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SPECTROSCOPY

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وکنترول فارمسی کیمیای دیپارتمنت ادویه

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Definition :

• Spectroscopy - The study of the interaction of electromagnetic radiation with matter .


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Electromagnetic radiation :

• An oscillating electric and magnetic field which travels through space

• A discrete series of “particles” that possess a specific energy but have no Mass

BOTH!


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Properties of Light• Light can be thought of as a wave or particle.

– The wavelength, , is the distance between crests of a wave (m)

– The frequency, , is the number of oscillations per second (Hz)

m/s 10998.2 8 ccsJ 10626.6 34 hE


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Introduction of Spectrometric Analyses

The study how the chemical compound interacts with different wavelengths in a given region of electromagnetic radiation is called spectroscopy or spectrochemical analysis.

The collection of measurements signals (absorbance) of the compound as a function of electromagnetic radiation is called a spectrum.


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Energy Absorption

The mechanism of absorption energy is different in the Ultraviolet, Infrared, and Nuclear magnetic resonance regions. However, the fundamental process is the absorption of certain amount of energy.

The energy required for the transition from a state of lower energy to a state of higher energy is directly related to the frequency of electromagnetic radiation that causes the transition.


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Regions of the electromagnetic spectrum :


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Interaction of e.m.r. with Matter

• Interaction of electromagnetic radiation with matter– The wave-length, , and the wave number, v’, of e.m.r. changes with the

medium it travels through, because of the refractive index of the medium; the frequency, v, however, remains unchanged

– Types of interactions

• Absorption• Reflection• Transmission• Scattering• Refraction

– Each interaction can disclose certain properties of the matter

– When applying e.m.r. of different frequency (thus the energy e.m.r. carried) different type information can be obtained .

refraction

transmission

absorption

reflection scattering


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Absorption and Emission of Photons


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Wave Number (cycles/cm)

X-Ray UV Visible IR Microwave

200nm 400nm 800nm

Wavelength (nm)

Spectral Distribution of Radiant Energy


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V = Wave Number (cm-1)

Wave Length

C = Velocity of Radiation (constant) = 3 x 1010 cm/sec.

= Frequency of Radiation (cycles/sec)

The energy of photon:

h (Planck's constant) = 6.62 x 10-27 (Ergsec)

V =C

E = h = hC

C

= C =

Electromagnetic Radiation


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Visible

Ultra violet

Radio

Gamma ray

Hz

cmcm-1Kcal/mol eV

Type

Quantum Transition

Type

spectroscopy

Type

Radiation

Frequency

υ

Wavelength

λ

Wave

Number VEnergy

9.4 x 107 4.9 x 106 3.3 x 1010 3 x 10-11 1021

9.4 x 103 4.9 x 102 3.3 x 106 3 x 10-7 1017

9.4 x 101 4.9 x 100 3.3 x 104 3 x 10-5 1015

9.4 x 10-1 4.9 x 10-2 3.3 x 102 3 x 10-3 1013

9.4 x 10-3 4.9 x 10-4 3.3 x 100 3 x 10-1 1011

9.4 x 10-7 4.9 x 10-8 3.3 x 10-4 3 x 103 107

X-ray

Infrared

Micro-wave

Gamma ray emission

X-ray absorption, emission

UV absorption

IR absorption

Microwave absorption

Nuclear magnetic resonance

Nuclear

Electronic (inner shell)

Molecular vibration

Electronic (outer shell)

Molecular rotation

Magnetically induced spin states

Spectral Properties, Application and Interactions of Electromagnetic Radiation


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Atomic Spectra

• Shell structure & energy level of atoms– In an atom there are a number of shells and of subshells

where e-’s can be found – The energy level of each shell & subshell are different

and quantised• The e-’s in the shell closest to the nuclei has the lowest

energy. The higher shell number is, the higher energy it is• The exact energy level of each shell and subshell varies with

substance

• Ground state and excited state of e-’s– Under normal situation an e- stays at the lowest possible

shell - the e- is said to be at its ground state– Upon absorbing energy (excited), an e- can change its

orbital to a higher one - we say the e- is at an excited state.

n = 1

n = 2

n = 3, etc.

energy

E

groundstate

Excitedstate

En

erg

y

n=1

n=2

n=3

n=4

1s2s2p

3s3p

4s3d4p

4d4f


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Atomic Spectra

• Electron excitation– The excitation can occur at different

degrees • low E tends to excite the outmost e-’s first• when excited with a high E (photon of high

v) an e- can jump more than one levels• even higher E can tear inner e-’s away from

nuclei

– An e- at its excited state is not stable and tends to return its ground state

– If an e- jumped more than one energy levels because of absorption of a high E, the process of the e- returning to its ground state may take several steps, - i.e. to the nearest low energy level first then down to next …

n = 1

n = 2

n = 3, etc.

energy

E

En

erg

y

n=1

n=2

n=3

n=4

1s2s2p

3s3p

4s3d4p

4d4f


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Atomic Spectra

• Atomic spectra– The level and quantities of energy

supplied to excite e-’s can be measured & studied in terms of the frequency and the intensity of an e.m.r. - the absorption spectroscopy

– The level and quantities of energy emitted by excited e-’s, as they return to their ground state, can be measured & studied by means of the emission spectroscopy

– The level & quantities of energy absorbed or emitted (v & intensity of e.m.r.) are specific for a substance

– Atomic spectra are mostly in UV (sometime in visible) regions

n = 1

n = 2

n = 3, etc.

energy

E

En

erg

y

n=1

n=2

n=3

n=4

1s2s2p

3s3p

4s3d4p

4d4f


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Absorption Spectroscopy Introduction

A.) Absorption: electromagnetic (light) energy is transferred to atoms, ions, or molecules in the sample. Results in a transition to a higher energy state.

