number only give NMR Spectra.

Purcell and Bloch were awarded Nobel prize in 1952. Objectives Structure elucidation Drug design MRI

THEORY & PRINCIPLE:

THEORY & PRINCIPLE Spinning charge with magnetic moment ( μ ) proportional to the spin MASS NUMBER ATOMIC NUMBER SPIN QUANTUM NUMBER ODD ODD OR EVEN 1/2, 3/2,…HALF INTEGRAL

ODD ODD INTEGRAL EVEN EVEN 0


APPLIED FREQUENCY=PRECESSIONAL FREQUENCY


E=h =B 0 / 2 E= hB 0 / 2

RELAXATION PROCESS:

RELAXATION PROCESS Process of transition from excited state to ground state Radiation emission Radiation less transition spin lattice or longitudinal relaxation process spin-spin or transverse relaxation

process

INSTRUMENTATION:

INSTRUMENTATION

Parts of nmr instrument :

Parts of nmr instrument Sample holder permanent magnets Magnetic coils Sweep generator Radio frequency generator Radio frequency receiver AND read out

NMR EQUIPMENT:

NMR EQUIPMENT

MAKERS OF NMR EQUIPMENT :

MAKERS OF NMR EQUIPMENT Major nmr instrument makers include Oxford instruments, Bruker Spinlock srl General electric Jeol , kimble chase Philips, siemens ag , varian inc and agilent technologies.

SOLVENT REQUIREMENTS:

SOLVENT REQUIREMENTS Should not contain hydrogens . Chemical inertness Magnetic isotropy Volatility Easily available and inexpensive Examples CCl 4 ,deuterated methanol,deuterated

chloroform,deuterated acetic acid,deuterated tri flouro acetic acid,deuterated dimethyl sulfoxide .

NMR Spectrum :

NMR Spectrum A Spectrum of Absorption of Radiation Vs. Applied Magnetic Strength is called as NMR Spectrum . Downfield The shift of an NMR signal to the left on the chart paper. Upfield The shift of an

NMR signal to the right on the chart paper.

Interpretation of nmr spectra: :

Interpretation of nmr spectra: Number of signals: How many different types of protons in the molecule. Position of signals (chemical shift): electronic environment of each proton Intensities of different signals:

How many protons of each type. Splitting pattern: environment of absorbing proton w.r.t neighboring protons

Broad peaks :

Broad peaks Homogenous field Transverse Relaxation time Magic angle Nmr OTHER SOURCES Paramagnetic ions nuclei with quadruple moments

Why should the proton nuclei in different compounds behave differently in the NMR experiment ? :

Why should the proton nuclei in different compounds behave differently in the NMR experiment ?

SPLITTING OF SIGNALS:

SPLITTING OF SIGNALS DUE TO SPIN –SPIN COUPLING OF ABSORBING AND NEIGHBORING PROTONS NUMBER OF PEAKS =NO.OF VICINAL PROTONS +1 i.e N=n+1

COUPLING CONSTANT:

COUPLING CONSTANT The distance between the peaks in a given multiplet of the magnitude of splitting effect - COUPLING CONSTANT ( J ) Depends only on molecular structure. Size determined by Number

and kind of intervening chemical bonds Spatial relations between the protons TYPE OF PROTONS J VARIES FROM Depends on GEMINAL PROTONS 0-20Hz Bond angle and Overall structure VICINAL

PROTONS 2-18Hz Spatial relations And whole structure

FACTORS INFLUENCING COUPLING CONSTANT :

FACTORS INFLUENCING COUPLING CONSTANT Geminal coupling constant Increasing bond angle - more + ve Electronegative substituent - more + ve Neighboring pi bonds - more – ve Vicinal coupling constant

Increasing Dihedral angle - more + ve Electronegative substituent - less + ve bond angle - less + ve Decoupling :Irradiation of protons or groups of equivalent protons with sufficiently intense radio

frequency energy to eliminate completely the observed coupling to the neighboring protons .

CHEMICAL SHIFT:

CHEMICAL SHIFT TMS-TETRA METHYL SILANE REFERENCE STANDARD UNITS - PARTS PER MILLION Shift in the NMR frequency due to the electronic molecular orbital coupling to the external magnetic field.

Shielding and desheilding:

Shielding and desheilding SHIELDED EFFECT When a proton is present inside circulating magnetic field or closer to an electropositive atom, more applied magnetic field is required to cause excitation

DESHEILDED EFFECT When a proton is present outside such circulating magnetic field or when it is attached to an electronegative atom, less applied magnetic field is sufficient for excitation

Factors affecting chemical shift:

Factors affecting chemical shift Inductive effect Anisotropy Hydrogen bonding CAUSES OF CHEMICAL SHIFT Positive Negative shielding effects

INDUCTIVE EFFECT:

INDUCTIVE EFFECT If the electron density about a proton nucleus is relatively high, the induced field due to electron motions will be stronger than if the electron density is relatively low – sheilding occurs UPFEILD SIGNAL The chemical shift increases with the electro negativity of X. Electron withdrawing groups - reduce electron density and cause desheilding. E.g. dimethyl ether more downfield signal >

ethane

ANISOTROPY- π Electron Functions :

ANISOTROPY- π Electron Functions Pi-electrons are more polarizable than are sigma-bond electrons. Therefore pi-electron movement produces strong secondary fields that perturb nearby nuclei. This kind

of spatial variation is called anisotropy , and it is common to non spherical distributions of electrons. STRUCTURE RANGE CHO 9.5-10 VINYL (C=C) 4-8 AROMATICS 6-9 ACETYLENIC(C C) 1.5-3.5


Carbon - carbon double bond deshields vinylic hydrogens and shifts their signal downfield (to the left) to a larger δ value


carbon-carbon triple bond shields an acetylenic hydrogen and shifts its signal upfield (to the right) to a smaller δ value.


Aromatic ring deshields aromatic hydrogens and shifts their signal downfield (to the left) to a yet larger δ value - extra inductive effect is known as the ring current Hydrogen bonding - deshields and shift to

downfeild

Nuclear overhauser effect:

Nuclear overhauser effect In study of molecular geometry Tells whether two protons are in close proximity

Proton exchange reaction:

Proton exchange reaction Describes the fact that in a given period of time, a single -OH proton may attach to a number of different ethyl alcohol molecules. The rate in pure alcohol ethyl alcohol is slow, but increased in acidic or basic impurities. Very slow - the expected multiplicity of hydroxyl group is

observed. Rapid - a single sharp signal is observed. It causes spin decoupling.

C 13 NMR:

C 13 NMR The 13 C nucleus is magnetically active as that of H-nucleus and has a spin quantum number ½.The natural abundance of 13 C is only 1.1% CHARACTERISTIC FEATURES OF 13 C NMR The chemical

shift of the CMR is wider( δ is 0-240ppm relative to TMS) in comparison to PMR( δ is 0-14ppm relative to TMS). Two types proton de coupled : each magnetically non equivalent carbon gives a single sharp peak

that does undergo further splitting. proton-coupled spectra: the signal for each carbon or a group of magnetically equivalent carbon is split by proton bonded directly to that carbon and the n+1 rule is

followed.

APPLICATIONS OF C13 NMR:

APPLICATIONS OF C13 NMR CMR is a noninvasive and nondestructive method,i.e,especially used in repetitive In-vivo analysis of the sample without harming the tissues . CMR of biological materials allows

for the assessment of the metabolism of carbon , which is so elementary to life on earth. The low natural abundance of 13C nuclei (1.1%) can be made use of tagging a specific carbon position by

selective C-13 enrichment, which the signal intensities and helps in tracing the cellular metabolism . Labelling is more convenient means of followimg the metabolism specific carbons throughout the

metabolism. Labelling of 13C nucleus at multiple carbon sites in the same molecule was possible, as result homonuclear 13C-13C coupling provides novel biochemical information .

2D NMR:

2D NMR It is a set of nmr methods which give data plotted in a space defined by two frequency axes rather than one. Types correlation spectroscopy (COSY), J-spectroscopy, exchange spectroscopy (EXSY),

Nuclear Overhauser effect spectroscopy (NOESY)

Stages :

Stages Four stages Preparation period- where a magnetization coherence is created through a set of RF pulses Evolution period : a determined length of time during which no pulses are delivered and the

nuclear spins are allowed to freely precess (rotate) Mixing period : where the coherence is manipulated by another series of pulses into a state which will give an observable signal Detection period : in which

the free induction decay signal from the sample is observed as a function of time,

COSY NMR:

COSY NMR CORELATION SPECTROSCOPY (COSY ) In standard COSY, the preparation (p1) and mixing (p2) periods each consist of a single 90° pulse separated by the evolution time t1, and the resonance signal

from the sample is read during the detection period over a range of times t2. Used to identify spins which are coupled to each other. Diagonal peaks have the same frequency coordinate on each axis and

appear along the diagonal of the plot correspond to the peaks in a 1D-NMR experiment cross peaks have different values for each frequency coordinate and appear off the diagonal indicate couplings between

pairs of nuclei.


COSY spectrum of codeine

APPLICATIONS:

APPLICATIONS MEDICINE –MAGNETIC RESONANCE IMAGING DETERMINES THE REGIONS OF ACTIVITY WHEN RF IS APPLIED IN PRESENCE OF MAGNETIC FEILD

QUALITATIVE ANALYSIS:

QUALITATIVE ANALYSIS Number of NMR signals =number of equivalent protons Chemical shift=type of hydrogen atoms Spin-spin splitting=arrangement of groups Area of peaks = number of hydrogen nuclei

QUANTITAVE ANALYSIS Molar ratio of components in mixture. Isomeric components Assay of components


Metabolomics- disease states and toxic results . NMR is used to generate metabolic fingerprints from biological fluids to obtain information about disease states or toxic insults. Chemistry- structure

elucidation of compounds . structural data can be elucidated by observing spin-spin coupling , a process

by which the precession frequency of a nucleus can be influenced by the magnetization transfer from nearby nuclei. Non-destructive testing- nucleic acids,DNA ,RNA . analyzing samples non-destructively


Acquisition of dynamic information-collective motion in proteins and DNA. Dynamic information including the low-frequency collective motion in proteins and DNA, Eg .: Ca 2+ -calmodulin system Data

acquisition in the petroleum industry measure rock porosity, estimate permeability from pore size distribution and identify pore fluids (water, oil and gas) Process control and process optimization in oil

refineries and petrochemical Plants. Variation observed in these spectra with changing physical and chemical properties is modeled to yield predictions on unknown samples


Mining, polymer production, cosmetics and food manufacturing as well as coal analysis - TIME DOMAIN NMRs. Absolute hydrogen content values , rheological information, and component composition. Earths

field NMR Molecular structure can be observed more clearly at low fields and low frequencies MISCILLANEOUS Study of optical purity & study of molecular interactions Determination of Hydrogen

bonding, Iodine value, Moisture analysis. Surface chain length determination Structure of complexes like SOF6. Structure of polyethylene Keto enol tautomerism Ligand isomerism , elemental analysis.

RECENT ADVANCES:

RECENT ADVANCES PROTIEN DYNAMICS Internal motions to be probed with exquisite time and spatial resolution. (Anthony Mittermaier and Lewis E. KayScience 14 April 2006: Vol. 312 no. 5771 pp. 224-228)

NMR Expanding its Role in Rational Drug Design NMR+HTS- determine the receptor-bound conformations of small organic ligands. NMR +liquid chromatography -quantitative and qualitative

analysis of complex mixtures including metabolites extracted from body fluids and extracts containing various natural products ( Current Medicinal Chemistry, Volume 8, Number 6, May 2001 , pp. 627-

650(24)

REFERENCES:

REFERENCES Instrumental Methods of Chemical Analysis by GR. Chatwal & Sham K. Anand pg.no-2.18-2.25. Elementary Organic Spetroscopy by Y.R. Sharma pg.no-5.11-5.26. Text book of quantative

Chemical Analysis by Vogels . "Principles of NMR". Process NMR Associates. Retrieved 2009-02-23. Organospectroscopy by william kemp pg.no-114-294 Website 1952 Nobel Prize for Physics at

Nobelprize.org Rabi, J.R. Zacharias, S. Millman , P. Kusch (1938). "A New Method of Measuring Nuclear Magnetic Moment". Physical Review www.wikipedia.org

NUCLEAR MaGNETIC RESONANCE SPECTROSCOPY :

NUCLEAR MaGNETIC RESONANCE SPECTROSCOPY PRESENTED BY SRAVYA.K (MPHARMACY ) PHARMACEUTICAL ANALYSIS AND QUALITY ASSURANCE AVANTHI INSTITUTE OF PHARMACEUTICAL

SCIENCES

Aim :

Aim To understand the details of how N m r works Objective Structural elucidation drug design

INTRODUCTION TO NMR :

INTRODUCTION TO NMR N m r spectroscopy is a powerful analytical technique used to characterize organic molecules by identifying carbon-hydrogen frame works within molecules. NMR = Nuclear:

properties of atomic nuclei Magnetic: magnetic field is required Resonance: interaction magnetic field and radio frequency The source of energy in NMR is radio waves which have long wavelengths, and thus

low energy and frequency

N m r spectroscopy range :

N m r spectroscopy range There is no NMR signal for the nuclei having even atom number or even mass number The radio frequency radiation has the frequency range of 4-600 MHz corresponded to the

wavelength region of 75-0.5 m.

NMR Theory :

NMR Theory That means that the nucleus acts as a small magnet Nuclei with odd atomic number (1H, 13C, 15N, etc.) have half integer nuclear spin.

NMR THEORY :

NMR THEORY In the absence of a magnetic field these nuclei point in random directions. In the presence of a magnetic field B0 they will align either parallel or anti-parallel to the magnetic field. The parallel

orientation is lower in energy.

NMR Theory :

NMR Theory Depending on the environment of a nucleus and the interactions it makes, a specific amount of energy will be required to make it resonant. The amount of energy absorbed for flipping is

detected by the NMR spectrometer.

Spin quantum of various nuclei :

Spin quantum of various nuclei

Principle :

Principle When energy in the form of radio frequency is applied and when l Absorption of energy occurs and N m r signal is recorded

Principle :

Principle The nuclei are said to be in resonance and the energy they emit when flipping from the high to the low energy state can be measured

FLOW CHART OF NMR INSTRUMENTATION :

FLOW CHART OF NMR INSTRUMENTATION According to resolution NMR spectroscopy can be a. low resolution/wide line instrument b. high resolution instrument

INSTRUMENTATION :

INSTRUMENTATION

:

Chapter 13 14 The NMR Spectrometer =>

Sample holder and probe :

Sample holder and probe Sample cell consists of 5mm out side diameter glass tube containing 500-650µLof liquid Contain air turbine to spin the sample. Houses the coils that permit excitation

& detection of NMR signal.

Magnet :

Magnet It is the heart of NMR instrument. Sensitivity & resolution of spectrometer are dependent on strength & quality of magnet. Sensitivity & resolution increases with increase in field strength. Field strength must be homogeneous and reproducible. Two types of magnets one is permanent magnet

other one is convectional electro magnet

Super conducting selnoids :

Super conducting selnoids Wounded from niobium-tin, niobium-titanium. Operates in liquid helium crystal at temp.4K & used in high resolution instrument. Magnet attain field as large as 23T & frequency of 1GHz Most S.S. are filled with nitrogen. Advantage-High stability, -simplicity-high field strength ,-small

size

RADIOFREQUENCY GENERATOR /TRANSMITTER :

RADIOFREQUENCY GENERATOR /TRANSMITTER Used to generate R/F radiation & consists of a coil. To obtain max. interaction of R/F radiation with sample, the coil wound around sample cell. The coil wound at 90 to magnetic field to achieve max. resonance. The frequency of 60, 90, 100, 220, 300,400 MHz are

used.

Radio Frequency Detector :

Radio Frequency Detector Measure the un absorbed radiofrequency. When R/F passes through the magnetised sample ,then-absorption-dispersion of signal occurs. So , it should capable of distinguish

absorption signal from dispersion signal. Two method of detection used

Recorder :

Recorder Give spectrum as a plot of strength of resonance signal on Y-axis & strength of M/F on X-axis. Strength of resonance signal is proportional to number of nucleus at that particular field strength. Area

under peak is direct measure of resonating nuclei.

