Spectroscopy:

Spectroscopy is a branch of science dealing with the study of interaction of electromagnetic radiation with matter. The most important consequences of such interaction is that energy is absorbed or emitted by the matter in discrete amount. The absorption processes known throughout the electromagnetic spectrum ranging from gamma region to radio region.

Spectroscopy is one of the most powerful tools available for the study of atomic and molecular structure and is used in the analysis of a wide range of samples.

The study of spectroscopy can be carried under following heads:

1. Atomic spectroscopy -deals with the interaction of electromagnetic radiations with atoms which are most commonly in their lowest energy state called the ground state.

2. Molecular spectroscopy -This deals with the interaction of electromagnetic radiation with molecules.

ELECTROMAGNETIC RADIATION:It is a form of energy that is transmitted through space at on enormous velocity. Wave properties of Electromagnetic Radiation:

A) Wavelength -It is the distance between two successive maxima on an electromagnetic wave. (Lambda).

B) Frequency -The number of wavelength units passing through a given point in unit time is called frequency of radiation.

C) Wave number -The number of waves per centimetre in vacuum.

ELECTROMAGNETIC SPECTRUM:

The entire range over which electromagnetic radiation exists is known as electromagnetic spectrum. The major characteristics of various spectrum region are outlined as follows:

A) Gamma region: this lies between 0.02 to 1A.

B) X-ray Region: this lies between 1 to 10 A.

C) Visible and Ultraviolet region:

Vacuum ultraviolet: 1-180 nm.

Ultraviolet: 180-400nm.

Visible: 400-750 nm.

D) Infrared region:

E) Microwave region:

F) Radio Frequency region:

Types of Spectroscopical techniques: A) Visible spectrophotometry: It is considere to be one of the oldest physical methods used for quantitative analysis and structural elucidation. This method is mainly used for quantitative analysis and serves as a useful auxiliary tool for structural elucidation. The wavelength of visible radiation starts at 8000 Å and ends at 4000 Å.B) Ultraviolet spectroscopy: The wavelength range of UV radiation starts at 4000 Å and ends at 2000 Å. Region between 2000 Å -4000Å is known as near ultraviolet region. The region below 2000 Åis called the far or vacuum ultraviolet region.

Ultraviolet spectroscopy:When a molecule absorbs ultraviolet radiation of frequency v per sec, the electron in that molecule undergoes transition from a lower to a higher energy level or molecular orbital. Three distinct types of electrons are involved in organic molecules:

a) σ electrons

b) Π electrons

c) n electrons

Types of transitions:-

1) n to π * transition: these types of transition are shown by unsaturated molecules.

2) σ to σ *: these transitions can occur in such compounds in which all the electrons are involved in single bonds and there are no lone pairs of electrons.

3) n to σ* transition :These transitions are generally appeare at longer wavelengths in the near ultraviolet (180-200nm) region.

4) π to π* transitions :This transition can in principle occur in any molecule having a pi electron system.

Some terms from UV visible spectroscopy:Chromophore: It may be define as any group which exhibits absorption of electromagnetic radiations in the visible or ultraviolet region.

Auxochrome: It is a group which itself does not act as a Chromophore but when attache to a Chromophore it shifts the adsorption maximum towards longer wavelength alongwith an increase in the intensity of absorption.

Shifts in position and intensity of absorption:1) Bathochromic shift or red shift: It involves the shift of absorption maximum towards longer wavelength because of the presence of certain groups such as OH and NH2 called auxochromes or by change in solvents. 2) Hypsochromic shift or blue shift: It involves the shift of absorption maximum towards shorter wavelength.

INSTRUMENTATION:

1. Radiation Source: The following requirements of a radiation source:

a) It must be stable

b) It must be of sufficient intensity

c) It must supply continues radiation

Sources:i. tungsten lamp

ii. Hydrogen discharge lamp

iii. Deuterium lamp

iv. Xenon discharge lamp

2. Monochromator:

The monochromator is used to disperse the radiation according to the wavelength. The dispersing element disperses the heterochromatic radiation into its component wavelength. The dispersing elements may be a prism or grating.

3. Detector:

There are three common types of detectors which are widely used in UV spectrophotometer.

i. Barrier layer cell: This cell is also known as photovoltaic cell.ii. Photocell: It consists of a high sensitive cathode in the form of a half cylinder of metal which is contained in an evacuated tube. The anode is also present in the tube which is fixed more or less along the axis of the tube.iii. Photomultiplier tube: A Photomultiplier tube is generally used as a detector.

4. Sample cells: The cells should fulfill three main conditions –a. Must be uniform in construction b. Material should be inertc. Must transmit light of the wavelength used

Qualitative applicationUV absorption spectroscopy can characterize those types of compounds which absorb UV radiation. These compounds are with unbounded electrons or those with the conjugated double beam system such as aromatic compounds. Identification is done by comparing the absorption spectrum with the spectra of known compounds. A record of UV absorption curves is found in certain reference books.

In order to record UV absorption spectrum, the usual practice is to measure the amount of radiation absorbed at various wavelengths by moving the slit across the dispersed beam of life light leaving the monochromator. Then a curve is plotted between wavelength and degree of absorption.