- Transition can be change in electronic levels, vibrations, rotations, translation, etc.

- Concentrate on Molecular Spectrum in UV/Vis (electronic transition)

- Power (P): energy of a beam that reaches a given area per second

- Intensity (I): power per unit solid angle

- P and I related to amplitude2

Eo

E1h Energy required of photon to give this transition:

hE= E1 - Eo

(excited state)

(ground state)


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B.) Terms:

1.) Beer’s Law: A = bc

The amount of light absorbed (A) by a sample is dependent on the path length (b), concentration of the sample (c) and a proportionality constant (– molar absorptivity)

Amount of light absorbed is dependent on frequency ()

c

Absorbance is directly proportional to concentration Fe+2

Increasing Fe+2 concentration


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B.) Terms:

1.) Beer’s Law: A = bc

Transmittance (T) = P/Po %Transmittance = %T = 100T

Absorbance (A) = log10 Po/P

No light absorbed- % transmittance is 100% absorbance is 0

All light absorbed- % transmittance is 0% absorbance is infinite


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Relationship Described in Terms of Beer’s Law

A = Absorbance = bc = -log(%T/100)

= molar absorptivity: constant for a compound at a given frequency () units of L mol-1 cm-1

b = path length: cell distance in cm

c = concentration: sample concentration in moles per liter.

Therefore, by measuring absorbance or percent transmittance at a given frequency can get information related to the amount of sample (c) present with an identified and .

Note: law does not hold at high concentrations, when A > 1


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is a measure of the amount of light absorbedper unit concentration at a particular .

Molar absorptivity is a constant for a particular substance, so if the concentration of the solution is halved, so is the

absorbance at sufficiently dilute concentrations.

Molar AbsorptivityA = lc

A

concentration


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25


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30


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32


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Cuvettes (sample holder)

• Polystyrene– 340-800 nm

• Methacrylate– 280-800 nm

• Glass– 350-1000 nm

• Suprasil Quartz– 160-2500 nm


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50


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UV-Visible Spectrophotometry

بنفش – ماورای سپکتروفوتومتریدید قابل


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UV-Visible Spectrophotometry

The absorption of ultraviolet and visible radiation by molecules are dependent upon the electronic structure of the molecule.

So the ultraviolet and visible spectrum are called electronic spectrum.


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What does the absorbed light (electromagnetic radiation)

do to the molecule?

high energy UV – ionizes electrons

low energy UV and visible – promotes electrons to higher energy orbitals(absorption of visible light leads to a colored solution)

IR – causes molecules to vibrate (more later)

700 nm 400 nm

IR UV

visibleEnergy increasing


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UV/visible light absorption

In organic molecules, electronic transitions to higher energy molecular orbitals – double bonds: *

In transition metals, hydrated ions as Cu++ have splitting of d orbital energies and electronic transitions – weak absorption

In complexed transition metals, charge transfer of electrons from metal to ligand as Cu(NH3)4

++ – strong absorption

Valence electrons


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Electronic Excitation

The absorption of light energy by organic compounds in the visible and ultraviolet region involves the promotion of electrons in , , and n-orbitals from the ground state to higher energy states. This is also called energy transition. These higher energy states are molecular orbitals called antibonding.


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Ene

rgy

*

*

n

*

*

n

*

n

*

Antibonding

Antibonding

Nonbonding

Bonding

Bonding


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Electronic Molecular Energy Levels

The higher energy transitions ( *) occur a shorter wavelength and the low energy transitions (*, n *) occur at longer wavelength.


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and * orbitals and * orbitals


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Electronic Transitions in Organic Molecules

http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm#uv1


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and* orbitals


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and * orbitals


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Electronic Transitions: *

The * transition involves orbitals that have significant overlap, and the probability is near 1.0 as they are “symmetry allowed”.


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* transitions - Triple bonds

Organic compounds with -C≡C- or -C≡N groups, or transition metals complexed by C≡N- or C≡O ligands, usually have “low-lying” * orbitals


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Electronic Transitions: n *

The n-orbitals do not overlap at all well with the * orbital, so the probability of this excitation is small. The of the n* transition is about 103 times smaller than for the * transition as it is “symmetry forbidden”.


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UV Activity

h


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Excited States


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Chemical Structure & UV Absorption

What is chromophore ?

•Chromophore is a functional group which absorbs a characteristic ultraviolet or visible region.