NMR SPECTRUM :

NMR SPECTRUM A spectrum of absorption of radiation vs applied magnetic strength is called as NMR spectrum The number of signals : Show how many different kinds of protons are present The location of

the signals: Show how shielded or de shielded the proton is

N M R parameters employed for determining structure :

N M R parameters employed for determining structure 1. Chemical Shift Indices: Determining secondary structure. 2. J-coupling: Determine dihedral angles. ( Karplus equation) 3. Nuclear Overhauser Effect (NOE): Determine inter-atomic distances (NOE µ R-6) 4. Residual dipolar coupling: Determine bond

orientations. 5. Relaxation rates (T1, T2 e t c): Determine macromolecular dynamics

DIFFERENT ISOTOPES USED IN NMR :

DIFFERENT ISOTOPES USED IN NMR Carbon-13 is a naturally occurring isotope of carbon that has nuclear spin. It is used in NMR spectroscopy to identify different carbon atoms environments within a molecule.

OTHER TYPES:

Applications :

Applications Chemistry: 1H, 13C Bio-sciences: 1H, 13C, 15N, 19F, 31P, etc. Medicine: 1H, 17O Based on nuclei available for 3-D structure

APPLICATIONS IN CHEMISTRY :

APPLICATIONS IN CHEMISTRY Chemical analysis : A matured technique for chemical identification and conformational analysis of chemicals whether synthetic or natural.

APPLICATIONS IN BIOSCIENCE :

APPLICATIONS IN BIOSCIENCE Chemical shift mapping – structural and functional information on the binding modes and site positions High throughput compound screening and drug design

Applications In Medicine :

Applications In Medicine NMR principle is applied in obtaining clinical images and of studying tissue metabolism in vivo. Image courtesy of James Danckert http://www.arts.uwaterloo.ca/~jdancker/fMRI

COMMERCIALLY AVAILABLE NMR AND THEIR APPLICATIONS :

COMMERCIALLY AVAILABLE NMR AND THEIR APPLICATIONS Biological complex Dynamic Nuclear Polarization (DNP) experiments at 263 GHz/ 400 MHz used biological complexes. The new AVANCE III HD

is the Ultimate NMR Platform for Life Science and Materials Research, Pharma /Biotech, Chemistry, Metabolics , Nutritional Science and Molecular Diagnostics Research HPLC-SPE-NMR in pharmaceutical

development: capabilities of identified drug impurity and metabolites Other types of N m r A .Multi-dimensional NMR Spectroscopy B. Solid-state NMR spectroscopy r

References :

References Skoog Holler. Crouch. Page-551-598 Inst. method of chemical analysis.page619-736 Pharma analysis by Kasture page 222- 230 NMR Spectroscopy, Basic Principles and Applications, by Roger S.

Macomber http://www.cis.rit.edu/htbooks/nmr/

MASS SPECTROMETRY


MASS SPECTROMETY PRESENTED BY D.SHWETHA 08FDIR0011 1


CONTENTS INTRODUCTION PRINCIPLE INSTRUMENTATION RECORDING MASS SPECTROGRAM APPLICATIONS OF MASS SPECTROMETRY 2


INTRODUCTION Mass spectrum is an analytical which can provide information concerning molecular structure of organic and inorganic compounds. It is used to determine the molecular weight as high as 4000. It based on sample principle, it is very complex and expensive instrument. The analytical chemist

is attracted to the mass spectrometry mainly by its speed and reliability. 3


PRINCIPLE Mass spectra is also called as positive ion spectra or line spectra. We use electron bombardment to convert neutral molecules to a positive charged one. Also there is no ground or excited state like other types of spectroscopy. Obtaining mass spectra consists of 2 types Conversion of neutral molecule into a charged molecule, preferably to a positively charged molecule. Separation of positively

charged fragments formed, based on their masses, by using electrical or magnetic field or both. The Sample is bombarded with high energy electron beam (70eV), where an electron is knocked off from

every molecule. Hence the molecules become positively charged. 4


When a positive potential (accelerating potential) is applied, as molecules are positively charged, they get repelled and travel with great speed, in straight path. Potential energy = Kinetic energy of molecule

eV =1/2 mv 2 Where e=charge of ion V=acceleration voltage m=mass v=velocity after acceleration When a magnetic field or electrical field applied, the positive charged fragments which were travelling in straight path, now travels in curved path. When they travel in a curved path under the influence of

magnetic field, the fragments are separated into different masses because the radius of curvature depends upon their respective masses. Under the magnetic field, Hev = mv 2 /r v = reH/m 5


Where r = radius of ion path H = strength of magnetic field e = charge of ion v = velocity after acceleration m = mass substituting the values of the first equation (i.e. eV = 1/2mv2) eV=1/2

*m*(reH/m) 2 m/e = H 2 r 2 /2v m/e is directly proportional to r 2 (H , v maintain constant). Therefore mass is directly proportional to (radius of ion path) 2 since, e = 1. 6


INSTRUMENTATION The instrumentation of Mass Spectrometer consists the following components. The inlet system (or sample handling system) The ion source ( or ionization chamber) The electrostatic

accelerating system The magnetic field The ion separator (analyzer) Ion detectors 7


INLET SYSTEM HEATED INLET SYSTEM: Gases and less volatile liquids, the liquids vaporized externally an then slowly introduced into the ionization source. DIRECT INLET SYSTEM: Solids , nonvolatile liquids,

unstable compounds directly introduced into the ion source. NON VOLATILE LIQUIDS: steroids, carbohydrates polymeric substances etc.. 8


ION SOURCE From the inlet system the sample is introduced into ionization chamber where a beam of electrons put across the molecules of the sample. The ion sources are of different type Electron impact

ion source Chemical ionization MALDI(Matrix-assisted laser desorption ionization) 9


ELECTRON IMPACT IONISATION In this an electrically heated filament A produces thermal electrons which are then accelerated by anode A'. In this way A beam of electrons which intersects of flow of

simple molecules is produced, resulting in the formation of positively charged ions. Then these ions are with -drawn by the electric field which exist between the repeller plate C and first accelerated plate B.

The intermediate plate B' helps to focus the ion beam and second accelerated plate C gives a final acceleration to the ions. The energy of electron beam is controlled by potential on anode A’. 10


11


The intermediate plate B' helps to focus the ion beam and second accelerated plate C gives a final acceleration to the ions. The energy of electron beam is controlled by potential on anode A'. If the

energy of the electron beam is low there occurs only the production of singly charged molecular ions, resulting in a mass spectrum having almost a single peak corresponding to the mass of the origin

molecule. M + e - M + + 2e - If the energy of the electron beam is increased, this yields highly excited ion which may produce fragments if it is complex or may knock out the second electron. M + e - M 2+

+ 2e - M 2+ + e - M 3+ + 2e - 12


CHEMICAL IONISATION In this technique a reaction gas like methane is introduced along the sample to be analyzed by mass spectrometer in the ionization chamber. When a beam of electrons passed through

the ionization chamber, the reaction gas (methane) undergoes ionization to produce ions which react further with neutral molecules to form products. The products so formed are reactive species and can

interact with the sample molecules to form positive ions. 13


14


MATRIX ASSISTED LASER DESORPTION In this technique low concentration of the analyte is uniformly dispersed in a solid or liquid matrix deposited on the metal plate. The metal plate put in vaccum

chamber and laser beam focused on the sample. Then matrix and the sample strongly absorb the laser radiation. Then the sample gets ionized. The most common type of mass analyzer used with the is the

time of flight analyzer. Various types of matrix Nicotinic acid matrix - to analyte the proteins glycoproteins Ferulic acid matrix to analyte the proteins and Caffieic acid matrix oligonucleotides

Succinic acid – to analyte the proteins. 15


16


THE ELECTROSTATIC ACCELERATING SYSTEM The positive ions formed in the ionization chamber are withdrawn by the electric field which exist between the first accelerating plate B and the second repeller plate C. A strong electrostatic field between B and C of 400 to 4000V accelerates the ions of masses m1

m2 m3…….. to their final velocities. The ions which escape through the slit D consist of a collimated ribbon of ions having velocities and kinetic energies given by eV = ½ m v = ½ m v = ½ m v ………..

Whenever the mass spectrometer is to record the spectrum, the second accelerator is charged to an initial potential of 4000volts.Then this charge is permitted to leak off to ground at a controlled rate over

a period of 25 minutes . 17


MAGNETIC FIELD As the accelerated particles from the electrical field enter the magnetic field enter the magnetic field, the force of magnetic field requires them to move in a curved path. The radius of this

curvature, r, is dependent upon the mass, m, the accelerating voltage, V, the electron charge, e, and the strength of the magnetic field, H. It is the two properties m/e and r upon which mass spectrometry is

based. The mass to charge ratio and the radius to the curvature are interdependent, where as a change in either the accelerating potential or the magnetic field will change m/e and r.. All particles greater or

less m/e ratio will strike the side of the separation tube and will be neutralized. 18


THE ION SEPARATOR It is the part of mass spectrometer which separates the ions according to their masses. An analyzer must possess the following characteristics. It should have a high resolution. It must have high rate of transmission of ions. There are different types of analyzers are there they are Single

focusing magnetic analyzer Double focusing analyzer Quadrupole mass spectrometer Time of flight systems 19


Single Focusing Magnetic Analyzer It has a horse shoe shaped which is evacuated. It has a sample inlet, electron bombarding source and accelerating plates on one end. At the other end, collector slit is present. At the curvature of the tube, there is a provision to apply electrical or magnetic field. 20


2.Double focusing mass analyzer Double beam instrument where two ion beam from independent sources pass side by through a common mass analyzer and are detected by separate collectors are

available . 21


3.Quadrupole Mass Spectrometry A quadrupole mass filter consists of four parallel metal rods arranged as in the figure below. Two opposite rods have an applied potential of (U+Vcos (wt)) and the other two

rods have a potential of -(U+Vcos(wt)), where U is a dc voltage and Vcos(wt) is an ac voltage. The applied voltages affect the trajectory of ions traveling down the flight path centered between the four rods. For given dc and ac voltages, only ions of a certain mass-to-charge ratio pass through the quadrupole filter

and all ions are thrown out of their original path. 22


Quadrupole mass spectrometers consist of an ion source, ion optics to accelerate and focus the ions through an aperture into the quadrupole filter, the quadrupole filter itself with control voltage supplies,

an exit aperture, an ion detector, detection electronics, and a high-vacuum system. 23


4.Time-of-Flight Mass Spectrometry (TOF-MS) A time-of-flight mass spectrometer uses the differences in transit time through a drift region to separate ions of different masses. It operates in a pulsed mode so

ions must be produced or extracted in pulses. This schematic shows ablation of ions from a solid sample with a pulsed laser. The reflection is a series of rings or grids that act as an ion mirror. This mirror

compensates for the spread in kinetic energies of the ions as they enter the drift region and improves the resolution of the instrument. The output of an ion detector is displayed on an oscilloscope as a

function of time to produce the mass. 24


25


ION DETECTORS 1.The Faraday Cup or Cylinder The Faraday cup or cylinder electrode detector is very simple. The basic principle is that the incident ion strikes the dynode surface which emits electrons and induces a current which is amplified and recorded. The Faraday cup is a relatively insensitive detector

but is very robust. 26


2.The Electron Multiplier Electron multipliers are probably the most common means of detecting ions, especially when positive and negative ions need to be detected on the same instrument. Their are two

types of electron multiplier. 1.A Faraday cup uses one dynode and as a result produces one level of signal amplification. One type of electron multiplier has series of dynodes maintained at increasing

potentials resulting in a series of amplifications. 2.The other type has a curved continuous dynode where amplifications occur through repeated collisions with the dynode surface. 27


28


RECORDING OF MASS SPECTROGRAM Mass spectrogram is generally represented by a bar graph in which intensity on the ordinates is plotted against m/e ratio on the abscissa. But this method is

cumbersome. Therefore, the data are often represented by a graph which plots relative abundance on the ordinate and m/e ratio on the abscissa. The relative abundance of the fragment is given as the

percent intensity of a given peak relative to the most intense peak in the spectrum. Time required to obtain a complete mass spectrum depends on the particular instrument. The time varies from 20 min to

1 second. 29


Resolution of Mass Spectrometer The ability of a mass spectrometer to distinguish between the ions of nearly equal masses is termed as the ''resolution'' of the instrument. For many inorganic and organic

application, the resolution is expressed as follows Resolution = m/ /\ m Where m and /\ m are the mass numbers of two neighboring peaks of equal intensity in the mass spectrum. The resolution of the

instrument is generally decreased due to the following factors Distribution of kinetic energies produced in the electron beam. Variation in the accelerating voltage Variation in the magnetic field Poorly

collimated ion beam Space charge of the ion beam Width of the ion beam as determined by the slits Pressure in the spectrometer 30


TYPES OF IONS PRODUCED IN MASS SPECTROMETRY Molecular ions The observed % abundance (we usually refer to intensity when we are speaking of signals) of the suspected molecular ion must

correspond to expectations based on the assumed molecular structure. Molecules containing p- or non-bonding electrons are less likely to fragment readily and will often yield intense signals for M+ ions in the spectrum. Conversely, highly branched molecules (which can generate relatively unstable tertiary radical cations) or molecules that can lose neutral stable radicals (like halides) will give less intense or

completely absent signals for M+. The relative abundance of the molecular ion will usually correspond to the following sequence: Aromatic and conjugated compounds > alicyclic compounds > sulfides >

unbranched hydrocarbons > mercaptans > ketones > aldehydes > amines > amides > esters > ethers > carboxylic acids > branched hydrocarbons > alcohols. 31


Base peak If an electron beam of energy of 70eV is used in a mass spectrometer, the molecular ion is produced by the loss of a single electron which undergoes splitting to form many fragments, and the parent peak in mass spectrum is called the base peak and the heights of all other peaks are measured

with respect to it .Generally the ion abundances are expressed in terms of the base peak. Multiple charged ions In mass spectrometer the ions are generally carrying a single positive charge. However

sometimes doubly charged or even triply charged ions are found in the mass spectrum. The doubly and triply charged ions are recorded at a half or a third of the m/e value of the singly charged ions. The

formation of these multiply charged ions are more common in heteroaromatic molecules . 32


Negative ions In addition to positive ions negative ions may be formed from electron bombardment of the sample. The formation of negative ions is very rare but these can be produced in three ways : AB+e

----------- A + B - (dissociation resonance capture) AB+e ----------- AB - (resonance capture) AB+e -----------A + + B - +e - (ion pair production) Rearrangement ions In some cases, fragments are observed which are not a part of the original molecule. These are known as rearrangement ions which are formed from the

molecular ions by redistribution of atoms or group of atoms at the moment of decomposition of the molecular ion. 33


Metastable ions The life time of an ion may be so small that it undergoes decomposition during its passage between the source and collector units in the spectrometer .The ions resulting from the

decomposition between the source region and the magnetic analyzer are called metastable ions which appear in the spectrum as broad peaks at non-integral mass numbers. The relationship between the

apparent m/e of the metastable ion and its parent is given by the following formulation. m1 + m2 + + m0 The metastable ion is observed at a mass m* which is related to m and m by the eqation. m*= m2 2 /

m1 Where m is the mass of parent ion, m the mass of daughter (metastable) ion , m the mass of the neutral fragment and m* apparent mass of the metastable ion . 34


Fragmentation The molecular ions are energetically unstable, and some of them will break up into smaller pieces. The simplest case is that a molecular ion breaks into two parts - one of which is another positive ion, and the other is an uncharged free radical. The uncharged free radical won't produce a line on the mass spectrum. Only charged particles will be accelerated, deflected and detected by the mass

spectrometer. These uncharged particles will simply get lost in the machine - eventually, they get removed by the vacuum pump. The ion, X + , will travel through the mass spectrometer just like any

other positive ion - and will produce a line on the stick diagram. 35


All sorts of fragmentations of the original molecular ion are possible - and that means that you will get a whole host of lines in the mass spectrum. For example, the mass spectrum of pentane looks like this: 36


General rules for interpretation of mass spectra The exact molecular weight 2.The Isotope effect 3.Nitrogen rule 4.Ring rule 37


APPLICATION OF MASS SPECTROMETRY 2.Isotopic abundance 1.Molecular mass determination 3.Quantitative analysis of mixtures 4.Distinction between the cis and trans-Isomer 5.Determination of

Ionization Potential 6.Bonding 7.Reaction Kinetics 8.Impurity Detection 9.Identification of the unknown compound 10.Characterization of polymers 38