In UV absorption spectroscopy, the absorption curve is curve is influenced by the whole molecule as well as by the particular group that contains the absorption electrons. This creates difficulty in identifying the presence of any one particular group that contains the absorbing electrons. This creates difficulties in identifying the presence of any one particular group. Due to this reason, UV absorption method is less useful than infrared and NMR methods. UV absorption spectroscopy is generally used for characterizing aromatic compounds and conjugated olefins.

Infrared spectroscopy:It is one of the most powerful analytical technique which offers the possibility of chemical identification. One of the advantage of infrared spectroscopy technique is that it provides useful information of structure quickly, without tiresome evaluation method.The technique is based on fact that chemical substance shows marked selective absorption in the infrared region. After absorption of IR radiation, the molecules of a chemical substance vibrate at many rates of vibration, giving rise to close-packed-absorption bands, called an IR absorption spectrum. Various bands present in the spectrum which will corresponds to the characteristic functional groups and bonds. Thus, an IR spectrum of a chemical substance is a fingerprinting for its identification.

Range of infrared region:a. the photographic region-ranges from visible to 1.2µb. the very near infrared region: ranges from 1.2 to 2.5µc. the near infrared region:2.5 to25µd. The far infrared region: 25 to 300-400µ.

Requirements for infrared spectroscopy absorption: 1. Correct wavelength wavelength of radiation: a molecule absorbs radiation only when the natural frequency of vibration of some part of molecule is same as the frequency of the incident radiation.2. Electric dipole: A molecule can only absorb IR radiation when its absorption causes a change in its electric dipole (dipole moment).

Instrumentation:The main parts of IR spectrometer is as follows:

1. IR radiation source2. monochroators3. sample cells and sampling of substance4. detectors

IR radiation source:Requirements:

a. intense enough for detectionb. steadyc. extend over the desired wavelength

1) Incandescent lamp- used in near infrared instruments.2) Nernst glower- glower is generally heated between temperatures 1000-1800° C.it

provides maximum radiation at about 7100 per cm (1.4µ)3) Globar source – it is rod of sintered silicon carbide. When it is heated to temperature

between 1300 and 1700 °C, it strongly emits radiation in IR radiation. It emits maximum radiation at 5200 per cm.

4) Mercury arc- It is high pressured mercury arc lamp used in far infrared region.

Monochromators:a) Prism monochromators: Sodium chloride is probably the most common prism salt.b) Grating monochromator:

Sample cells and sampling of substanceAs infrared spectroscopy has been used for the characterization of solid, liquid or gas samples, it is evident

DetectorsThere are three categories of detector:

Thermocouple Pyro electric Photo conducting

Thermocouples: consist of a pair of junctions of different metals; for example, two pieces of bismuth fused to either end of a piece of antimony. The potential difference (voltage) between the junctions changes according to the difference in temperature between the junctions

Pyroelectric detectors: are made from a single crystalline wafer of a pyroelectric material, such as triglycerine sulphate. The properties of a pyroelectric material are such that when an electric field is applied across it, electric polarisation occurs (this happens in any dielectric material). In a pyroelectric material, when the field is removed, the polarisation persists. The degree of polarisation is temperature dependant. So, by sandwiching the pyroelectric material between two electrodes, a temperature dependent capacitor is made. The heating effect of incident IR radiation causes a change in the capacitance of the material. Pyroelectric detectors have a fast response time. They are used in most Fourier transform IR instruments.

Photoelectric detectors: such as the mercury cadmium telluride detector comprise a film of semiconducting material deposited on a glass surface, sealed in an evacuated envelope. Absorption of IR promotes non conducting valence electrons to a higher, conducting, state. The electrical resistance of the semiconductor decreases. These detectors have better response characteristics than pyroelectric detectors and are used in FT-IR instruments - particularly in GC - FT-IR.

Application:1. Identification of functional group and structure elucidationEntire IR region is divided into group frequency region and fingerprint region. Range of group frequency is 4000-1500 cm-1 while that of finger print region is 1500-400 cm-1.

2. Identification of substancesIR spectroscopy is used to establish whether a given sample of an organic substance is identical with another or not. This is because large number of absorption bands is observed in the IR spectra of organic molecules and the probability that any two compounds will produce identical spectra is almost zero. So if two compounds have identical IR spectra then both of them must be samples of the same substances.

3. Studying the progress of the reactionProgress of chemical reaction can be determined by examining the small portion of the reaction mixture withdrawn from time to time.

4. Detection of impurities:IR spectrum of the test sample to be determined is compared with the standard compound. If any additional peaks are observed in the IR spectrum, then it is due to impurities present in the compound.

5. Quantitative analysisThe quantity of the substance can be determined either in pure form or as a mixure of two or more compounds. In this, characteristic peak corresponding to the drug substance is chosen and log I0/It of peaks for standard and test sample is compared. This is called base line technique to determine the quantity of the substance.