REFERENCES 1. Instrumental Methods of Chemical Analysis by GURDEEP R CHATWAL, SHAM K. ANAND. 2. Text Book of Pharmaceutical Analysis, Third Edition, Dr. S. RAVI SANKAR. 3. www.google.com 39

Mass Spectrometry: Methods & Theory presented by reza nemati UPM:

Mass Spectrometry: Methods & Theory presented by reza nemati UPM

Proteomics Tools:

Proteomics Tools Molecular Biology Tools Separation & Display Tools Protein Identification Tools Protein Structure Tools

Mass Spectrometry (MS):

Mass Spectrometry (MS) Introduce sample to the instrument Generate ions in the gas phase Separate ions on the basis of differences in m/z with a mass analyzer Detect ions

How does a mass spectrometer work?:

How does a mass spectrometer work? Ionization method MALDI Electrospray (Proteins must be charged and dry) Mass analyzer MALDI-TOF MW Triple Quadrapole AA seq MALDI-QqTOF AA seq and MW QqTOF AA seq and protein modif. Create ions Separate ions Detect ions Mass spectrum Database

analysis


Generalized Protein Identification by MS Artificial spectra built Artificially trypsinated Database of sequences (i.e. SwissProt) Spot removed from gel Fragmented using trypsin Spectrum of fragments

generated MATCH Library


Methods for protein identification

Typical Mass Spectrometer:

Typical Mass Spectrometer


LC/LC-MS/MS-Tandem LC, Tandem MS

Positive or Negative Ion Mode?:

Positive or Negative Ion Mode? If the sample has functional groups that readily accept H+ (such as amide and amino groups found in peptides and proteins) then positive ion detection is used- PROTEINS

If a sample has functional groups that readily lose a proton (such as carboxylic acids and hydroxyls as found in nucleic acids and sugars) then negative ion detection is used- DNA

Amino Acid Residue Masses:

Amino Acid Residue Masses Glycine 57.02147 Alanine 71.03712 Serine 87.03203 Proline 97.05277 Valine 99.06842 Threonine101.04768 Cysteine 103.00919 Isoleucine113.08407 Leucine 113.08407

Asparagine114.04293 Aspartic acid 115.02695 Glutamine 128.05858 Lysine 128.09497 Glutamic acid 129.0426 Methionine 131.04049 Histidine 137.05891 Phenylalanine 147.06842 Arginine 156.10112

Tyrosine 163.06333 Tryptophan 186.07932 Monoisotopic Mass

Amino Acid Residue Masses:

Amino Acid Residue Masses Glycine 57.0520 Alanine 71.0788 Serine 87.0782 Proline 97.1167 Valine 99.1326 Threonine 101.1051 Cysteine 103.1448 Isoleucine 113.1595 Leucine 113.1595 Asparagine

114.1039 Aspartic acid 115.0886 Glutamine 128.1308 Lysine 128.1742 Glutamic acid 129.1155 Methionine 131.1986 Histidine 137.1412 Phenylalanine 147.1766 Arginine 156.1876 Tyrosine 163.1760

Tryptophan 186.2133 Average Mass

PMF on the Web:

PMF on the Web ProFound http://129.85.19.192/profound_bin/WebProFound.exe MOWSE http://srs.hgmp.mrc.ac.uk/cgi-bin/mowse PeptideSearch

http://www.narrador.embl-heidelberg.de/GroupPages/Homepage.html Mascot www.matrixscience.com PeptIdent http://us.expasy.org/tools/peptident.html

MS-MS & Proteomics:

MS-MS & Proteomics Provides precise sequence-specific data More informative than PMF methods (>90%) Can be used for de-novo sequencing (not entirely dependent on databases) Can be used to ID

post-trans. modifications Requires more handling, refinement and sample manipulation Requires more expensive and complicated equipment Requires high level expertise Slower, not generally high

throughput Advantages Disadvantages


Limitations of Proteomics -solubility of indiv. protein differs -2D gels unable to resolve all proteins at a given time -most proteins are not abundant (ie kinases) - proteins not in the database cannot be

identified -multiple runs can be expensive -proteins are fragile and can be degraded easily -proteins exist in multiple isoforms -no protein equivalent of PCR exists for amplification of small samples

MASS SPECTROMETRY:

MASS SPECTROMETRY A.Solairajan, M.Pharm,1 st year, S.B.C.P. 11-Apr-12 Solairajan 1


11-Apr-12 Solairajan 2 What is Mass Number? Zoo Zoo

Mass number:-:

Mass number:- The Mass number (A), also called atomic mass number or nucleon number is the total number of protons and neutrons in an atomic nucleus. 11-Apr-12 Solairajan 3

Example:-:

Example:- Carbon atom have 6 protons and 6 neutrons in the centre of the nucleus, 2 electrons situated in the inner orbital where as other 4 electrons are outside the orbital. We can represent carbon atom

like 11-Apr-12 Solairajan 4 Mass Number(A) No. of protons(Z) Carbon Atom:-

Common elements and Mass number:

Common elements and Mass number 11-Apr-12 Solairajan 5


Introduction Basic principles Instrumentation Ion formation & types Fragmentation process Fragmentation pattern 11-Apr-12 Solairajan 6


Chemical ionisation MS (CIMS) Field ionisation MS (FIMS) Fast atom bombardment MS (FAB MS) Matrix assisted laser desorption/ ionisation MS (MALDI-MS) Gas chromatography MS (GC-MS) Interpretation of

spectra Applications in pharmacy 11-Apr-12 Solairajan 7


WHAT IS MASS SPECTROMETRY ? Mass spectrometry is an instrumental technique in which sample is converted to rapidly moving positive ions by electron bombardment and charged particles are separated

according to their masses. WHAT IS MASS SPECTRUM ? Mass spectrum is a plot of relative abundance against the ratio of mass/charge(m/e). 11-Apr-12 Solairajan 8

Simple mass spectrometry:

Simple mass spectrometry 11-Apr-12 Solairajan 9


Three principle behind mass spectra To measure relative molecular masses. To know the fragmentation of the molecules. Comparison of mass spectra with known compounds . 11-Apr-12 Solairajan 10

BASIC PRINCIPLES:

BASIC PRINCIPLES 11-Apr-12 Solairajan 11


Loss of electron from a molecule leads to radical cation . 11-Apr-12 Solairajan 12 e- Molecular ion 15 eV 70 eV


Electron removed from molecule orbital having lowest ionization potential (IP). In general n < π < σ 1 eV = 23 Kcal/mol 11-Apr-12 Solairajan 13 Compounds Ionization potential CH 4 12.6 eV C 2 H 4 10.52 eV CH

3 NH 2 10.3 eV

Components of mass spectrometer:

Components of mass spectrometer Inlet system Ion source Ionisation methods Mass Analysers Ion Detectors Vacuum System 11-Apr-12 Solairajan 14


11-Apr-12 Solairajan 15

INSTRUMENTATION:

INSTRUMENTATION 11-Apr-12 Solairajan 16

Inlet System:

Inlet System SOLIDS SAMPLES with lower vapour pressure directly inserted into the ionization chamber and volatilization is controlled by heating the probe . LIQUIDS are handled by hypodermic needles

injection through a silicon rubber dam . GASES SAMPLES are leaked into the ionisation chamber directly by the help of mercury manometer. 11-Apr-12 Solairajan 17 Inlet system


The ion source is the part of the mass spectrometer that ionizes the material under analysis (the analyte ). The ions are then transported by magnetic or electric fields to the mass analyzer. Molecular

ions are formed when energy of the electron beam reaches to 10-15 eV . Fragmentation of the ion reaches only at higher bombardment energies at 70 eV . 11-Apr-12 Solairajan 18 Ion sources:- Ionisation


Ionisation Method Divided Into Two Categories . Gas phase ionisation (gases and vapour ) S amples are ionised outside the ion source. T his technique include, 1.Electron impact ionization (EIS) 2.Chemical

ionization.(CI) 3.Field ionisation .(FI) Desorption technique (liquid and solid) S amples are ionised inside the ion source. T his technique include, 1. Field desorption.(FD) 2. Fast atom bombardment.(FAB) 3.

Laser desorption .(LD) 11-Apr-12 Solairajan 19 Ionisation

Ionizing agent in MS:

Ionizing agent in MS 11-Apr-12 Solairajan 20 Ionisation

IONIZATION METHOD IN MS:

IONIZATION METHOD IN MS 11-Apr-12 Solairajan 21 IONISATION METHOD COMPOUNDS MASS RANGE Electron impact ionisation Thermally volatile and stable 500 Da Chemical ionisation Thermally volatile and Stable 500 Da Electro spray ionisation Polar and Basic 70000 Da Fast atom bombardment Peptides

7000 Da Field ionisation Thermally volatile 1000 Da MALDI Large Biomolecules 3,00,000 Da Plasma desorption Neutral compounds 500 Da APCI Thermally liable 1000 Da SIMS Same as FAB 300-13000 Da

Laser desorption Elemental analysis 500 Da

Electron impact ionisation:-:

Electron impact ionisation :- A beam of electrons passes through a gas-phase sample and collides with neutral analyte molecules (M) to produce a positively charged ion or a fragment ion. Generally electrons

with energies of 70 eV are used to form a fragment ions. The positive ions are collected in focusing plates and passed to mass analyzer. 11-Apr-12 Solairajan 22 Ionisation


11-Apr-12 Solairajan 23 Ionisation

Electrospray ionisation:-:

Electrospray ionisation :- The ESI source consists of a very fine needle and a series of skimmers. A sample solution is sprayed into the source chamber to form droplets. When droplets carry charge exit

the capillary end, as the solvent evaporates, the droplets disappear leaving highly charged analyte molecules. 11-Apr-12 Solairajan 24 Ionisation

Chemical ionisation:-:

Chemical ionisation :- Chemical Impact Ionisation between interactions of sample with large amount of reagent gas. Commonly used reagent gases include methane,ammonia,isobutane . Oxygen and

hydrogens are used in Negative ion chemical ionisation in MS. 11-Apr-12 Solairajan 25 Ionisation


The vaporised sample is introduced into the mass spectrometer with an excess of a reagent gas (methane) at pressure of about 1 torr . The excess carrier gas is ionized by electron impact to the

primary ions CH 4 .+ and CH 3 + . These may react with the excess methane to give secondary ions. 11-Apr-12 Solairajan 26 CI contd … Ionisation


In this method the molecule pass through sharp metal anode carrying an electric field of 10 10 v m -1 Electrons are analysed in primary focusing cathode slit. ADV :- abundance of molecular ions. DISADV :-

lower resolution. 11-Apr-12 Solairajan 27 Field ionisation :- Ionisation


Useful for nonvolatile and thermolabile compounds. Sample is applied to field ion emitter and the solvent allowed to evaporate. Evaporated sample that leads to chemical ionisation or EIS. Example:-

Nucleotides & Quarternary ammonium compounds . 11-Apr-12 Solairajan 28 Field desorption:- Ionisation


Argon gas ionised by hot filament and focused beam that bombards the sample . Beam impinges the sample, a series of molecular reactions occur and analyse in MS analyser . Ex:- Insulin,Amino

glycosides,Phospholipids . 11-Apr-12 Solairajan 29 Fast atom bombardment:- Ionisation


Sample is coated with a high energetic fragment Californium 252. This fission fragment desorbs positive,negative , and neutral molecules. 252 cf generates 10 12 power at 10,000k, this may ionise the

target molecule. This method involves the interaction of laser beam with sample to produce both vaporisation and ionisation . The vaporised sample passed to mass spectrometers for analysis. Appl :-used for elemental analysis . 11-Apr-12 Solairajan 30 Plasma desorption:- Laser desorption:- Ionisation


MALDI is a LIMS method of vaporizing and ionizing and sample molecules are dispersed in a solid matrix such as nicotinic acid. A UV laser pulse ablates the matrix which carries some of the large molecules into

the gas phase in an ionized form so they can be extracted into a mass spectrometer. 11-Apr-12 Solairajan 31 MALDI MALDI:- Ionisation

:

Atmospheric pressure chemical ionisation (APCI) is an analogous ionisation method to chemical ionisation (CI). Corona discharge is used to ionize the analyte in the atmospheric pressure region. 11-

Apr-12 Solairajan 32 APCI:- Ionisation

SIMS:- :

SIMS:- Secondary ion mass spectrometry (SIMS) is based on the observation that charged particles (Secondary Ions) are ejected from a sample surface when bombarded by a primary beam of heavy

particles. Primary beam species useful in SIMS include Cs+, O2+, O , Ar +, and Ga + 11-Apr-12 Solairajan 33 Ionisation


An ion, after leaving ion source, the ions are separated according to their m/e ratio. In this area, the ions are accelerated by both electrostatic and magnetically Types:- Magnetic sector mass analysers Double

focussing analysers Quadrupole mass analysers Time of Flight analysers (TOF) Ion trap analyser Ion cyclotron analyser 11-Apr-12 Solairajan 34 MASS ANALYSERS Mass Analyzer


m/z= H 2 r 2 /2V H Magnetic field R Radius of the curvature V Applied voltage 11-Apr-12 Solairajan 35 Magnetic sector mass analyser :- Mass Analyzer

DOUBLE FOCUSSING ANALYSERS:

DOUBLE FOCUSSING ANALYSERS It contains two analysers namely Electrostatic analyser Magnetic sector analyser . 11-Apr-12 Solairajan 36 Mass Analyzer

QUADRUPOLE MASS ANALYSER:

QUADRUPOLE MASS ANALYSER The quadrupole consists of two pairs of parallel rods with applied DC and RF voltages. Ions are scanned by varying the DC/ Rf quadrupole voltages. 11-Apr-12 Solairajan 37

Mass Analyzer


11-Apr-12 Solairajan 38 Mass Analyzer


TOF analyzer – ions are accelerated through a flight tube and the time of fight to the detector is measured. Typical flight times are 1 to 50 μ s. 11-Apr-12 Solairajan 39 TIME OF FLIGHT ANALYSER Mass

Analyzer


The quadrupole ion trap typically consists of a ring electrode and two hyperbolic end cap electrodes. As the radio frequency voltage is increased, the orbits of heavier ions become stabilised,and passed into

the detector. 11-Apr-12 Solairajan 40 ION TRAP ANALYSER Mass Analyzer


11-Apr-12 Solairajan 41 ION CYCLOTRON ANALYSER:- Mass Analyzer Fourier transform-ICR mass spectrometry, is a type of mass analyzer for determining the mass-to-charge ratio (m/z) of ions based on

the cyclotron frequency of the ions in a fixed magnetic field. The ions are trapped in a Penning trap(a magnetic field with electric trapping plates) where they are excited to a larger cyclotron radius by an

oscillating electric field perpendicular to the magnetic field.


The excitation also results in the ions moving in phase (in a packet). The signal is detected as an image current on a pair of plates which the packet of ions passes close to as they cyclotron. The resulting signal is called a free induction decay (FID), transient or interferogram that converts signal. The useful signal is

extracted from this data by performing a Fourier transform to give a mass spectrum. 11-Apr-12 Solairajan 42




Faraday cup Electron Multiplier photomultiplier Micro Channel Plate 11-Apr-12 Solairajan 44 DETECTORS:- Detector


The basic principle is that the incident ion strikes the dynode surface which emits electrons and induces a current which is amplified and recorded. The dynode electrode is made of a secondary emitting

material like CsSb, GaP or BeO. It is ideally suited to isotope analysis. 11-Apr-12 Solairajan 45 Faradaycup Faradaycup :- Detector


Electron multipliers are the most common especially when positive and negative ions need to be detected on the same instrument. Dynodes made up of copper-beryllium which transduces the initial ion current ,and electron emitted by first dynode are focused magnetically from dynode to the next.

Final cascade current is amplified more than million times . 11-Apr-12 Solairajan 46 Electron multipliers Electron Multipliers Detector


The dynode consists of a substance( a scintillator ) which emits photons(light). The emitted light is detected by photo multiplier tube and is converted into electric current. These detectors are useful in

studies on metastable ions 11-Apr-12 Solairajan 47 Photomultipliers:- Detector


11-Apr-12 Solairajan 48 Micro channel Plate:- Detector


All mass spectrometers need a vacuum to allow ions to reach the detector without colliding with other gaseous molecules or atoms. If such collisions did occur, the instrument would suffer from reduced

resolution and sensitivity. 11-Apr-12 Solairajan 49 Vacuum system:-

TYPES OF PEAKS IN MS:

TYPES OF PEAKS IN MS Molecular ion peak Fragment ions peak Rearrangement ions peak Metastable ion peaks Multicharged ions Base peak Negative ion peak Molecular ion Peak:- When a sample is

bombarded with electrons of 9 to 15 eV energy, the molecular ion is produced, by loss of single electron. M e - M + + 2 e - 11-Apr-12 Solairajan 50


Fragment ions Peak:- when an energy is given further more upto 70 eV , fragment ions produced, it have lower mass number. Rearrangement ion Peak:- Recombination of fragment ion is known as

Rearrangement Peaks. Metastable ion Peak:- The ions resulting from the decomposition between the source region and magnetic analyser are called as Meta stable ions.These appear as broad peaks called

Metastable ion Peaks. Multicharged ions:- Ions may exist with 2 or 3 charges instead of usual single charge.The peaks due to these charged ions are known as Multicharged ion peaks. 11-Apr-12 Solairajan

51


Base Peak:- The largest peak in the mass spectrum corresponding to the most abundant ion or most intense peak in the spectrum is called as Base Peak. Negative ion Peak:- Negative ions are formed from

electron bombardment of sample. These results due to the capture of electron by a molecule during collision of molecules 11-Apr-12 Solairajan 52 Fragment ion peak

FRAGMENTATION:

FRAGMENTATION Fragmentation is a type of chemical dissociation. Fragmentation takes place by a process of heterolysis or homolysis . Types of Fragmentation:- Collision induced dissociation(CID)

Electron capture dissociation(ECD) Electron transfer dissociation(ETD) Electron detachment dissociation(EDD) Photo dissociation Infrared multiphoton dissociation(IRMPD) Blackbody infrared radiative dissociation(BIRD) Surface induced dissociation(SID) Charge remote fragmentation Higher

energy C-trap dissociation(HCD) 11-Apr-12 Solairajan 53


Collision Induced Dissociation Molecular ions are accelerated by electrical potential to high kinetic energy and then allowed to collide with neutral molecules like He,N or Ar . Collision between these

molecules leads to bond breakage and formation of fragment ions. These fragment ions are analysed by mass spectrometer. Example:- Triple quadrupole spectrometer produces CID fragments. 11-Apr-12

Solairajan 54


11-Apr-12 Solairajan 55 SORI-CID: -(Sustained Off-Resonance Irradiation Collision-Induced Dissociation ) It is one of CID technique used in Fourier transform ion cyclotron resonance mass spectrometry. In this method accelerating ions in cyclotron motion and increasing the pressure resulting collisions produce

CID fragments.

Electron Capture Dissociation:-:

Electron Capture Dissociation:- It is a method of fragmenting gas phase ions for tandem mass spectrometric analysis (structural elucidation). ECD involves the direct introduction of low energy

electrons to trapped gas phase ions. Electron-capture dissociation typically involves a multiply protonated molecule M interacting with a free electron to form an odd-electron ion. 11-Apr-12

Solairajan 56

Electron Transfer Dissociation:-:

Electron Transfer Dissociation :- ETD induces fragmentation of cations by transferring electrons to them . Example:-peptides or proteins . Electron Detachment Dissociation:- EDD is a method for fragmenting

anionic species in mass spectrometry. 11-Apr-12 Solairajan 57

Photo Dissociation:-:

Photo Dissociation:- Photodissociation is a chemical reaction in which a chemical compound is broken down by photons . IRMPD:- Absorption of multiple infra red photons by a molecule and leads to

dissociation. BIRD:- Long interaction of molecule with radiation field like carbon dioxide laser . Surface-induced dissociation:-(SID) It is a technique used in mass spectrometry to fragment molecular ions in the

gas phase by collision of an ion with a surface under high vacuum . 11-Apr-12 Solairajan 58


Charge Remote Fragmentation:- It is a type of covalent bond breaking that occurs in a gas phase ion in which the cleaved bond is not adjacent to the location of the charge. This fragmentation can be

observed using tandem mass spectrometry . Higher-energy C-trap dissociation:- (HCD) It is a fragmentation technique, used for peptide modification analysis. Immonium ions generated via HCD pinpoint modifications such as phospho tyrosine. An added octopole collision cell facilitates de novo

sequencing. 11-Apr-12 Solairajan 59

Fragmentation of the Molecular ion:

Fragmentation of the Molecular ion Fragmentation of a molecular ion, M, produces a radical and a cation . -Only the cation is detected by MS. 11-Apr-12 Solairajan 60

Description of Fragmentation process:- :

Description of Fragmentation process:- Fragmentation of the odd electron molecular ion (M .+ ) may occur by Homolytic or Heterolytic cleavage of a single bond. 11-Apr-12 Solairajan 61

Mass interpretation:

Mass interpretation Fragmentation rules Mclafferty rearrangement Alpha cleavage Beta cleavage Nitrogen rule Retro diels alder reaction IHD 11-Apr-12 Solairajan 62

Fragmentation rules:- (9 rules):

Fragmentation rules:- (9 rules) Rule:-1 The height of the M .+ peak decreases with increasing degree of branching. 11-Apr-12 Solairajan 63

Rule:-2 :

Rule:-2 The height of the M .+ Peak decreases with increasing molecular weight. Example:- Fatty molecules, steroids. 11-Apr-12 Solairajan 64

Rule:-3:

Rule:-3 The cleavage is favored at alkyl substituted carbons leads to formation of a carbocation . 11-Apr-12 Solairajan 65

Rule:-4:

Rule:-4 Double bonds, cyclic structures and aromatic rings stabilize M .+ and increase the probability of its appearance. 11-Apr-12 Solairajan 66 Molecular ion peak & Base peak

Rule:-5:

Rule:-5 Double bonds favor allylic cleavage to give the resonance stabilized cation. 11-Apr-12 Solairajan 67

Rule:-6:

Rule:-6 Saturated rings tend to lose alkyl side chains at the α bond (see rule 3) Unsaturated rings can undergo a Retro-Diels-Alder reaction 11-Apr-12 Solairajan 68

Rule:-7:

Rule:-7 Alkyl substituted aromatic compounds are cleaved preferably at the β bond to the ring, giving the resonance stabilized benzyl ion (or) tropyllium ion. 11-Apr-12 Solairajan 69

Rule:-8:

Rule:-8 C-C bonds next to hetero atom are frequently cleaved, leaving the charge on the hetero atom (resonance stabilization). 11-Apr-12 Solairajan 70

Rule:-9:

Rule:-9 Cleavage is often associated with elimination of small stable, neutral molecules, such as CO,olefins,water,ammonia,H 2 S,HCN,ketene or alcohols (often with rearrangements) Ex:- Mclafferty

rearrangement 11-Apr-12 Solairajan 71

Mclafferty Rearrangement:-:

Mclafferty Rearrangement:- Mclafferty arrangement can occur in ketones,aldehydes,carboxylic acids, and esters . In this rearrangement a radical center in molecular ion derived from a lone pair or pi bond,

removes hydrogen from the Gamma position( γ ) , a pi bond is formed between the β and γ position, and the bond between the α and β positions is broken. 11-Apr-12 Solairajan 72

α cleavage:

α cleavage 11-Apr-12 Solairajan 73 Alpha cleavage in mass spectrometry is a characteristic fragmentation of the molecular ion derived from carbonyl compounds, in which the bond linking the carbonyl carbon to the atom occupying an alpha position breaks. It is an expected pathway

for carbonyl compounds,ethers,halides,alcohols,and amines .



β cleavage:

β cleavage Beta cleavage in mass spectrometry is a characteristic fragmentation of the molecular ion derived from some organic compounds, most notably alcohols, ethers, and amines, in which the

bond connecting alpha- and beta-carbons break . 11-Apr-12 Solairajan 75



Retro-Diels-Alder reactions:-:

Retro-Diels-Alder reactions:- Retro Diels-Alder fragmentation occurs in 3-cyano-cyclohexene, lets first look at the fragmentation of cyclohexene . First ionization occurs and electrons from the double bond

transfer to an adjacent carbon and an electron from the bond between the 3 and 4 carbons transfers to form a second double bond that is conjugate with the first one. These rearrangements cleave the

molecule between the 3 and 4 carbon and 5 and 6 (where another electron is transferred to form a double bond between the 4 and 5 carbons). This leaves an olefin and a diene . 11-Apr-12 Solairajan 77


11-Apr-12 Solairajan 78 6 1 2 3 4 5 4 5 Diene Olefin

Nitrogen rule:-:

Nitrogen rule:- The nitrogen rule states, that a molecule that has no or even number of nitrogen atoms has an even nominal mass , whereas a molecule that has an odd number of nitrogen atoms has an odd

nominal mass . 11-Apr-12 Solairajan 79 Example:-1 Example:-2

Contd….:

Contd …. The molecular ion appears at m/z 121, indicating an odd number of nitrogen atoms in the structure . 11-Apr-12 Solairajan 80 Odd number of molecular ion

IHD:-:

IHD:- In a hydrocarbon where all carbon atoms have only single bonds and no rings are involved, the compound would have maximum number of H atoms. If any of the bonds are replaced with double or

triple bonds, there would be deficiency of H atoms. By calculating the index of hydrogen deficiency(IHD), we can calculate molecular formula and how many multiple bonds and rings are involved. IHD is also

called the Degree of Unsaturation . A double bond and ring each counts as one IHD. A triple bond counts as two IHD . 11-Apr-12 Solairajan 81


11-Apr-12 Solairajan 82 Example:-1 Example:-2 CH2=CH2



Mass Spectrum of compounds:-:

Mass Spectrum of compounds:- Alkane :- 11-Apr-12 Solairajan 84 Base Peak Molecular ion peak


Fragmentation of Cyclo Hexane:- C 6 H 12 + = 84 ( Molecular ion Peak ), C 4 H 8 + = 56 ( Base Peak ), (M-28) C 6 H 9 + = 69 (Fragment ion Peak), (M-15) C 3 H 7 + = 43 (Fragment ion Peak), (M-41) C 2 H 5 + = 29

(Daughter ion Peak), CH 3 + = 15 (Daughter ion Peak). 11-Apr-12 Solairajan 85

Alcohol:

Alcohol Possible Fragmentations are:- Mol.wt-46 C2H5OH+ =46 (Molecular ion peak) CH 3 O + = 31 (Base Peak) CHO + = 27 (Fragment ion Peak) CH 3 + = 15 (Daughter ion Peak) 11-Apr-12 Solairajan 86 Base

Peak Molecular ion Peak Fragment ion Peak Daughter ion Peak

Aldehyde:-:

Molecular formula:-C 6 H 12 O Molecular Weight:-100 C 6 H 12 O + = 99 (Molecular ion Peak) C 3 H 8 + = 44 (Base Peak) C 4 H 9 + = 57 (Fragment ion Peak) C 2 H 5 + = 29 (Fragment ion Peak) 11-Apr-12

Solairajan 87 Aldehyde :- Fragment ion Peak Base Peak- Mclafferty rearrangement Molecular ion Peak- α cleavage



Amide:-:

Amide:- Molecular wt :- 87, Molecular formula :- C 4 H 9 NO C 4 H 9 NO + = 87 (Molecular ion Peak), C 2 H 5 NO + = 59 (Base Peak) 11-Apr-12 Solairajan 89 Fragment ion Peak- α , β cleavage Base Peak-

Mclafferty rearrangement Molecular ion Peak- β cleavage



Amine:-:

Amine:- Molecular wt:-59 Mol.formula :-C 3 H 9 N 11-Apr-12 Solairajan 91 Molecular ion Peak- β -H transfer Base Peak- β -H transfer



Ester:

Ester Mol.wt :-102, Mol.formula :-C 5 H 10 O 2 11-Apr-12 Solairajan 93 Base Peak- α cleavage Molecular ion Peak α -cleavage



Ether:

Ether Mol.wt :-130 Mol.formula :-C 8 H 18 O 11-Apr-12 Solairajan 95 Base Peak-ipso cleavage α cleavage Molecular ion Peak- α cleavage



GC-MS:

GC-MS Gas chromatography–mass spectrometry ( GC-MS ) is a method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. Applications of GC-MS include :- Drug detection, Fire investigation, Environmental analysis,

Explosives investigation, and Identification of unknown samples. 11-Apr-12 Solairajan 97

GC-MS:

GC-MS 11-Apr-12 Solairajan 98

Tandem MS:-:

Tandem MS:- What is Tandem MS :- - Uses 2 (or more) mass analyzers in a single instrument. -One purifies the analyte ion from a mixture using a magnetic field. -The other analyzes fragments of the

analyte ion for identification and quantification . 11-Apr-12 Solairajan 99




Tandem mass spectrometry , also known as MS/MS or MS 2 , involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages. 11-Apr-12

Solairajan 101

Components of Tandem Mass Spectrometer:

Components of Tandem Mass Spectrometer 11-Apr-12 Solairajan 102 MS-1 Collision cell MS-2

Applications of Tandem MS:

Applications of Tandem MS Biotechnology & Pharmaceutical To determine chemical structure of drugs and drug metabolites. Detection/quantification of impurities, drugs and their metabolites in biological

fluids and tissues . Analysis of liquid mixtures Fingerprinting Nutraceuticals /herbal drugs/tracing source of natural products or drugs Clinical testing & Toxicology Inborn errors of metabolism, cancer, diabetes,

various poisons, drugs of abuse, etc. 11-Apr-12 Solairajan 103

MALDI-MS:

MALDI-MS Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry allowing the analysis of biomolecules ( biopolymers such

as DNA, proteins, peptides and sugars ) and large organic molecules (such as polymers, dendrimers and other macromolecules ). 11-Apr-12 Solairajan 104


MALDI is based on the bombardment of sample molecules with a laser light to bring about sample ionisation . The sample is pre-mixed with a highly absorbing matrix compound for the most consistent

and reliable results. The matrix transforms the laser energy into excitation energy for the sample, which leads to sputtering of analyte and matrix ions from the surface of the mixture. Most commercially

available MALDI mass spectrometers now have a pulsed nitrogen laser of wavelength 337 nm. 11-Apr-12 Solairajan 105

Common matrix in MALDI:

Common matrix in MALDI 11-Apr-12 Solairajan 106 Matrix Solvent Applications 2,5-dihydroxy benzoic acid Acetonitrile,water,methanol,acetone,CHcl 3 Peptides,Nucleotides , oligo nucleotides 3,5-

dimethoxy-4-hydroxycinnamic acid Acetonitrile , water,acetone , CHcl 3 Peptides,proteins,lipids 4-hydroxy-3-methoxycinnamic acid Acetonitrile, water, propanol Proteins Picolinic acid Ethanol Oligo

nucleotides





Applications of MS:-:

Applications of MS:- Elucidation of the structure of the organic and biological molecules. Determination of molecular mass of peptides, proteins, and Oligonucleotides. Monitoring gases in patients breath during surgery. Identification of drugs abuse and metabolites of drugs of abuse in blood, urine, and

saliva. Analyses of aerosol particles. Determination of pesticides residues in food . 11-Apr-12 Solairajan 109

References:-:

References:- Instrumental methods of chemical analysis by willard Organic spectroscopy by William kemp Spectroscopic identification of organic compounds by Silverstein Instrumental analysis by skoog

Wikipedia ww2.chemistry.gatech.edu/class/4341-6371/ fahrni /set02.pdf 11-Apr-12 Solairajan 110

CONTENTS:

CONTENTS Introduction Principle Working of the mass spectrometer Instrumentation Theory of mass spectrometry Applications [email protected]

INTRODUCTION:

INTRODUCTION J. J. Thomson (1913) separated the isotopes 20 Ne and 22 Ne Atlantic Refining Company (1942), first commercial use This technique resolves ionic species by their m/e ratio Francis William

Aston won the 1922 Nobel Prize in Chemistry for his work in mass spectrometry Replica of an early mass spectrometer


Some of the modern techniques of mass spectrometry were devised by Arthur Jeffrey Dempster and F.W. Aston in 1918 and 1919 respectively. In 1989, half of the Nobel Prize in Physics was awarded to

Hans Dehmelt and Wolfgang Paul for the development of the ion trap technique in the 1950s and 1960s. In 2002, the Nobel Prize in Chemistry was awarded to John Bennett Fenn for the development of

electrospray ionization (ESI) and Koichi Tanaka for the development of soft laser desorption (SLD) and their application to the ionization of biological macromolecules, especially proteins. The earlier

development of matrix-assisted laser desorption/ionization (MALDI) by Franz Hillenkamp and Michael Karas has not been so recognized despite the comparable (arguably greater) practical impact of this

technique, particularly in the field of protein analysis. This is due to the fact that although MALDI was first reported in 1985, it was not applied to the ionization of proteins until 1988,after Tanaka's report.