Mass spectroscopy:INTRODUCTIONMass spectrometry was first described by physicists in the late 1880s. WilhelmWien, for example, was the first to demonstrate in 1898 that superimposed electric and magnetic fields can deflect positive ions and his work was preceded by the work of physicist Eugen Goldstein, who in 1886 discovered a new kind of radiation, “Kanalstrahlen” and reached the conclusion that these “new” rays were merely positively charged particles. Mass Spectrometry is a powerful technique for identifying unknowns, studying molecular structure, and probing the fundamental principles of chemistry. Applications of mass spectrometry include identifying and quantitating pesticides in water samples, it identifying steroids in athletes, determining metals at PPQ (Parts Per Quadrillion) levels in water samples. Mass spectrometry is essentially a technique for "weighing" molecules. Obviously, this is not done with a conventional balance or scale. Instead, mass spectrometry is based upon the motion of a charged particle, called an ion, in an electric or magnetic field. The mass to charge ratio (m/z) of the ion affects this motion. Since the charge of an electron is known, the mass to charge ratio a measurement of an ion's mass. Typical mass spectrometry research focuses on the formation of gas phase ions the chemistry of ions, and applications of mass spectrometry. This review articles cover the basics of mass spectrometry instrumentation of a mass spectra.

Figure 1. Mass Spectrometer Block Diagram

THE MASS SPECTROMETERIn order to measure the characteristics of individual molecules, a mass spectrometer converts them to ions so that they can be moved about and manipulated by external electric and magnetic fields. The three essential functions of a mass spectrometer and the associated components.1. A small sample of compound is ionized, usually to cations by loss of an electron.2. In the Ion Source the ions are sorted and separated according to their mass and charge. The Mass Analyzer3. In the Mass Analyzer the separated ions are then detected and tallied, and the results are displayed on a chart the Detector.

IONIZATION TECHNIQUESA variety of ionization techniques are used for mass spectrometry. Most ionization techniques excite the neutral analyte molecule which then ejects an electron to form a radical cation (M+ other ionization techniques involve ion molecule reactions that produce adduct ions (M H+). The most important considerations are the physical state of the analyte and the ionization energy. Electron ionization and chemical ionization are only suitable for gas phase ionization. Fast atom bombardment, secondary ion mass spectrometry, electrospray, and matrix assisted laser desorption are used to ionize condensed phase samples.

Electron IonizationElectron Ionization (EI) is the most common ionization techniqueused for mass spectrometry. EI works well for many gas phase molecules, but it does have some limitations. Although the mass spectra are very reproducible and are widely used for spectral libraries, EI causes extensive fragmentation so that the molecular ion is not observed for many compounds. Fragmentation is useful because it provides structural information for interpreting unknown spectra.

Chemical IonizationChemical Ionization (CI) is a “soft” ionization technique thatproduces ions with little excess energy. As a result, less fragmentation is observed in the mass spectrum. Since this increases the abundance of the molecular ion, the technique is complimentary to 70eV EI. CI is often used to verify the molecular mass of an unknown. Only slight modifications of an EI source region are required for CI experiments.

In Chemical Ionization the source is enclosed in a small cell with openings for the electron beam, the reagent gas and the sample. The reagent gas is added to this cell at approximately 10 Pa (0.1torr) pressure. This is higher than the 10-3 Pa (10-5torr) pressure typical for a mass spectrometer source. At 10-3 Pa the mean free path between collisions is approximately 2 meters and ion-molecule reactions are unlikely. In the CI source, however, the mean free path between collisions is only 10-4 meters and analyte molecules undergo many collisions with the reagent gas. The reagent gas in the CI source is ionized with an electron beam to produce a cloud of ions. The reagent gas ions in this cloud react and produce adduct ions like CH5+, which are excellent proton donors.

Fast Atom Bombardment and Secondary Ion Mass SpectrometryFast AtomBombardment (FAB) and Secondary Ion Mass Spectrometry (SIM S) both use high energy atoms to sputter and ionize the sample in a single step. In these techniques, a beam of rare

gas neutrals (FAB) or ions (SIM S) is focused on the liquid or solid sample. The impact of this high energy beam causes the analyte molecules to sputter into the gas phase and ionize in a single step. The exact mechanism of this process is not well understood, but these techniques work well for compounds with molecular weights up to a few thousand Dalton. Since no heating is required, sputtering techniques (especially FAB) are useful for studying thermally labile compounds that decompose in conventional inlets

Atmospheric Pressure Ionization and Electrospray IonizationAtmospheric Pressure Ionization (API) sources ionize the sample at atmospheric pressure and then transfer the ions into the mass spectrometer. These techniques are used to ionize thermally labile samples such as peptides, proteins and polymers directly from the condensed phase. The sample is dissolved in an appropriate solvent and this solution is introduced into the mass spectrometer. With conventional inlets the solvent increases the pressure in the source region of the mass spectrometer. In addition, Joule-Thompson cooling of the liquid as it enters the vacuum causes the solvent droplets to freeze. The frozen clusters trap analyte molecules and reduce the sensitivity of the experiment.API sources introduce the sample through a series of differentially pumped stages. This maintains the large pressure difference between the ion source and the mass spectrometer without using extremely large vacuum pumps. In addition a drying gas is used to break up the clusters that form as the solvent evaporates. Because the analyte molecules have more momentum than the solvent and air molecules, they travel through the pumping stages to the mass analyzer.