WHAT IS MASS SPECTROMETRY:

WHAT IS MASS SPECTROMETRY It is an analytical technique for the determination of the elemental composition of a sample or molecule [email protected]


MS is a powerful analytical technique Identify Unknown Compounds quantify known materials Elucidation of structural and chemical properties Requires minute Quantities (<Pg ) Identification of analyte molecules at very low concns in complex matrices High Sensitivity, Selectivity & Specificity- Provides valuable information in various branches of science Chemistry, Physics, Biology, Medicine,

Material Science, Environment, Forensic Science, Geochemistry, Archeology, Astronomy etc. [email protected]

Did you know that mass spectrometry is used to...:

Did you know that mass spectrometry is used to... Detect and identify the use of steroids in athletes Monitor the breath of patients by anesthesiologists during surgery Determine the composition of

molecular species found in space Determine whether honey is adulterated with corn syrup Locate oil deposits by measuring petroleum precursors in rock [email protected]


Monitor fermentation processes for the biotechnology industry Detect dioxins in contaminated fish Determine gene damage from environmental causes Establish the elemental composition of

semiconductor materials [email protected]

principle:

principle The MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measurement of their mass-to-charge ratios. [email protected]

How the mass analyzer works:

How the mass analyzer works a sample is loaded onto the MS instrument, and undergoes vaporization. the components of the sample are ionized by one of a variety of methods (e.g., by impacting them with an electron beam), which results in the formation of charged particles (ions) the positive ions are then

accelerated by an electric field computation of the mass-to-charge ratio ( m/z ) of the particles based on the details of motion of the ions as they transit through electromagnetic fields, and detection of the

ions, which in step 4 were sorted according to m/z . [email protected]

WHAT IS THE PROCESS IN MASS?:

WHAT IS THE PROCESS IN MASS? [email protected]

Basic Components:

Basic Components Sample Introduction System Volatilizes the sample and introduces it to the ionization chamber under high vacuum Ion Source Ionizes the sample (fragmentation may occur) and accelerates

the particles into the mass analyzer Mass Analyzer (or Mass Separator) Separates ionized particles based on their mass-to-charge ratio (m/e - )

Basic Components cont…:

Basic Components cont… Detector - Ion Collector Monitors the number of ions reaching detector per unit time as a current flow Signal Processor Amplifies the current signal and converts it to a DC Voltage Vacuum Pump System A very high vacuum (10 -4 to 10 -7 torr ) is required so that the generated ions

are not deflected by collisions with internal gases

INSTRUMENTATION:

INSTRUMENTATION Sample Introduction Systems Batch Inlet Direct Probe Chromatography Interface (GC-MS) Inductively Coupled Plasma (ICP) [email protected]


Electron ionization chemical ionization electrospray ionization matrix-assisted laserdesorption /ionization glow discharge field desorption (FD) fast atom bombardment (FAB) Thermospray

desorption/ionization on silicon (DIOS) Direct Analysis in Real Time (DART) atmospheric pressure chemical ionization (APCI) secondary ion mass spectrometry (SIMS) spark ionization and thermal

ionization (TIMS). Ion source technologies


Mass analyzer technologies Time-of-flight Quadrupole Quadrupole ion trap Linear quadrupole ion trap Fourier transform ion cyclotron resonance Orbitrap Detectors electron multiplier Faraday cups

Microchannel plate detectors


Batch Inlet Sample is volatilized externally and allowed to “leak” into the ion source Good for gas and liquid samples with boiling points < 500 °C Direct Probe Good for non-volatile liquids, thermally unstable

compounds and solids Sample is held on a glass capillary tube, fine wire or small cup Sample Introduction Systems


Chromatography Interface (GC-MS) The MS is used both quantitatively and qualitatively Major interface problem – carrier gas dilution Jet separator (separates analyte from carrier gas) Inductively Coupled

Plasma (ICP) Operates somewhat like a nebulizer in an AAS Also ionizes the sample in argon stream (at very high temperatures, >6000 °C) Only a small amount of analyte is utilized (< 1%)

Electron ionization :

Electron ionization Electron ionization ( EI , formerly known as electron impact ) is an ionization method in which energetic electrons interact with gas phase atoms or molecules to produce ions. This technique

is widely used in mass spectrometry, particularly for gases and volatile organic molecules [email protected]


The following gas phase reaction describes the electron ionization process : where M is the analyte molecule being ionized, e - is the electron and M +• is the resulting ion. Diagram representing an

electron ionization ion source

Chemical Ionization (CI) Ion Source:

Chemical Ionization (CI) Ion Source A modified form of EI Higher gas pressure in ioniation cavity ( 1 torr ) Reagent gas (1000 to 10000-fold excess) added; usual choice is methane, CH 4 Reagent gas is directly ionized instead of analyte Gentle; little fragmentation; even-electron ions produced more stable than

odd-electron ions produced in EI Excess energy of excited ions removed by many ion-reagent gas collisions

Chemical Ionization Reactions:

Chemical Ionization Reactions Reagent gas ionization: CH 4 CH 4 + +e – (also CH 3 + , CH 2 + ) Secondary reactions: CH 4 + + CH 4 CH 5 + + CH 3 CH 3 + + CH 4 H 2 + C 2 H 5 + (M+29) Tertiary

reactions CH 5 + + MH CH 4 + MH 2 + (M+1) proton exchange CH 3 + + MH CH 4 + M + (M–1) hydride exchange CH 4 + + MH CH 4 + MH + (M) charge exchange

Fast Atom Bombardment:

Fast Atom Bombardment Ion source for biological molecules Ar ions passed through low pressure Ar gas to produce beam of neutral ions Atom-sample collisions produce ions as large as 25,000 Daltons

glow discharge:

glow discharge Sputtering of the cathode material (the sample) by an argon plasma. Ionisation of the elements of the sample in the plasma. Extraction and acceleration of ions. Ions separation with a magnetic sector ( Mattauch Herzog configuration). Ions detection by a Faraday cup or an electron

multiplier

Matrix-Assisted Laser Desorption/Ionization (MALDI):

Matrix-Assisted Laser Desorption/Ionization (MALDI) Analyte mixed with radiation-absorbing material and dried Sample ablated with pulsed laser Often coupled to time-of-flight (TOF) detector Excellent for

larger molecules, e.g. peptides, polymers

MASS ANALYZERS:

MASS ANALYZERS Quadrupole Analyzer Ions forced to wiggle between four rods whose polarity is rapidly switched Small masses pass through at high frequency or low voltage; large masses at low

frequency or high voltage Very compact, rapid (10 ms) R = 700-800

TOF Time of Flight Mass Analyzer:

TOF Time of Flight Mass Analyzer Separates ions based on flight time in drift tube Positive ions are produced in pulses and accelerated in an electric field (at the same frequency) All particles have the

same kinetic energy Lighter ions reach the detector first Typical flight times are 1-30 µsec

Time of Flight Mass Analyzer:

Time of Flight Mass Analyzer Separation Principle All particles have the same kinetic energy In terms of mass separation principles: V particle = Her/m Hold H,e , and r constant V particle = 1/m (constant) V

particle is inversely proportional to mass

Quadrupole Ion Trap:

Quadrupole Ion Trap Ions follow complex trajectories between two pairs of electrodes that switch polarity rapidly Ions can be ejected from trap by m/z value by varying the frequency of end cap

electrodes

Detectors:

Detectors electron multiplier Faraday cups Microchannel plate detectors [email protected]

Electron multiplier:

Electron multiplier Continuous dynode electron multiplier An electron multiplier (continuous dynode electron multiplier) is a vacuum-tube structure that multiplies incident charges. In a process called

secondary emission, a single electron can, when bombarded on secondary emissive material, induce emission of roughly 1 to 3 electrons. If an electric potential is applied between this metal plate and yet

another, the emitted electrons will accelerate to the next metal plate and induce secondary emission of still more electrons. This can be repeated a number of times, resulting in a large shower of electrons all

collected by a metal anode, all having been triggered by just one.

Faraday cup:

Faraday cup A Faraday cup is a metal (conductive) cup designed to catch charged particles in vacuum. The resulting current can be measured and used to determine the number of ions or electrons hitting

the cup. The Faraday cup is named after Michael Faraday who first theorized ions around 1830. Schematic of a Faraday cup

Faraday cup cont..:

Faraday cup cont.. When a beam or packet of Ions hits the metal it gains a small net charge while the ions are neutralized. The metal can then be discharged to measure a small current equivalent to the number of impinging ions. Essentially the faraday cup is part of a circuit where ions are the charge

carriers in vacuum and the faraday cup is the interface to the solid metal where electrons act as the

charge carriers (as in most circuits). Faraday cup with an electron-suppressor plate in front By measuring the electrical current (the number of electrons flowing through the circuit per second) in the metal part

of the circuit the number of charges being carried by the ions in the vacuum part of the circuit can be determined.

Micro-channel plate (MCP):

Micro-channel plate (MCP) It is a planar component used for detection of particles (electrons or ions) and impinging radiation (ultraviolet radiation and X-rays). It is closely related to an electron multiplier, as

both intensify single particles or photons by the multiplication of electrons via secondary emission. However, because a micro channel plate detector has many separate channels, it can additionally

provide spatial resolution.


A micro-channel plate is a slab made from highly resistive material of typically 2 mm thickness with a regular array of tiny tubes or slots (microchannels) leading from one face to the opposite, densely

distributed over the whole surface. The microchannels are typically approximately 10 micrometers in diameter (6 micrometer in high resolution MCPs) and spaced apart by approximately 15 micrometers;

they are parallel to each other and often enter the plate at a small angle to the surface (~8° from normal).

References:

References Skoog, Instrumental analysis, cengage learning , Indian edition. www.youtube.com www.google.com www.wikipedia.com [email protected]

OVERVIEW OF MASS SPECTROMETRY :

OVERVIEW OF MASS SPECTROMETRY M. Anitha Sri (Y11MPH448) I/II M.Pharmacy, Industrial pharmacy CHALAPATHI INSTITUTE OF PHARMACEUTICAL SCIENCES.

Contents :

Contents Introduction Instrumentation Mass Spectrum Resolution Determination of molecular formula Data analysis and interpretation Applications 2

INTRODUCTION :

INTRODUCTION A mass spectrometer is an instrument that measures the masses of individual molecules. Three Basic functions: 1. creating gaseous ion fragments from the samples 2. separating them according to their mass-to-charge ratio 3. records the relative abundance of each ionic species

present Also known as positive ion spectra or line spectra. 3

Slide 4:

Block diagram of Components of Mass Spectrometer

INSTRUMENTATION :

INSTRUMENTATION Inlet system Ion source Electrostatic accelerating system Magnetic field Ion separator Ion collector and Detector Vacuum system 5

Inlet system :

Inlet system Direct vapor inlet Direct insertion probe Gas chromatography(GC-MS) Liquid chromatography(LC-MS) Particle Beam Interface Thermospray Interface Electrospray Interface

Desorption techniques(FAB and LSIMS) 6

Direct Vapour Inlet :

Direct Vapour Inlet Gases or volatile liquids Method is Molecular leak or Molecular pumping The sample can be introduced through a septum port or through a valve port. 7

Direct Insertion Probe :

Direct Insertion Probe 8 Solids and liquid samples. Autoprobe.

GC-MS :

GC-MS Most common technique for introducing samples. Several different interface designs are used to connect these two instruments. The MS coupled to the GC should be capable of high resolution. Highly

specific. 9

Slide 10:

10

LC-MS :

LC-MS Used for Thermo labile compounds. Several interfaces are used to connect LC and MS. 11

Particle Beam Interface :

Particle Beam Interface Solvent is removed from an aerosol of LC effluent The resulting analyte is analysed in the ion source Known as MAGIC (Monodisperse Aerosol Generator Interface for

Chromatography) 12

Thermospray :

Thermospray Involves simply heating the tip of the entry tube to promote vaporisation. Through the centre of the stainless steel tube, passes a small diameter tube which carries the column eluent. The

tube projects slightly beyond the end of the heater cap which is situated in a cartridge heater together with a thermocouple. 13

Electro Spray :

Electro Spray 14 Sample is dissolved in a solvent and pumped through a narrow capillary. Voltage is applied to the capillary tip and the sample is dispersed into an aerosol, aided by a coaxially introduced

nebulising gas.

ESI :

ESI 15 The charged droplets diminish in size by solvent evaporation assisted by a flow of drying gas. Eventually charged sample ions, free from solvent, are released from the droplets, which pass through

the orifice into an intermediate vacuum region and from these through a small aperture into the analyser of the MS.

Ionisation Techniques :

Ionisation Techniques 16

Electron Impact :

Electron Impact 17

Chemical Ionisation :

Chemical Ionisation Chemical interaction between reagent gas ions and analyte molecule. Two-step process. CH4 + e- = CH4+ + 2e- Secondary ions of reagent gas are produced, which react with the analyte molecules. The mechanism may be proton transfer, hydride abstraction or charge transfer. CH4+ + MH = CH4 + MH+ CH5+ + MH = CH4 + MH2+ CH3+ + MH = CH4 + M+ Reagent gases: Argon, Helium, Nitrogen.

18

Field Ionisation :

Field Ionisation 19 Ions are formed under the influence of high electric field produced by applying high voltages. On the surface of fine tube, many hundreds of projecting carbon microtips are present. These

extract the electron from the sample and ionise the sample molecules.

Fast Atom Bombardment :

Fast Atom Bombardment 20 High energy primary beam is directed at a target surface to obtain high yield of secondary ions. Primary beam may be ions, electrons, photons or neutral atoms. SIMS may be

dynamic or static.

MALDI :

MALDI Two step process. Desorption is triggered by a laser beam. The second step is ionization. Nitrogen laser of 337 nm wavelength is used. Sinapinic acid is used as matrix for proteins and α-cyano-4-

hydroxycinnamic acid for peptides. 21

Choosing an Ionisation technique :

Choosing an Ionisation technique 22

Electrostatic Accelerating system :

Electrostatic Accelerating system 23 The positive ions formed in the ionisation chamber are accelerated by pairs of accelerator plates to impart velocities to the ions. Ions are sorted acc. to m/e ratio based on 3

properties: energy, velocity and momentum. The beam from the slits of these plates consists of a collimated ribbon of ions having equal energies. K.E = eV = ½ m1v12 = ½ m2v22……….

Magnetic Field :

Magnetic Field eV = ½ mv2 F = HeV HeV = mv2/r m/e = H2r2/2V 24 e = charge m= mass v = velocity V = voltage F = Magnetic force H = Magnetic field strength r = radius

Ion Separator :

Ion Separator Single Focussing Double Focussing Cycloidal Quadrupole TOF MS/MS Radio Frequency 25

Single Focussing :

Single Focussing 26

Single Focussing :

Single Focussing 27

Double Focussing :

Double Focussing 28

Cycloidal :

Cycloidal 29

Quadrupole :

Quadrupole 30

Time of Flight :

Time of Flight The time-of-flight (TOF) mass analyzer separates ions in time as they travel down a flight/drift tube. This is a very simple mass spectrometer that uses fixed voltages and does not require a magnetic field. The greatest drawback is that TOF instruments have poor mass resolution, usually less

than 500. 31

MS/MS :

MS/MS 32 Hybrid MS. The two analysers are separated by a field free collision chamber, which contains an inert gas.

Radio Frequency :

Radio Frequency 33 An assembly of grids is employed to select ions acc. to their velocities. Alternative grids are connected to a radiofrequency source and the other grids are connected to a steady potential.

It is simple in construction and doesn’t require a magnet.

Ion collector and Detector :

Ion collector and Detector Detection of ions is based on their charge Detectors monitors the ion current, amplifies it and the signal is transmitted to the data system where it is recorded in the form of mass

spectra. Types of Detectors: Faraday Cup Collector. Electron Multiplier Channel Electron Multiplier Array The detection is either by pulse counting or analog measurement. 34

Faraday-Cup :

Faraday-Cup 35 Ions enter the cup and transfer their charge to the cup. Secondary electrons are generated. No. of secondary electrons generated depends on several factors: mass of ions energy of

ions charge on the ions Angle of incidence material of cup nature of the ion.