Electron Spray Ionization (ESI)ESI is the most common API application. It has undergone remarkable growth in recent years and is frequently used for LC/M S of thermally labile and high molecular weight compounds. The electrospray is created by applying a large potential between the metal inlet needle and the first skimmer in an API source. The mechanism for the ionization process is not well understood and there are several different theories that explain this ionization process. One theory is that as the liquid leaves the nozzle, the electric field induces a net charge on the small droplets. As the solvent evaporates, the droplet shrinks and the charge density at the surface of the droplet increases. The droplet finally reaches a point where the columbic repulsion from this electric charge is greater than the surface tension holding it together. This causes the droplet to explode and produce multiply charged analyte ions. A typical ESI spectrum shows a distribution of molecular ions with different charge numbers.

Matrix Assisted Laser Desorption/Ionization:Matrix Assisted Laser Desorption/Ionization (MALDI) is used to analyze extremely large molecules. This technique directly ionizes and vaporizes the analyte from the condensed phase. MALDI is often used for the analysis of synthetic and natural polymers, proteins, and peptides. Analysis of compounds with molecular weights up to 200,000dalton is possible and this high mass limit is continually increasing. In MALDI, both desorption and ionization is induced by a single laser pulse. The sample is prepared by mixing the analyte and a matrix compound chosen to absorb the laser wavelength. This is placed on a probe tip and dried. A vacuum lock is used to insert the probe into the source region of the mass spectrometer. A laser beam is then focused on this dried mixture and the energy from a laser pulse is absorbed by the matrix. This energy ejects

analyte ions from the surface so that a mass spectrum is acquired for each laser pulse. The mechanism for this process is not well understood and is the subject of much controversy in the literature. This technique is more universal (works with more compounds) than other laser ionization techniques because the matrix absorbs the laser pulse. With other laser ionization techniques, the analyte must absorb at the laser wavelength. Typical MALDI spectra include the molecular ion; some multiply charged ions, and very few fragments.

MASS ANALYZERSAfter ions are formed in the source region they are accelerated into the mass analyzer by an electric field. The mass analyzer separates these ions according to their m/z value. The selection of a mass analyzer depends up on the resolution, mass range, scan rate and detection limits required for an application. Each analyzer has very different operating characteristics and the selection of an instrument involves important tradeoffs. Analyzers are typically described as either continuous or pulsed. Continuous analyzers include quadrupole filters and magnetic sectors. These analyzers are similar to a filter or monochromator used for optical spectroscopy. They transmit a single selected m/z to the detector and the mass spectrum is obtained by scanning the analyzer so that different mass to charge ratio ions are detected. While a certain m/z is selected, any ions at other m/z ratios are lost, reducing the S/N for continuous analyzers. Single Ion Monitoring (SIM) enhances the S/N by setting the mass spectrometer at the m/z for an ion of interest. Since the instrument is not scanned the S/N improves, but any information about other ions is lost. Pulsed mass analyzers are the other major class of mass analyzer. These are less common but they have some distinct advantages.

QuadrupoleThe quadrupole mass spectrometer is the most common mass analyzer. Its comp act size, fast scan rate, high transmission efficiency, and modest vacuum requirements are ideal for small inexpensive instruments. Most quadrupole instruments are limited to unit m/z resolution and have a mass range of m/z 1000. Many bench top instruments have a mass range of m/z 500 but research instruments are available with mass range up to m/z 4000.

Figure 3.Quadrupole Mass Analyzer

Electric Sector/Double Focusing Mass SpectrometersAn electric sector consists of two concentric curved plates. A voltage is applied across these plates to bend the ion beam as it travels through the analyzer. The voltage is set so that the beam follows the curve of the analyzer. The radius of the ion trajectory (r) depends upon the kinetic energy of the ion (V) and the potential field (E) applied across the plates.

r=2V/E

Time-of-FlightThe time-of-flight (TOF) mass analyzer separates ions in time as they travel down a flight 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. These instruments have high transmission efficiency; no upper m/z limit, very low detection limits, and fast scan rates. For some applications these advantages outweigh the low resolution. Recent developments in pulsed ionization techniques and new instrument designs with improved resolution have renewed interest in TOF-MS.

Figure 4: Time-of-Flight Mass Spectrometer.

Ion Cyclotron ResonanceThe Ion Cyclotron Resonance (ICR) mass spectrometer uses a super conducting magnet to trap ions in a small sample cell. This type of mass analyzer has extremely high mass resolution and is also useful for tandem mass spectrometry experiments. These instruments are very expensive and are typically used for specialized research applications. The ICR traps ions in a magnetic field that causes ions traveling a circular path. This is similar to the path of an ion in a magnetic sector, but the ions are not traveling as fast and the magnetic field is stronger. As a result the ions are contained in the small volume of the trap .The ions cyclotron frequency is the angular frequency of an ion's orbit. This frequency is determined by the magnetic field strength (B) and the m/z value of the ion.

DETECTORSDetection of ions is based upon their charge or momentum. For large signals a faraday cup is used to collect ions and measure the current. Older instruments used photographic plates to measure the ion abundance at each mass to charge ratio. Most detectors currently used amplify

the ion signal using a collector similar to a photomultiplier tube. This amplifying Molecular structure is important for understanding mass spectral inter pretation. To get the most from this section, draw out the structures of the molecules discussed. During the discussion find which bonds break and calculate the mass of the fragments. Actively reading this section will result in a much greater understanding of and appreciation for mass spectrometry. 24 detectors include: electron multipliers, channel electrons and multichannel plates. The gain is controlled by changing the high voltage applied to the detector. A detector is selected for its speed, dynamic range, gain, and geometry. Some detectors are sensitive enough to detect single ions.

VACUUM SYSTEMAll 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 to 10Pa (10 to 10torr) depending upon the geometry of the instrument This-2-5-4-7high vacuum requires two pumping stages. The first stage is a mechanical pump that provides rough vacuum down to 0.1 Pa (10torr). The second stage uses diffusion pumps turbo molecular pumps to provide high vacuum. ICR instruments have even higher vacuum requirements and often include a cryogenic pump for a third pumping stage. The pumping system is an important part of any mass spectrometer but a detailed discussion is beyond the scope of this paper.

DATA SYSTEMThe final component of a mass spectrometer is the data system. This p art of the instrument has undergone revolutionary changes in the past twenty years. It has evolved from photographic plates and strip chart recorders to data systems that control the instrument, acquire hundreds of spectra in a minute and search tens of thousands of reference spectra to identify an unknown. Because these systems are evolving so rapidly, a thorough discussion is not included in this paper. Interested readers should study the manuals for their instrument.

INTERPRETATIONAlthough mass spectrometry is a very sensitive instrumental technique, there are other techniques with picogram detection limits. In addition to sensitivity, however, mass spectrometry also is also useful for identifying the chemical structure of this picogram sample. Since the mass spectrum is a fingerprint of the molecular structure, comparison to a computer databases can be used to identify an unknown compound. This is often done using Probability Based Matching (PBM), a popular pattern recognition technique. Although these computer searches are convenient and powerful, it is important to understand how to interpret a mass spectrum. A computer only compares the unknown spectrum to the library spectra and offers a selection of compounds in the database that produce similar spectra. This computer search is very useful and it makes interpretation much easier, but there are limits to the computer search.

APPLICATIONSIsotope ratio MS: isotope dating and trackingMass spectrometer to determine the 16O/18O and 12C/13C isotope ratio on biogenous carbonate. Mass spectrometry is also used to determine the isotopic composition of elements within a sample. Differences in mass among isotopes of an element are very small, and the less

abundant isotopes of an element are typically very rare, so a very sensitive instrument is required. These instruments sometimes referred to as isotope ratio mass spectrometers (IR-MS).

Trace gas analysisSeveral techniques use ions created in a dedicated ion source injected into a flow tube or a drift tube: selected ion flow tube (SIFT-MS), and proton transfer reaction (PTR-MS), are variants of chemical ionization dedicated for trace gas analysis of air, breath or liquid headspace using well defined reaction time allowing calculations of analyte concentrations from the known reaction kinetics without the need for internal standard or calibration.

Atom probeAn atom probe is an instrument that combines time-of-flight mass spectrometry and field ion microscopy (FIM) to map the location of individual atoms.

PharmacokineticsPharmacokinetics is often studied using mass spectrometry because of the complex nature of the matrix (often blood or urine) and the need for high sensitivity to observe low dose and longtime point data. The most common instrumentation used in this application is LC-MS with a triple quadrupole mass spectrometer. Tandem mass spectrometry is usually employed for added specificity. There is currently considerable interest in the use of very high sensitivity mass spectrometry for micro dosing studies, which are seen as a promising alternative to animal experimentation.

Protein characterizationMass spectrometry is an important emerging method for the characterization and sequencing of proteins. The two primary methods for ionization of whole proteins are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). In keeping with the performance and mass range of available mass spectrometers, two approaches are used for characterizing proteins. In the first, intact proteins are ionized by either of the two techniques described above, and then introduced to a mass analyzer. This approach is referred to as "top-down" strategy of protein analysis. In the second, proteins are enzymatically digested into smaller peptides using proteases such as trypsin or pepsin, either in solution or in gel after electrophoretic separation. Other proteolytic agents are also used. The collection of peptide products are then introduced to the mass analyzer approach.

Glycan analysisMass spectrometry (MS), with its low sample requirement and high sensitivity, has been predominantly used in glycobiology for characterization and elucidation of glycan structures.

Space explorationAs a standard method for analysis, mass spectrometers have reached other planets and moons. Two were taken to mars by the Viking program.

Respired gas monitorMass spectrometers were used in hospitals for respiratory gas analysis beginning around 1975 through the end of the century. Some are probably still in use but none are currently being manufactured.