Electron Multiplier :

Electron Multiplier 36 A metal plate called conversion dynode that converts the impinging ions to electrons is present. Ion beams strikes the conversion dynode. Secondary electrons are produced by the

electron multiplier.

Channel Electron Multiplier Array :

Channel Electron Multiplier Array 37 Composed of a regular close packed array of channels in a flat plate of semiconducting material. Inside of each channel is coated with a secondary electron emissive

material, thus each channel constitutes an independent electron multiplier.

Vacuum system :

Vacuum system All mass spectrometers operate at very low pressure (high vacuum). This reduces the chance of ions colliding with other molecules in the mass analyzer. Any collision can cause the ions to react, neutralize, scatter, or fragment. All these processes will interfere with the mass spectrum. To

minimize collisions, experiments are conducted under high vacuum conditions, typically 10-2 to 10-5 Pa (10-4 to 10-7 torr) depending upon the geometry of the instrument. This high vacuum requires two

pumping stages. The first stage is a mechanical pump that provides rough vacuum down to 0.1 Pa (10-3 torr). The second stage uses diffusion pumps or turbo molecular pumps to provide high vacuum. 38

General Fragmentation Patterns :

General Fragmentation Patterns Simple Direct cleavage Retro-Diels Alder Reaction Hydrogen Transfer Rearrangement Mc lafferty rearrangement 39

Mc-Lafferty rearrangement :

Mc-Lafferty rearrangement 40 Involves intramolecular migration of γ-hydrogen from electron rich center to electron deficit center followed by cleavage at β position resulting in the formation of neutral

alkene. Common in ketones, esters and carboxylic acids

Types of ions :

Types of ions Molecular ion Fragment ions Rearrangement ions Multiply charged ions Negative ions Metastable ions Pseudomolecular or Quasi ions 41

Metastable ions :

Metastable ions Ions formed in the analyser after moving away from the ionisation chamber. Gives broad bands. Formed at non-integral mass numbers. Mass of metastable ion is calculated by: m* =

m22/m1 m* = mass of metastable ion m1 = mass of molecular ion m2 = mass of daughter ion 42

Derivitisation :

Derivitisation For some ionisation techniques, the compound should be derivitised before being analysed. Derivitisation is the use of chemicals to modify the analyte, usually to reduce its polarity. Often OH and NH groups are reacted with silylating reagents, or acetic anhydride, to form compounds with O-

Si, N-Si, O-C or N-C bonds instead. The derivative then lacks the ability to form hydrogen bonds and is more volatile than the analyte was. Mass spectrometry is a gas phase technique; irrespective of the

nature of the sample the analysis is on gaseous ions, hence the need for volatility. 43

Mass Spectrum :

Mass Spectrum The mass spectrum is presented in terms of ion abundance vs. m/e ratio (mass) The most abundant ion formed in ionization gives rise to the tallest peak on the mass spectrum – this is the

base peak 44 base peak, m/e 43

Slide 45:

All other peak intensities are relative to the base peak as a percentage If a molecule loses only one electron in the ionization process, a molecular ion is observed that gives its molecular weight – this is

designated as M+ on the spectrum 45 M+, m/e 114

Slide 46:

In most cases, when a molecule loses a valence electron, bonds are broken, or the ion formed quickly fragment to lower energy ions. The masses of charged ions are recorded as fragment ions by the

spectrometer – neutral fragments are not recorded ! 46 fragment ions

Resolution :

Resolution 47 Adjacent peaks must be clearly separated. The valley between the two adjacent peaks should not be more than 10% of the height of the larger peak. R = Mn/Mn - Mm

Determination of Molecular Formula :

Determination of Molecular Formula Nitrogen Rule Rule Of Thirteen When a molecular mass, M+, is known, a base formula can be generated from the following equation: M/13 = n + r/13 the base formula being: CnHn+r Index of Hydrogen Deficiency: HDI = n-r+2 / 2 Ring rule: For the molecule CwHxNyOz, R =

w + 1 + y-x/2 48

Data Analysis from mass spectrum :

Data Analysis from mass spectrum The molecular ion peak in aromatic compounds is relatively much intense. Conjugated olefins show more intense molecular ion peak as compared to the corresponding

non-conjugated olefins with same no. of unsaturation. The relative abundance of the saturated hydrocarbon is more than the corresponding branched chain compound. In aromatic compounds, the substituent groups like -OH, -OR, -NH2 increase the relative abundance and –NO2, -CN decrease the

relative abundance. 49

Slide 50:

Absence of molecular ion peak in the mass spectrum means that the compound under examination is highly branched or tertiary alcohol. In case of Chloro or Bromo compounds, isotope peaks(M+ + 2) are also formed along with the molecular ion peak. Isotope peak is not observed when Fluorine or Iodine

atom is present in the compound. 50

Computerised matching of spectra with spectral libraries :

Computerised matching of spectra with spectral libraries The computer can compare a mass spectrum it has determined with the spectra in the databases of the libraries. The output is a table called “HIT LIST”.

Hit list includes the name of each compound that the computer has used for matching, its molecular weight, molecular formula, probability that the spectrum of the test compound matches the spectrum in

the data base. The probability is determined by the no. of peaks and their intensities that can be matched. 51

Applications :

Applications Determination of molecular mass and structure. Determination of Isotopic abundance. Distinction between isomers. Determination of Ionisation potential and Bond Dissociation energies.

Detection of presence of impurities. Identification of unknown compound. 52

References :

References U.S.P. Y.R. SHARMA. ELEMENTARY ORGANIC SPECTROSCOPY, PRINCIPLES AND CHEMICAL APPLICATIONS. 4th ed. S.CHAND. 2007. D.A.SKOOG, F.J. HOLLER, S.R.CROUCH. PRINCIPLES OF

INSTRUMENTAL ANALYSIS. 6th ed. THOMPSON BROOKS. 2007. D.L.PAVIA, G.M.LAMPMAN, G.S.KRIZ. INTRODUCTION TO SPECTROSCOPY. 3rd ed. THOMPSON BROOKS. 2001. G.R.CHATWAL, S.K.ANAND. INSTRUMENTAL METHODS OF CHEMICAL ANALYSIS. 5th ed. HIMALAYA PUBLISHING HOUSE. 2002.

H.HWILLARD, L.L.MERRITT, J.A.DEAN, F.A.SETTLE. INSTRUMENTAL METHODS OF ANALYSIS. 7th ed. CBS PUBLISHERS.

CHEMICAL IONIZATION MASS SPECTROMETRY [CIMS] :

CHEMICAL IONIZATION MASS SPECTROMETRY [CIMS] An Assignment Submitted to Singhania University Towards Partial Fulfilment of Semester-I of Master of Pharmacy Presented By:- Vinod Kumar Dhakar

School of Pharmacy & Medical Science Singhania University Pacheri Bari, Jhunjhunu (Raj) 2008

Key Points :

Key Points Introduction Need of chemical ionization Chemical Ionization Advantages How it works ? Types of Chemical Ionization References

Introduction :

Introduction Chemical Ionization (CI) was introduced by Field and Munson (1966). Their work stemmed from earlier observations that some molecules introduced into an EI source at high pressure would

generate ions of the type [M+H]+ rather than the conventional M+ molecular ion. This process, which became known as "self - CI" generated stable molecular species.

Need of chemical ionization :

Need of chemical ionization Spectra generated by electron impact (EI) may suffer from the disadvantages of excessive fragmentation and lack of a molecular ion, problems which can some time be

overcome by lowering the electron impact energy that means use of chemical ionization technique.

Chemical Ionization :

Chemical Ionization Chemical ionization (CI) is an ionization technique used in mass spectrometry. Chemical ionization is a lower energy process than electron ionization. The lower energy yields less

fragmentation, and usually simpler spectra. A typical CI spectra has an easily identifiable molecular ion.

Advantages :

Advantages More abundant peak related to molecular ion. Simpler fragmentation patterns Easy application of gas chromatography-mass spectrometry interfacing. Since methane can be used not only

reactant gas but also as the carrier gas in the gas chromatography

How it works ? :

How it works ? In a CI experiment, ions are produced through the collision of the analyte (sample) with ions of a reagent (or ionizing) gas that are present in the ion source. Some common reagent gases

include: methane, ammonia, and isobutane. Inside the ion source, the reagent gas is admitted in several thousand fold excess compared to the analyte (sample).

Slide 8:

The pressure within the ion chamber is about 1mg Hg (1 Torr). The mixture is subjected to electron bombardment (100eV). Electrons entering the source will preferentially ionize the reagent gas The

resultant collisions with other reagent gas molecules will create secondary ions, which in turn interact in a specific manner with molecule of the sample Positive and negative ions of the analyte are formed by

reactions with secondary ion.

Slide 9:

If we consider methane as a typical reagent gas, electron impact first removes an electron from the molecule to give CH4+ & CH3+ (Primary ion ), which is then involved in ion-molecule reaction to yield

the secondary ions. Then positive and negative ions of the analyte (sample) are formed by reactions with secondary ions.

Slide 10:

Steps of chemical ionization 1.Primary Ion Formation CH4+ e -----? CH4+ + 2e CH4+ -----? CH3+ + H.

Slide 11:

2.Secondary Reagent Ions CH4+ + CH4 --------? CH5+ + CH3 CH3+ + CH4 --------? C2H5+ + H2 C2H5++ CH4 --------? C3H5 + 2H2

Slide 12:

3.Product ion Formation CH5+ + MH---------? MH2+ + CH4 MH + C2H5+ ------? MH2+ + C2H4 MH + C2H5+ ------? M+ + C2H6

Types of Chemical Ionization :

Types of Chemical Ionization Gas phase chemical ionization Negative chemical ionization (NCI) Positive chemical ionization (PCI)

Applications :

Applications Used in the identification of mixture of drugs in body fluid. Used for the detection of Morphine, related alkaloids and their metabolites in the urine. Used for the investigation of complex

mixture of compound, particularly from biological fluids. Used for analysis of drugs from urine in overdose patient.

References :

References Beckett, A.H., Stenlake J.B.; “Practical Pharmaceutical Chamistry”.fourth edition- part two. Kemp William, “Organic Spectroscopy". Third edition; Palgrave, New York. Silverstein,R.M and Basler,

G.C. (1968) “Spectrometric Identification of Organic Compounds”, Wiley, New York.

AAS

Atomic Absorption Spectroscopy:

1 Atomic Absorption Spectroscopy Prof Mark A. Buntine School of Chemistry Dr Vicky Barnett University Senior College


2 “This material has been developed as a part of the Australian School Innovation in Science, Technology and Mathematics Project funded by the Australian Government Department of Education, Science and

Training as a part of the Boosting Innovation in Science, Technology and Mathematics Teaching (BISTMT) Programme .” Atomic Absorption Spectroscopy

Slide 3:

Professor Mark A. Buntine Badger Room 232 [email protected]


4 Atomic Absorption Spectroscopy AAS is commonly used for metal analysis A solution of a metal compound is sprayed into a flame and vaporises The metal atoms absorb light of a specific frequency,

and the amount of light absorbed is a direct measure of the number of atoms of the metal in the solution

Atomic Absorption Spectroscopy: An Aussie Invention:

5 Atomic Absorption Spectroscopy: An Aussie Invention Developed by Alan Walsh (below) of the CSIRO in early 1950s.

Electromagnetic Radiation:

6 Electromagnetic Radiation Sinusoidally oscillating electric (E) and magnetic (M) fields. Electric & magnetic fields are orthogonal to each other. Electronic spectroscopy concerns interaction of the

electric field (E) with matter.

The Electromagnetic Spectrum:

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7 The Electromagnetic Spectrum Names of the regions are historical. There is no abrupt or fundamental change in going from one region to the next. Visible light represents only a very small fraction of the

electromagnetic spectrum. 10 20 10 18 10 16 10 14 10 12 10 8 -rays X-rays UV IR Micro- wave Frequency (Hz) Wavelength (m) 10 -11 10 -8 10 -6 10 -3 Visible 400 500 600 700 800 nm

The Visible Spectrum:

8 The Visible Spectrum l < 400 nm, UV 400 nm < l < 700 nm, VIS l > 700 nm, IR

The Electromagnetic Spectrum:

9 The Electromagnetic Spectrum Remember that we are dealing with light. It is convenient to think of light as particles (photons). Relationship between energy and frequency is:

Energy & Frequency:

10 Energy & Frequency Note that energy and frequency are directly proportional. Consequence: higher frequency radiation is more energetic. E.g. X-ray radiation ( = 10 18 Hz): 4.0 x 10 6 kJ/mol IR radiation (

= 10 13 Hz): 39.9 kJ/mol (h = 6.626 x 10 -34 J.s)

Energy & Wavelength:

11 Energy & Wavelength Given that frequency and wavelength are related: =c/ Energy and wavelength are inversely proportional Consequence: longer wavelength radiation is less energetic eg. -ray radiation ( = 10 -11 m): 1.2 x 10 7 kJ/mol Orange light ( = 600 nm): 199.4 kJ/mol (h = 6.626 x 10 -

34 J.s c = 2.998 x 10 8 m/s)

Absorption of Light:

12 Absorption of Light When a molecule absorbs a photon, the energy of the molecule increases. Microwave radiation stimulates rotations Infrared radiation stimulates vibrations UV/VIS radiation

stimulates electronic transitions X-rays break chemical bonds and ionize molecules Ground state Excited state photon

Absorption of Light:

13 Absorption of Light When light is absorbed by a sample, the radiant power P (energy per unit time per unit area) of the beam of light decreases. The energy absorbed may stimulate rotation, vibration or

electronic transition depending on the wavelength of the incident light.


14 Atomic Absorption Spectroscopy Uses absorption of light to measure the concentration of gas-phase atoms. Since samples are usually liquids or solids, the analyte atoms must be vapourised in a flame (or

graphite furnace).

Absorption and Emission:

15 Absorption and Emission Ground State Excited States Absorption Emission Multiple Transitions

Absorption and Emission:

16 Absorption and Emission Ground State Excited States Absorption Emission

Atomic Absorption:

17 Atomic Absorption When atoms absorb light, the incoming energy excites an electron to a higher energy level. Electronic transitions are usually observed in the visible or ultraviolet regions of the

electromagnetic spectrum.

Atomic Absorption Spectrum:

18 Atomic Absorption Spectrum An “absorption spectrum” is the absorption of light as a function of wavelength. The spectrum of an atom depends on its energy level structure. Absorption spectra are

useful for identifying species.

Atomic Absorption/Emission/ Fluorescence Spectroscopy:

19 Atomic Absorption/Emission/ Fluorescence Spectroscopy


20 Atomic Absorption Spectroscopy The analyte concentration is determined from the amount of absorption.


21 Atomic Absorption Spectroscopy The analyte concentration is determined from the amount of absorption.


22 Emission lamp produces light frequencies unique to the element under investigation When focussed through the flame these frequencies are readily absorbed by the test element The ‘excited’ atoms are

unstable- energy is emitted in all directions – hence the intensity of the focussed beam that hits the detector plate is diminished The degree of absorbance indicates the amount of element present Atomic

Absorption Spectroscopy


23 Atomic Absorption Spectroscopy It is possible to measure the concentration of an absorbing species in a sample by applying the Beer-Lambert Law: e = extinction coefficient


24 Atomic Absorption Spectroscopy But what if e is unknown? Concentration measurements can be made from a working curve after calibrating the instrument with standards of known concentration.

AAS - Calibration Curve:

25 AAS - Calibration Curve The instrument is calibrated before use by testing the absorbance with solutions of known concentration. Consider that you wanted to test the sodium content of bottled water. The following data was collected using solutions of sodium chloride of known concentration

Concentration (ppm) 2 4 6 8 Absorbance 0.18 0.38 0.52 0.76

Calibration Curve for Sodium:

26 Calibration Curve for Sodium Concentration (ppm) A b s o r b a n c e 2 4 6 8 0.2 0.4 0.6 0.8 1.0

Use of Calibration curve to determine sodium concentration {sample absorbance = 0.65}:

27 Use of Calibration curve to determine sodium concentration {sample absorbance = 0.65} Concentration (ppm) A b s o r b a n c e 2 4 6 8 0.2 0.4 0.6 0.8 1.0 Concentration Na + = 7.3ppm


28 Atomic Absorption Spectroscopy Instrumentation • Light Sources • Atomisation • Detection Methods

Light Sources:

29 Light Sources Hollow-Cathode Lamps (most common). Lasers (more specialised). Hollow-cathode lamps can be used to detect one or several atomic species simultaneously. Lasers, while more sensitive,

have the disadvantage that they can detect only one element at a time.