Found mostly in the operating room, they were a part of a complex system, in which respired gas samples from patients undergoing anesthesia were drawn into the instrument through a valve mechanism designed to sequentially connect up to 32 rooms to the mass spectrometer. A computer directed all operations of the system. The data collected from the mass spectrometer was delivered to the individual rooms for the anesthesiologist to use.

NMRINTRODUCTION:Over the past fifty years Nuclear Magnetic Resonance spectroscopy, commonly referred to as NMR, has become the dominant method of analysis of organic compounds, because in many cases it provides a way to determine an entire structure using one set of analytical tests. It is also increasingly used in inorganic chemistry and biochemistry, where it also provides a lot of valuable structural information. NMR is a property of the nucleus of an atom, concerned with what is known as nuclear spin (I). This is equivalent to the nucleus acting like a tiny bar magnet. Isotopes can have a variety of values for I (including zero). In this NMR spectroscopy only nucleus containing odd mass number (A) or odd atomic number (Z) are most useful which includes hydrogen 1 (1H), carbon 13 (13C), fluorine 19 (19F) and phosphorus 31 (31P). Whereas nucleus containing even mass number (A) or even atomic number (Z) are not used , This includes 10B, 14N etc.Resonance frequency of a particle, substance is the key feature of NMR and is directly proportional to the strength of the applied magnetic field.The effectiveness of NMR can also be improved by using following techniques

1. Hyperpolarization2. Two-dimensional3. Three-dimensional4. Higher-dimensional multi frequency techniques

DEFINITIONSNuclear Magnetic Resonance (NMR) is a spectroscopy technique which is based on the transition of Electromagnetic radiations in a radio frequency region 4 to 900 MHz by nuclei of atoms in the presence of magnetic field. In this radio frequency radiations are used to induce transitions between different nuclear spin states of samples in a magnetic field. When proton (hydrogen) is studied then it is called as Proton Magnetic Resonance (PMR). When other nuclei like 13C, 9F, 35Cl etc. are studied then it is called as NMR.

PRINCIPLE:Nuclei that exhibit the NMR phenomenon are those which have the spin quantum number I greater than 0 (I>0). A nucleus with an odd mass or an odd atomic number possess a nuclear spin, due to spinning a magnetic field is generated along the axis. The spin quantum number I of the nuclei as follows:

Mass number (A) Atomic number (Z) Spin quantum number (I)

Odd odd or even 1/2, 3/2, 5/2…Even even 0Even Odd 1, 2, 3…

Table-1: The spin quantum numberI is associated with the mass number (A) and atomic number (Z) of the nuclei

Without externally applied magnetic field, the nuclear spins are random in all directions. But when externally magnetic field is applied; the nucleus align themselves by creating magnetic momentum.

Fig-1: Orientation of spinning nuclei in absence and presence of external magnetic fieldHence, nucleus spins on their own axis when placed in an external magnetic field resulting in a circular motion creating aprecessional orbit, with a frequency called precessional frequency. When energy in the form of radio frequency is applied and is equal to precessional frequency, then the transition of protons from lower energy (α state) to higher energy (β state) take places and NMR signals are recorded.

When application of radio frequency energy is stopped nucleus returns to ground state. Increasing in strength of magnetic field does not cause transition from lower energy (α state) to higher energy (β state). But it merely increases precessional frequency.

RELAXATION PROCESS:It is the process of transition from excited state to ground state where absorbed radio frequency energy can be lost by two ways:1. Radiation Emission: lost with emission of radio frequency radiations itself.

2. Radiation Transition: (without radiation) it occurs in two ways:a) Spin-lattice/ longitudinal relaxation process: where the energy is lost by means of translational/ vibrational/ radiation energy.b) Spin-spin/ Transverse relaxation process: where the energy is lost to neighboring nuclei.

NMR SPECTRUM:The NMR spectrum is a plot of, "intensity of NMR signals Vs frequency (magnetic field)". It is generally calibrated in units of frequency rather than in units of magnetic field strength.

INSTRUMENTATION:The Nuclear Magnetic Resonance Spectrophotometers are two types based on the parameters that are measured.1. Single coil spectrometers: It measures absorption.2. Double coil spectrometers: It measures resonant radiation.

NMR spectrometers can also be divided into low resolution or high resolution spectrometers, with the former being capable of quantitative element analysis and also being known as wide line spectrometer.The major components of NMR instrument are as follows:

Fig-4: Schematic diagram of NMR Spectrophotometer

Sample Holder:Usually the dimension of sample holder is 8.5cm in length and 0.3 mm diameter. Glass tubes are generally used as sample holder as these are more economic. The following ideal characteristics are there in the sample holder.

It should be sturdy.

It should be practical.

It should be cheap.

It should be transparent to radio frequency radiations.

It should be chemically inert.

Sample probe:It holds the sample tube in a magnetic field and rotates it along its axis, resulting in sharper lines with better resolution due to decrease in the effects of in homogeneities in the magnetic field. The probe may be either a single coil or system of coils depending upon the type of instrument.

Permanent Magnet:Permanent magnet or electromagnet has the important feature that it should give homogeneous magnetic field, i.e., the strength and direction of the magnetic field should not change from point to point. As the field strength is proportional to the chemical shifts, it must not be less than 20,000 guass.