Hollow-Cathode Lamps:

30 Hollow-Cathode Lamps Hollow-cathode lamps are a type of discharge lamp that produce narrow emission from atomic species. They get their name from the cup-shaped cathode, which is made from

the element(s) of interest.


31 Hollow-Cathode Lamps The electric discharge ionises rare gas (Ne or Ar usually) atoms, which in turn, are accelerated into the cathode and sputter metal atoms into the gas phase.


32 Hollow-Cathode Lamps


33 Hollow-Cathode Lamps The gas-phase metal atoms collide with other atoms (or electrons) and are excited to higher energy levels. The excited atoms decay by emitting light. The emitted wavelengths are

characteristic for each atom.


34 Hollow-Cathode Lamps M M * M + e M * M + Ar * M * M M * M * M + h n collision-induced excitation spontaneous emission

Hollow-Cathode Spectrum:

35 Hollow-Cathode Spectrum Harris Fig. 21-3: Steel hollow-cathode

Atomisation:

36 Atomisation Atomic Absorption Spectroscopy (AAS) requires that the analyte atoms be in the gas phase. Vapourisation is usually performed by: Flames Furnaces Plasmas

Flame Atomisation:

37 Flame Atomisation Flame AAS can only analyse solutions. A slot-type burner is used to increase the absorption path length (recall Beer-Lambert Law). Solutions are aspirated with the gas flow into a

nebulising/mixing chamber to form small droplets prior to entering the flame.

Flame Atomisation:

38 Flame Atomisation Harris Fig 21-4(a)

Flame Atomisation:

39 Flame Atomisation Degree of atomisation is temperature dependent. Vary flame temperature by fuel/oxidant mixture.

Furnaces:

40 Furnaces Improved sensitivity over flame sources. (Hence) less sample is required. Generally, the same temp range as flames. More difficult to use, but with operator skill at the atomisation step, more

precise measurements can be obtained.

Furnaces:

41 Furnaces

Furnaces:

42 Furnaces

Inductively Coupled Plasmas:

43 Inductively Coupled Plasmas Enables much higher temperatures to be achieved. Uses Argon gas to generate the plasma. Temps ~ 6,000-10,000 K. Used for emission expts rather than absorption expts due

to the higher sensitivity and elevated temperatures. Atoms are generated in excited states and spontaneously emit light.

Inductively Coupled Plasmas:

44 Inductively Coupled Plasmas Steps Involved: RF induction coil wrapped around a gas jacket. Spark ionises the Ar gas. RF field traps & accelerates the free electrons, which collide with other atoms and

initiate a chain reaction of ionisation.

Detection:

45 Detection Photomultiplier Tube (PMT). pp 472-473 (Ch. 20) Harris

Photomultiplier Tubes:

46 Photomultiplier Tubes Useful in low intensity applications. Few photons strike the photocathode. Electrons emitted and amplified by dynode chain. Many electrons strike the anode.

X RAY DIFFRACTION

X RAY X-RAY DIFFRACTIONby-Ms. Deepika Pandit, Mr. K.S. Rathore, Dr. Anju GoyalB.N. Girls College of Pharmacy, Udaipur-313001 (Raj.) :

X-RAY DIFFRACTIONby-Ms. Deepika Pandit, Mr. K.S. Rathore, Dr. Anju GoyalB.N. Girls College of Pharmacy, Udaipur-313001 (Raj.) INTRODUCTION TYPES BASIC PRINCIPLE X-RAY SOURCE

INTRUMNTATION X-RAY DIFFRACTION DIFFERACTION PATTERN APPLICATIONS

INTRODUCTION :

INTRODUCTION X-Ray spectroscopy is gradually getting importance because it belongs to a category of non destructive method of analysis. A variety of x-ray techniques and methods are in use. But we shall classify all method into 4 main category. These are- X-ray absorption X-ray emission X-ray fluorescence

X-ray diffraction

TYPES :

TYPES X-ray absorption: In these method, a beam of x-rays is allowed to pass through the sample and the attenuation or fraction of x-ray photons absorbed is considered to be a measure of the

concentration of the absorbing substance. X-ray emission: X-ray are obtained by employment of radioactive source whose decay process results in x-ray emission. X-ray fluorescence: In these methods, X-rays are generated within the sample and by measuring the wavelength and intensity of the generated x-rays, one can perform quantitative and qualitative analysis. X-ray diffraction method: These methods

are based on the scattering of x-ray by crystals. By these methods, one can identify the crystal structures of are extremely important as compared with x-ray absorption& x-ray fluorescence methods

BASIC PRINCIPLE :

BASIC PRINCIPLE In 1901 roentgen received a noble prize for the discovery of x- ray these rays exist in the region of 0.01-10nm but 0.08-0.2nm is most useful region for analytical purpose. In an atom the

electrons are arranged in layers or shell’s K-shell L- shell M-shell N-shell The electrons migrate from the vacant outer orbital to inside vacant orbit to fill up the vacant slot. The kind of energy generated leads to

origin of the x- rays, time scale is approximately 10-12 -10-4 sec The x-ray are generated by – Bombardment of metal target with beam of high energy electrons. Exposure of matter to primary x-rays

beam to generate secondary x-ray showing fluorescence. Use radioactive element which on disintegration leads to x-ray formation. From synchronization of radiation source but the last is most

expensive process

X-RAY SOURCE :

X-RAY SOURCE In x-ray instruments, sources are- Tube, Radioisotopes, Secondary fluarescencent sources. The most common source is a highly evacuated tube. The anode is heavy, hollow, water cooled

block of copper with a metal target plated. The metal having high melting point, good thermal conductivity and large atomic number (N). Such metal are silver, iron, copper, chromium, tungsten,

rhodium, cobalt, molybdenum.

INSTRUMENTATION :

INSTRUMENTATION X-ray generating equipment: X-ray tube Collimator: A series of closely spaced, parallel metal plates or by a bundle of tubes, 0.5mm or smaller in diameter. Filter: When the wavelength

of two spectral lines is nearly the same there is an element may be used as a filter to reduce the intensity of the line with the shorter wavelength.

Table of filters: :

Table of filters:

Slide 10:

Analyzing crystals The relationship between the wavelength of the x-ray beam, the angle of diffraction, θ, and the distance between each set of atomic planes of the crystal lattice, d, is given by the Bragg

condition: mλ = 2d sin θ Where, m= order of diffraction. The geometrical relationship are shown in fig4. For the ray diffracted by the second plane of the crystal, the distance CBD represents the additional

distance of travel in comparison with a ray reflected from the surface plane. Angles CAB and BAD are both equal to θ.Therefore, CB= BD =AB sin θ & CBD = 2 AB sin θ Where AB is the interplanar spacing, d.

Slide 11:

. The d-value should be small enough to make the angle 2θgreater than approximately 8° even at the shortest wavelength used A small d-spacing is also favorable for producing a larger dispersion δθ/δλ, of the spectrum, as seen by differentiating the Bragg equation: On the other hand, a small d value imposes an upper limit to the range of wavelengths that can be analyzed because at λ = 2d the angle 2θ becomes

180°.

Slide 12:

Detector: Photographic emulsion Ionizing chamber Geiger counter Scintillation counter Semiconductor detector

Reciprocal lattice concept :

Reciprocal lattice concept Diffraction phenomena are interpreted most conveniently with the aid of the reciprocal lattice concept. A plane can be represented by a line drawn normal to the plane. When a

normal is drawn to each plane in a crystal with a length inversely proportional to the interplanar spacing and the normal's are drawn from a common origin, the terminal points of these normal constitute a lattice array. This is called the reciprocal lattice because the distance of each point from the lattice is

reciprocal to the interplanar spacing of the planes that it represents.

Diffraction pattern :

Diffraction pattern Laue photographic method Bragg x-ray spectrometer method Rotating crystal method Powder method Laue photographic method: Transmission method: In this method the crystal is held stationary in a beam of x-rays , after passing through the crystal is diffracted and is recorded on a

photographic plate. Laue pattern can be used to orient crystals for solid state experiments. Back reflection method: This method provides similar information as the transmission method.

Slide 15:

Bragg’s x-ray spectrometer method: Using the Laue’s photograph, Bragg analyzed the structures of crystals of sodium chloride, KCl and ZnS. Bragg devised a spectrometer to measure the intensity of x-ray beam. The spectra obtained in this way can be employed for crystallographic analyses. This is based on

the Bragg’s equation: nλ =2d sin θ This equation gives the condition which must be satisfied for the reflection of x-rays from a set of atomic planes.

Slide 16:

Rotating crystal method: In this method monochromatic x-radiation is incident on a single crystal that is rotated about one of its axes. The reflected beams lie as spots on the surface of cones that are coaxial with the rotation axis. The diffracted beam directions are determined by intersection of the reciprocal lattice points with the sphere of reflection. By remounting the crystal successively about different axes,

one can determine the complete distribution of reciprocal lattice points. One mounting is sufficient if the crystal is cubic but two or more may be needed if the crystal has lower symmetry.

Slide 17:

Powder method: In these method the crystal is replaced by a large collection of very small crystals, randomly oriented, and a continuous cone of diffracted rays is produced. There are some important differences, with respect to rotating crystal method. The cone obtained with a single crystal are not

continuous because the diffracted beams occur only at certain points along the cone, whereas the cones with the powder method are continuous. The cone produced in the powder method is determined by

the spacing of prominent planes and are not uniformly spaced.

Application: :

Application: Determination of crystal structure by bragg’s law Determination of cis-trans isomerism: Particle size determination: Spot counting method: Broadening of diffraction lines: Low-angle scattering

Polymer characterization Determination of linkage isomers

Essential Parts of the Diffractometer:

Essential Parts of the Diffractometer X-ray Tube : the source of X Rays Incident-beam optics: condition the X-ray beam before it hits the sample The goniometer : the platform that holds and moves the

sample, optics, detector, and/or tube The sample & sample holder Receiving-side optics : condition the X-ray beam after it has encountered the sample Detector: count the number of X Rays scattered by the

sample

Instrumentation:

Production of X-Rays Collimator Monochromator Filter Crystal monochromator Detector Photographic methods Counter methods Instrumentation

The wavelength of X rays is determined by the anode of the X-ray source.:

The wavelength of X rays is determined by the anode of the X-ray source. Electrons from the filament strike the target anode, producing characteristic radiation via the photoelectric effect. The anode

material determines the wavelengths of characteristic radiation. While we would prefer a monochromatic source, the X-ray beam actually consists of several characteristic wavelengths of X rays.

K L M

Bragg’s law is a simplistic model to understand what conditions are required for diffraction. :

Bragg’s law is a simplistic model to understand what conditions are required for diffraction. For parallel planes of atoms, with a space d hkl between the planes, constructive interference only occurs when

Bragg’s law is satisfied. In our diffractometers, the X-ray wavelength l is fixed. Consequently, a family of planes produces a diffraction peak only at a specific angle q . Additionally, the plane normal must be

parallel to the diffraction vector Plane normal: the direction perpendicular to a plane of atoms Diffraction vector: the vector that bisects the angle between the incident and diffracted beam The space

between diffracting planes of atoms determines peak positions. The peak intensity is determined by what atoms are in the diffracting plane. q q d hkl d hkl

XRD-Methods:

XRD-Methods Laue photographic method Braggs X-Ray spectrometer Rotating crystal method Powder method

Laue photographic method :

Laue photographic method In his first experiments, Max von Laue (Nobel Prize in Physics in 1914) used continuous radiation (with all possible wavelengths) to impact on a stationary crystal. With this

procedure the crystal generates a set of diffracted beams that show the internal symmetry of the crystal . In these circumstances, and taking into account Bragg's Law , the experimental constants are the interplanar spacings d and the crystal position referred to the incident beam. The variables are the

wavelength λ and the integer number n : n λ = 2 d hkl sin θ nh,nk,nl Thus, the diffraction pattern will contain (for the same spacing d ) the diffracted beams corresponding to the first order of diffraction

( n=1 ) of a certain wavelength, the second order ( n=2 ) of half the wavelength ( λ/2 ), the third order ( n=3 ) with wavelength λ/3 , etc. Therefore, the Laue diagram is simply a stereographic projection

of the crystal


The Laue method in transmission mode The Laue method in reflection mode Laue diagram of a crystal

Braggs X-Ray spectrometer :

Braggs X-Ray spectrometer


When x-rays are scattered from a crystal lattice, peaks of scattered intensity are observed which correspond to the following conditions: The angle of incidence = angle of scattering. The pathlength

difference is equal to an integer number of wavelengths. The condition for maximum intensity contained in Bragg's law above allow us to calculate details about the crystal structure, or if the crystal

structure is known, to determine the wavelength of the x-rays incident upon the crystal.

X-radiation for diffraction measurements is produced by a sealed tube or rotating anode.:

X-radiation for diffraction measurements is produced by a sealed tube or rotating anode. Sealed X-ray tubes tend to operate at 1.8 to 3 kW. Rotating anode X-ray tubes produce much more flux because they operate at 9 to 18 kW. A rotating anode spins the anode at 6000 rpm, helping to distribute heat over a larger area and therefore allowing the tube to be run at higher power without melting the target. Both sources generate X rays by striking the anode target wth an electron beam from a tungsten filament.

The target must be water cooled. The target and filament must be contained in a vacuum.

Rotating crystal method :

Rotating crystal method

Most of our powder diffractometers use the Bragg-Brentano parafocusing geometry.:

Most of our powder diffractometers use the Bragg-Brentano parafocusing geometry. A point detector and sample are moved so that the detector is always at 2 q and the sample surface is always at q to the

incident X-ray beam. In the parafocusing arrangement, the incident- and diffracted-beam slits move on a circle that is centered on the sample. Divergent X rays from the source hit the sample at different points

on its surface. During the diffraction process the X rays are refocused at the detector slit. This arrangement provides the best combination of intensity, peak shape, and angular resolution for the widest number of samples. F: the X-ray source DS: the incident-beam divergence-limiting slit SS: the

Soller slit assembly S: the sample RS: the diffracted-beam receiving slit C: the monochromator crystal AS: the anti-scatter slit


What is X-ray Powder Diffraction (XRD) X-ray powder diffraction (XRD) is a rapid analytical technique primarily used for phase identification of a crystalline material and can provide information on unit cell

dimensions. The analyzed material is finely ground, homogenized, and average bulk composition is determined.


Fundamental Principles of X-ray Powder Diffraction (XRD) Max von Laue, in 1912, discovered that crystalline substances act as three-dimensional diffraction gratings for X-ray wavelengths similar to the

spacing of planes in a crystal lattice. X-ray diffraction is now a common technique for the study of crystal structures and atomic spacing. X-ray diffraction is based on constructive interference of monochromatic

X-rays and a crystalline sample. These X-rays are generated by a cathode ray tube, filtered to produce monochromatic radiation, collimated to concentrate, and directed toward the sample. The interaction

of the incident rays with the sample produces constructive interference (and a diffracted ray) when conditions satisfy Bragg's Law ( n λ=2 d sin θ).


This law relates the wavelength of electromagnetic radiation to the diffraction angle and the lattice spacing in a crystalline sample. These diffracted X-rays are then detected, processed and counted. By

scanning the sample through a range of 2θangles, all possible diffraction directions of the lattice should be attained due to the random orientation of the powdered material. Conversion of the diffraction peaks to d-spacings allows identification of the mineral because each mineral has a set of unique d-spacings. Typically, this is achieved by comparison of d-spacings with standard reference patterns.


All diffraction methods are based on generation of X-rays in an X-ray tube. These X-rays are directed at the sample, and the diffracted rays are collected. A key component of all diffraction is the angle

between the incident and diffracted rays. Powder and single crystal diffraction vary in instrumentation beyond this.