Magnetic Coils:

It is employed for the production of NMR spectra. It is achieved by passing direct current either through the coils that are wound around the magnetic pole or through a pair of Helmholtz coils located on either side of the sample probe. The relationship between the resonance frequency of the nucleus and the strength of magnetic field (H0) is expressed as:

v = constant x H0From that equation, frequency is directly proportional to strength of magnetic field (H0). If H0is kept constant, the precession frequency is fixed. If radiofrequency is kept constant, the resonance frequency of the nucleus must be changed by varying H0

Magnetic Coils:If precession frequency is equal to applied frequency radiations, this results in nucleus to resonate. Sweep generator method, is used to vary the magnetic field and it is easier, than the variation of radio frequency.

Radio Frequency Generator:Radio Frequency Generator is also known as Radio Frequency Transmitter. In order to generate radio frequency radiation, radio frequency oscillator is used which irradiates the sample molecules. Due to applied radiofrequency, an energy difference occurs and the nuclei moves from ground state to excited state. The coil surrounding the sample results in resonance signals.

Radio Frequency Receiver:When the radio frequency radiation is passed through the magnetized sample two phenomena namely absorption and dispersion may occur. The observation of either absorption or dispersion will enable the resonance frequency to be determined.

Fig-5: Absorption and Dispersion signals

For the detection of resonance signal following methods are used:a) Radio Frequency Bridge is employed under single coil instruments. It allows absorption and dispersion signals to appear as an output of EMF across the bridge. Signals can be recorded mechanically.

b) This method employs a separate receiver coil which is sometimes called as crossed coil or nuclear induction method. In this the transmitter and receiver coils are arranged perpendicular to each other and to the direction of the magnetic field.

Amplifier:The absorption signal received from radio frequency receiver is extremely weak. Therefore, it requires considerable amplification before it is fed to a chart recorder in which amplifier is used for amplification of weak signals.

Read Out:The NMR spectra obtained from instruments are directly recorded via a computer or even mechanically.

SOLVENT SYSTEM USED IN NMR:A substance which is free of protons should be used as solvent. i.e., it should not give of its own absorption in NMR spectrum. It should be capable of dissolving at least 10% of substance.Examples:CCl4 - Carbon TetrachlorideCS2 - Carbon DisulfideCDCl3-DeuteriochloroformC6D6 - HexaDeuteriobenzeneD2O - Deuterium oxide

PARAMETERS EFFECTING NMR SPECTROSCOPY:1. Chemical Shift: A chemical shift is defined as the difference in parts per million (ppm) between the resonance frequency of the observed proton and tetramethylsilane (TMS) hydrogen.Orit is theShifting in positions of NMR absorptions(reference & sample) which arise due to shielding or deshielding of protons by electrons is called as chemical shift.Factors influencing Chemical Shift:The following are the factors which affects chemical shift:a) Inductive effectb) Van der waals deshieldingc) Anisotropic effectd) Hydrogen bonding

2. Spin Spin Coupling:The interaction between the spins of neighboring nuclei in the molecule may causes splitting of NMR. This is called as spin spin coupling. This is related to the number of possible combinations of the spin orientations of the neighboring protons.

3. COUPLING CONSTANT (J):The distance between the adjacent lines of multiplet is a measure of splitting effect known as coupling constant. It is expressed in Hz. Coupling constant is a measure of spin spin coupling.

INTERPRETATION OF NMR SPECTRA:NMR spectrum of a substance gives very valuable information about its molecular structure. This information is gathered as follows:1. The number of signals in the PMR spectrum tells us how many kinds of protons in different chemical environments are present in the structure under examination.2. The position of signals gives information about each kind of protons.3. The intensities of different signal give information about the presence of relative number of protons of different kinds.4. The splitting of signals tells us about the absorbing proton with respect to neighboring protons.

Fig-8: NMR Spectrum of Ethanol

When the spectrum is recorded for ethanol it gives Triplet, quartet and singlet peak because of the presence of three kinds of protons. The triplet is because of three equivalent methyl protons and splits into a triplet because of the two neighboring hydrogen’s on methylene group. The quarter is for two methylene protons and multiplicity of four peaks is due to coupling with the three methyl protons but the hydroxyl proton will not couple. Finally the singlet peak is due to hydroxyl proton which does not show any coupling with adjacent methylene protons.

APPLICATIONS: The two major areas where NMR has proven to be of critical importance are in the fields of medicine and chemistry, with new applications being developed daily.

1. Chemistry related applications:

I. Structural diagnosis: A large number of principles are known which will decide about structure of an unknown from its NMR spectrum. Some of these are outlined as follows:

The number of main NMR signals should be equal to number of equivalent protons in unknown compounds.

Chemical shift indicates types of H-atoms present

II. Quantitative Analysis:a) Assay of component or % purity:b) c)Hydrogen analysis:c) Hydrogen Bonding:d) Moisture analysis:

III. Determination of molar composition of the compound

IV. Identification of chemical reactionNMR is used to identify whether the chemical reaction is complete or not.E.g.:CH3OOHa + HOHb <-----> CH3COOHb+ HOHa

V. Study of Isotopes other than Protons:Nuclei having magnetic moments in addition to that of proton can be studied by using the nuclear magnetic resonance spectroscopy.

2. Medical related applications:NMR is used in medical field for diagnosis of human body in two ways:

Used for diagnosis for inborn errors of metabolism in urine like, phenyl ketonuria, Maple's urine syndrome.

Magnetic resonance imaging (MRI) is an important medical diagnostic tool used to study the function and structure of the human body which is very safe to administer

3. Other applications:NMR has also proven to be very useful in other fields such as non-destructive testing, data acquisition in the petroleum industry, process control, earth’s field NMR and magnetometers.

Non-destructive testing saves a lot of money for expensive biological samples and can be again used if more trials are needed to perform.

The petroleum industry uses NMR equipment to measure porosity of different rocks and permeability of different underground fluids.

Magnetometers are used to measure the various magnetic fields that are relevant.

The mass spectrum of aspirin:-

There is a definitive peak of molecule ions at 180, which is the mol mass of the molecule. Other fragments, such as 45 and 121 (180-59), lead the analyzer to assume that they are breakaways of the aromatic rings.

Principal ions at m/z 120, 43, 138, 92, 121, 39, 64, 63

NMR spectrum of Aspirin:

In a 13C NMR spectrum, every C atom produces an individual signal, which can then be used to formulate a sum formula. The 13C NMR spectrum ranges from 0 to 250 ppm, whereas these values can differ and they can roughly be divided in the following categories:Between 0 and 50 ppm: signals of C- atoms are found, which only have single bonds and are saturated with hydrogen atomsBetween approx. 50 and 100 ppm: C atoms are present, who have created a bond to a strongly electronegative atom such as chlorine or oxygen.Between approx. 100 and 150 ppm: the signals of aromatic C-atoms are present.Over 150 ppm: almost only the signals of C-atoms, which have formed a double bond to an O-atom, ketones, aldehydes, esters and carboxylic acids.

The interpretation of a 1H NMR spectrum It is done similarly: the 1H NMR spectrum usually lies between 0 and 10 ppm. There are also very specific regions, where one can await signals of specific hydrogen nuclei:Between 0 and 3 ppm: present are only the signals that give evidence of H atoms that had created single bonds to C atomsbetween 3 and 6 ppm: present are the signals that give evidence of H-atoms that had created double bonds to C-atoms or the signals of H-atoms that had bonded themselves to strong electronegative atoms such as O and N atoms.Over 6 ppm: present are only signals of H-atoms that had bonded themselves to aromatic C-atoms or of H-atoms that are located in a powerful electron pulling environment such as aldehydes and carboxylic acids.

Using this method, you can identify 1H-NMR and 13C-NMR spectra as acetylsalicylic acid, aspirin.

The result shows that a signal around 2 ppm is present, which was produced by a CH3group. Plus, the signals around 7 ppm signify the presence of the 4 aromatic 1H nuclei. The multiplicity

of these signals is very complex due to the fact that there are many coupling partners available to bond with. This inevitably also leads to intensity of the aromatic signals being significantly smaller than those signals at 2 ppm. Lastly, the signal of the chemical shift of the H-atom of the carboxylic acid group is awaited to be at around 10 ppm. However, it isn't rare that such signals can't be found on a spectrum because of interaction processes and Hydrogen links that may render the signal useless.

The actual measurement of the 1H NMR spectrum of acetylsalicylic acid shows that the awaited result has been confirmed:

The 13C NMR spectrum of acetylsalicylic acid generates nine signals. The signals of C=O - functions are awaited to be located at the highest chemical shift at over 150 ppm. Following are the six signals of the aromatic C shifts at around 120 ppm and then the signal of the CH3-group at 40 ppm.

The awaited result is also confirmed with the actual measurement of the 13C NMR spectrum.

The measured 13C NMR spectrum of acetylsalicylic acid:

Infrared Spectroscopical analysis of Aspirin :The next picture shows the IR spectrum of aspirin.

In the area between 2500 and 3000cm-1, the C-H and the O-H bonds can be recognized. By the three significant swinging areas at 1600, 1700, and 1780cm-1, one can positively acknowledge the presence of C=O and aromatic C=C swinging. The swinging at 1780cm-1 can be put together with the ester function and 1700cm can be put together with carboxylic acid.

Table 4d: IR Band Assignments of Acetylsalicylic Acid (Aspirin)

Ultraviolet Spectrum.

Ultraviolet visible spectrum of aspirin The ultraviolet visible spectrum (200 - 350 nm) of aspirin in acidic (0.1 N HCl), and neutral (methanol) solvent systems are shown in Fig. 2. The maximum ultraviolet absorption of aspirin was found at 230 and 278 nm in acidic (0.1 N HCl) and 225 and 276 nm in neutral (methanol) solvent systems, respectively. The obtained spectra and maximum absorption wavelength for aspirin in acidic solvent system was compared with the reference spectra of aspirin.