Applications of XRD :

Applications of XRD XRD is a nondestructive technique To identify crystalline phases and orientation To determine structural properties: Lattice parameters (10-4A), strain, grain size, expitaxy , phase

composition, preferred orientation (Laue) order-disorder transformation, thermal expansion To measure thickness of thin films and multi-layers To determine atomic arrangement Detection limits: ~3% in a two

phase mixture; can be ~0.1% with synchrotron radiation Spatial resolution: normally none


Applications X-ray powder diffraction is most widely used for the identification of unknown crystalline materials (e.g. minerals, inorganic compounds). Determination of unknown solids is critical to studies in geology, environmental science, material science, engineering and biology. Other applications include characterization of crystalline materials identification of fine-grained minerals such as clays and mixed layer clays that are difficult to determine optically determination of unit cell dimensions measurement

of sample purity


With specialized techniques, XRD can be used to: determine crystal structures using Rietveld refinement determine of modal amounts of minerals (quantitative analysis) make textural measurements, such as

the orientation of grains, in a polycrystalline sample characterize thin films samples by: determining lattice mismatch between film and substrate and to inferring stress and strain determining dislocation density and quality of the film by rocking curve measurements measuring superlattices in multilayered

epitaxial structures determining the thickness, roughness and density of the film using glancing incidence X-ray reflectivity measurements


Strengths and Limitations of X-ray Powder Diffraction (XRD)? Strengths Powerful and rapid (< 20 min) technique for identification of an unknown mineral In most cases, it provides an unambiguous mineral

determination Minimal sample preparation is required XRD units are widely available Data interpretation is relatively straight forward


Limitations Homogeneous and single phase material is best for identification of an unknown Must have access to a standard reference file of inorganic compounds (d-spacings, hkl s) Requires tenths of a gram of material which must be ground into a powder For mixed materials, detection limit is ~ 2% of sample For unit cell determinations, indexing of patterns for non-isometric crystal systems is complicated Peak

overlay may occur and worsens for high angle 'reflections'

X-Ray Diffraction :

X-Ray Diffraction PRESENTED BY: K.SANDHYARANI. B.PHARMACY IVYEAR. 1

CONTENTS:

CONTENTS INTRODUCTION. PRODUCTION OF X-RAYS. BRAGG’S EQUATION INSTRUMENTATION 2

ELECTROMAGNETIC SPECTRUM:

ELECTROMAGNETIC SPECTRUM 3

INTRODUCTION:

INTRODUCTION X-Rays : X-rays are short wave length electromagnetic radiations produced by the deceleration of high energy electrons or by electronic transitions of electrons in the inner orbital of atoms. X-ray region 0.1to100 A ˚ Analytical purpose 0.7 to 2 A ˚ More energetic (i.e. can penetrate

deeper into a material ). 4

PRODUCTION OF X-RAYS:

PRODUCTION OF X-RAYS The process of producing X-rays may be visualized in terms of Bohr’s theory of atomic structure. The atom is composed of nucleus and numerous electrons. The electrons are arranged

in shells.(K,L,M) When a high energy beam of electrons strike the target metal it knockout an electron completely from one of the inner shell(K,L,M) of that atom. 5


One of the outer electrons will fall into the vacated orbital, with the simultaneous emission of energy in the form of x-rays. The energy of x-ray is equal to the difference in energy between two levels involved.

If a K-shell looses its electron and it is replaced by the electron from the L-shell as a resulting X-ray is termed as K-X-ray . The energy of K- Xray is termed as E k . 6

EQUATION::

EQUATION: EK=EL-EK EL is the energy of L-shell. EK is the energy of K-shell. K-rays are divided into two types K-alpha, K-beta. If the electron falling into the K-shell comes from the closest shell,(L) it is K-alpha

X-rays. If the electron falling into the K-shell comes from the nearest shell ,(M)it is K-beta X-rays. 7

Theory of x-ray at atomic level :

Theory of x-ray at atomic level 8 Inner orbit Outer orbit 8


d A C B dSin The path difference between ray 1 and ray 2 = 2d Sin For constructive interference: n = 2d Sin Ray 1 Ray 2 Deviation = 2 Constructive interference of the reflected beams

emerging from two different planes will take place if the difference in path lengths of two rays is equal to whole number of wavelengths. BRAGG’s EQUATION 9

INSTRUMENTATION:

INSTRUMENTATION X-ray source:1.Crooke’s tube 2.Coolidge tube Collimator Monochromator-1.Filter type 2.Crystal type Detectors: a)Photographic methods b)Counter methods: 1.Geiger-muller counter

2.Proportional counter 3.Scintillation counter 4.Solid-state semi-conductor detector 5.Semi conductor detectors 10

INSTRUMENTATION OF XRD :

INSTRUMENTATION OF XRD 11


PRODUCTION OF X-RAYS: X-rays are generated when high velocity electrons impinge on a metal target. Approximately 1% of the total energy of the electron beam is converted into x-radiation. The remainder being dissipated as heat. Many types of x-ray tubes are available which are used for producing x-rays. 12

source

Generation of x-rays:

Generation of x-rays 13


Coolidge tube Called as hot cathode tube. Works with a very good quality vacuum (about 10 -4 Pa, The electrons are produced by thermionic effect from a tungsten filament heated by an electric current. There are two designs: 1.End-window tubes : Have thin "transmission target" to allow X-rays to pass

through the target 2.side-window tubes: An Electrostatic Lens to focus the beam onto a very small spot on the anode. A window designed for escape of the generated X-ray photons. Power 0.1 to 18 kW . 14


15

COLLIMATOR:

COLLIMATOR Inserted in the diffracted-beam to get a narrow x-ray beam. It consists two sets of closely packed metal plates seperated by a gap. The left end of the collimator shown is mounted on the X-ray

tube. The yellow-colored region at the left end determines the the size of the beam. The green region at the right end removes parasitic radiation. 16

Materials used:Nacl,LiF,quartz etc,. :

Materials used: Nacl,LiF,quartz etc,. Filter type A window that absorbs undesirable radiation and allows required wavelength to pass. Eg:Zr absorbs x-rays emitted by Mo. Crystal type Positioned in the x-ray beam so that the angle of the reflecting planes satisfied the Bragg’s equation for the required wave

length. Characteristics of a crystal: Mechanically strong and stable The mosaicity and resolution of the crystal, should be small. MONOCHROMATOR 17

DETECTORS:

DETECTORS 18


Counter methods 1. Geiger-muller counter: Filled with an inert gas like argon. Measures ionizing radiation . Detect the emission of nuclear radiation: alpha particles , beta particles or gamma rays Advantages: a)Trouble free b)Inexpensive Disadvantages: a)Cannot be used to measure energy of

ionizing radiation. b)Used for low counting rates c)Efficiency falls off below 1A 19


2. Proportional counter: Filled with heavier gas like xenon or krypton as it is easily ionized. Output pulse is dependent on intensity of X-rays falling on counter. Count the particles of ionizing radiation and measures their energy. Advantages: a)Count high rates with out significant error. Disadvantages:

a)Associated electronic circuit is complex. b)Expensive. 20


3.Scintillation counter: Measures X-rays of shorter wavelengths. The sensor , called a scintillator , consists of a transparent crystal , usually phosphor, plastic (usually containing anthracene ), or organic liquid that fluoresces when struck by ionizing radiation . The PMT is attached to an electronic amplifier

to count and possibly quantify the amplitude of the signals. Advantages: a)Count high rates. 21


4.Solid state semi-conductor detector: The electrons produced by X-ray beam are promoted into conduction bands and the current which flows is directly proportional to the incident X-ray energy.

Disadvantage: Maintainted at very low Temp to minimise the noise and prevent deterioration of the detector. 5.Semi-conductor detectors: Silicon-lithium drifted detector. The principle is similar to gas

ionization detector. Voltage of pulse=Q/C Application : In neutron activation analysis Semi-conductor detector 22

REFERENCES:

REFERENCES 1)Instrumental methods of chemical analysis ,B.K.sharma,17 th edition 1997-1998,GOEL publishing house.page no:329-359 2)Principles of instrumental analysis,5 th edition ,by Dougles

a.skoog,f.James holles,Timothy A.Niemen.page no:277-298 3)Instrumental methods of chemical analysis ,Gurudeep R.chatwal,sham k.anand,Himalaya publications page no:2.303-2.332 4) Instrumental

Methods Of Chemical Analysis – H. Kaur pg.no:727-729,737 5) http://www.scienceiscool.org/solids/intro.html 6) http://en.wikipedia.org/wiki/X-ray_crystallography 23

X-RAY DIFFRACTION:

X-RAY DIFFRACTION HEMANTH.GAMPALA, M.PHARM, PHARMACEUTICAL ANALYSIS . BY

Slide 2:

INTRODUCTION : ORIGIN OF X-RAYS INTERACTION OF X-RAYS WITH MATTER METHODS INSTRUMENTATION X-RAY DIFFRACTION METHODS APPLICATIONS

Slide 3:

INTRODUCTION: X-rays are a form of light. They are in fact more energetic than the visible light. X-radiation is part of the electromagnetic spectrum, just like visible light, radio waves, microwaves, etc.

Here's a schematic of the whole spectrum

Slide 4:

As the wavelengths of light decrease, they increase in energy. X-rays have smaller wavelengths and therefore higher energy than ultraviolet waves .

Slide 5:

OUT LINE ORIGIN OF X-RAYS: study about atom and its shells INTERACTION OF X-RAYS WITH MATTER Absorption 2. Diffraction 3 MAIN METHODS : x-ray absorption method x-ray diffraction method x-ray

fluorescence method

Slide 6:

ORIGIN OF X RAYS: High velocity of electrons bombarded on metal target x rays are produced.

Slide 7:

INTERACTION OF X RAY WITH MATTER: In 3ways: ABSORPTION: x rays Matter Loss energy by scattering Absorption take place. Incoming X-rays Secondary emission

Slide 8:

Follows beers law i = i o e- µ lp i 0 = intensity of incident x ray i = intensity after absorption l = thickness of material µ = mass absorption co-efficient p = density of absorption material.

Slide 9:

Mass absorption co-efficient: (µ ) is a measurement of how strongly a chemical species absorbs or scatters light at a given wavelength µ = CN/A λ C =Proportional constant N= Avogadro's number A=

Atomic weight λ = wavelength of x rays.

Slide 10:

2. Scattering and diffraction: concept: x ray beam substance Contain electrons Absorption Oscillation Emits EM radiations in form of waves Waves under go constructive interference Diffracted by crystal

plane.

Slide 11:

Sets Electron cloud into oscillation Sets nucleus (with protons) into oscillation Small effect neglected

Slide 12:

Oscillating of electrons with the incoming x-rays

Slide 13:

http://www.authorstream.com/Presentation/pravinasir-429738-expect-cloud-computing-revenue-generation-clouds-science-technology-ppt-powerpoint/

n λ = AP+PC AP = PC = l n λ = l + l =2l But triangle OPA, l = d sin θ n λ =2d sin θ = angle of incidence d = space between plans n = integer(1,2,3…etc) Millers: n λ =2d hki sin θ BRAGGS LAW

Slide 14:

METHODS: 1 . X-ray absorption method : imperfection of internal structure. A beam of x rays is passed Sample X ray photons absorbed by substance Measuring concentration of absorbing substance. Use :

elemental analysis, thickness measurement.

Slide 15:

2. X ray diffraction method: x rays fall on sample (crystal) Scattering of x rays. Use: crystal structure. 3.X ray fluorescence method: X ray fall on Sample Emits x ray beam.

Slide 16:

Wavelength: determine which element is present in sample. Intensity: determine how much is present. Use: qualitative and quantitative elemental analysis Above 3 methods are non destructive.

Slide 17:

INSTRUMENTATION: PRODUCTION OF X-RAYS COLLIMATOR MONOCHROMATOR filter crystal monochromator DETECTORS

Slide 18:

INSTRUMENTATION: Amplifier recorder

Slide 19:

Collimator: Close metal plates separated by small gap Use is to produce narrow beam(pencil rays) Monochromator : Absorbs the undesirable radiations and allows required wavelength to pass. Types: 1.filter: eg : zirconium 2.crystal: eg : sodium chloride , lithium fluoride. PRODUCTION OF X RAYS High

velocity of electrons bombarded on metal target x rays are produced

Slide 20:

DETECTORS Photographic. Counter method: types Geiger- muller tube counter Proportional counter Scintillation counter Solid state semiconductor counter Semiconductor

Slide 21:

Geiger muller tube counter : :

Slide 22:

Scintillation detector :

Slide 23:

X-RAY DIFFRACTION METHODS: investigation of internal structures. Laue photographic Bragg X-ray spectrometer Rotating crystal Power metho d

Slide 24:

.Laue photographic: 2 types: Transmission method Back reflection method 1 Transmission method:: In this method the crystal is held stationary in a beam of x-rays , after passing through the crystal is

diffracted and is recorded on a photographic plate.

Slide 25:

2.Back reflection method : This method provides similar information as the transmission method.

Slide 26:

Bragg’s X-ray spectrometer Bragg’s x-ray spectrometer method: Using the Laue’s photograph, Bragg analyzed the structures of crystals of sodium chloride, KCl . Bragg devised a spectrometer to measure

the intensity of x-ray beam. The spectra obtained in this way can be employed for crystallographic analyses. This is based on the Bragg’s equation: nλ =2d sin θ.

Slide 28:

For measurement of λ : Wave length of x rays can determine by following equation: 2dsin θ =n λ λ /d is lattice constant. Knowing d, wavelength λ can determined. Measurement of d: d = a∫ 2 /2 for simple

cubic lattice. d = a/2 Fcc crystal lattice . d = a∫ 3 /2 for bcc crystal lattice . simple cubic lattice bcc crystal lattice Fcc crystal lattice

Slide 29:

Where (a) can calculated by m wt Х no.of atoms in unit cell 1/3 a = Avogadro no. Х density for Nacl crystal. d is calculated Belongs to fcc crystal Four atom in a unit cell Its density is 2.18g/cc Molecular wt

is 58.5 Avogadro no is 6.02 Х 10 23

Slide 30:

58.5 Х 4 1/3 a = = 5.63 Х 10 -8 cm 6.02 Х 10 23 x 2.18 For fcc lattice: a 5.63 Х 10 -8 d = 2 = 2 = 2.815 Х 10 -8 cm d = 2.815A 0

Slide 31:

d100:d110:d111= 1 : 1 : 1 for simple cubic lattice ∫ 2 ∫ 3 d100:d110:d111= 1 : 1 : 1 for fcc crystal ∫ 2 ∫ 3 d100:d110:d111= 1 : 1 : 1 for bcc crystal ∫ 2 ∫ 3 Light is passed at angles 5.9 0 ,8.4 o ,5.20 0

DETERMINATION OF CRYSTAL STRUCTURE BY BRAGGS LAW: Ratio of spacing for the planes can be obtained. Space will be different for different crystals. For NACL crystal:

Slide 32:

d100:d110:d111 = 1 1 1 sin5.9 sin8.4 sin5.2 = 1 1 1 0.1028 0.146 0.0906 = 1: 0.704 : 1.155 = 1 : 1 : 1 ∫ 2 ∫ 3

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Rotating crystal: 2 types 1.Complete rotation method: occurs in a series of complete revolutions 2.Oscillation method: oscillated through an angle 15 to 20 o

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Powder method:

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Different cones for different reflections Cone of diffracted rays

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Compared to other methods the sample used in this method is low quantity that is 1mg. By using this formula, the crystal nature can be studied Θ = 360 x 1 / π r Where , Θ = Angle of incidence 360 =

scattering angle R = film radius. Used for: Cubic crystals Determination of complex structures of alloys and metals.

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APPLICATIONS: STRUCTURE OF CRYSTALS Non-destructive method Molecular structure and size of crystal. POLYMER CHARACTERISATION Powder method determines degree of crystalinity of the polymer. Non crystalline portion scatters the x ray beam gives continuous background. Crystaline portion causes

diffraction lines that are not continuous Amorphous materials: causes scattering. Crystal materials : causes diffraction

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STATE OF ANNEAL IN METALS Well annealed metals-sharp diffraction lines If subjected to hammering or bending-diffused diffraction pattern PARTICLE SIZE DETERMINATION a)Spot counting method: particles

above 5microns b)Broadening of diffraction lines particles of the range 30-1000A o

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APPLICATION TO COMPLEXES a)Determination of cis -trans isomerism b)Determination of linkage isomerism MISCELLANEOUS APPLICATIONS a)Soil classification based on crystallinity b)Analysis of

industrial dusts c)Assessment of weathering and degradation of natural and synthetic minerals d)Study of corrosion products e)Examination of tooth enamel and dentine f)Effects of diseases on bone

structure.

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