• Chromophoric Group ---- The groupings of the molecules which contain the electronic system which is giving rise to absorption in the ultra-violet region.


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Chromophore absorptions

Chromophore Example Excitation max, nm Solvent

C=C Ethene

171 15,000 hexane

CC 1-Hexyne

180 10,000 hexane

C=O Ethanal

n

290180

1510,000

hexanehexane

N=O Nitromethane

n

275200

175,000

ethanolethanol

C-X X=Br X=I

Methyl bromide

Methyl Iodide

n

n

205255

200360

hexanehexane


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Organic ChromophoresChromophore Transition max(nm) log()

Nitrile (-C≡N) to 160 <1.0

Alkyne (-C≡C-) to 170 3.0

Alkene (-C=C-) to 175 3.0

Alcohol (ROH) to 180 2.5

Ether (ROR) to 180 3.5

Ketone (-C(R)=O) to 180 3.0

to 280 1.5

Aldehyde (–C(H)=O) to 190 2.0

to 290 1.0

Amine (-NR2) to 190 3.5

Acid (-COOH) to 205 1.5

Ester (-COOR) to 205 1.5

Amide (-C(=O)NH2) to 210 1.5

Thiol (-SH) to 210 3.0

Nitro (-NO2) to 271 <1.0

Azo (-N=N-) to 340 <1.0


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Single Beam Spectrophotometer


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Dual Beam Spectrophotometer


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Sample Cells

UV Spectrophotometer

Quartz (crystalline silica)

Visible Spectrophotometer

Glass


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Cuvettes (sample holder)

• Polystyrene– 340-800 nm

• Methacrylate– 280-800 nm

• Glass– 350-1000 nm

• Suprasil Quartz– 160-2500 nm


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Components of an Instrument for UV/Vis Absorbance Measurements:

1.) Basic Design:

Hitachi Instruments U-3010

Light Source, selector, Sample cell holder, Detector (amplifier, recorder)


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a) Desired Properties of Components of UV/Vis:

Light Source Selector Creates Proper Narrow Bandpass:Stable: Selects Desired Constant P Large Light Throughput:

Good Precision Increase PIntense:

Increase PEasier to See Absorbance

Sample Cell Holder DetectorFixed Geometry: Stable

Constant b Sensitive to of InterestTransmits of Interest:

Increase P


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b) Light Sources UV/Vis (~ 200 – 800 nm):

1. Deuterium & Hydrogen Lamps (UV range)- continuous source, broad range of frequencies- based on electric excitation of H2 or D2 at Low pressure

40V Electric Arc

Electrode

Filament

D or H Gas2 2

Sealed Quartz Tube

In presence of arc, some of the electrical energy is absorbed by D2 (or H2) which results in the disassociation of the gas and release of light

D2 + Eelect D*2 D’ + D’’ + h(light produced)

Excited state


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2. Tungsten Filament Lamp (Vis – Near IR)- continuous source, broad range of frequencies- based on black body radiation:

heat solid filament to glowing, light emitted will be characteristic of temperature more than nature of solid filament

Low pressure (vacuum)

Tungsten Filament

Temperature Dependence of


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b) Wavelength Selectors:

1. Monochromator- separates frequencies () from polychromatic light in time or space.- allows only certain ’s to be selected and used.

i.) Dispersing Monochromator:

a) Prism: based on refraction of light and fact that different ’s have different values of refraction index (i) in a medium.


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UV vs. IR vs. NMR

• UV has broad peaks relative to IR & NMR

• UV has less information than IR & NMR

• UV spectra are easier to collect

• UV spectra are faster to collect

• UV spectrometers are cheaper

• UV spectra require only nanograms of material or chemicals


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Io I

Cell withPathlength, b,

containing solution

lightsource detector

blank where Io = I

concentration 2concentration 1

b

with sample I < Io

The process of light being absorbed by a solution

As concentration increased, less light was transmitted (more light absorbed).


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Some terminology

I – intensity where Io is initial intensity

T – transmission or %T = 100 x T(absorption: Abs = 1 – T or %Abs = 100 - %T)

T = I/ Io

A – absorbanceA = - log T = -log I/ Io


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Beer’s Law

A = abc

where a – molar absorptivity, b – pathlength, and c – molar concentration

See the Beer’s Law Simulator


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Analyze at what wavelength?Scan visible wavelengths from 400 – 650

nm (detector range) to produce an absorption spectrum (A vs. )

Crystal Violet Absorption Spectrum

0

0.2

0.4

0.6

0.8

1

1.2

1.4

200 250 300 350 400 450 500 550 600 650 700 750wavelength, nm

Abso

rban

ce

max

max - wavelength where maximum absorbance occurs

phototube detector range


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The BLANK

The blank contains all substances except the analyte.

Is used to set the absorbance to zero:Ablank = 0

This removes any absorption of light due to these substances and the cell.

All measured absorbance is due to analyte.


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Light source

Grating

Rotating the gratingchanges the wavelength going through the sample

slits

slits

Sample

filter

Phototube

The components of a Spec-20D

occluder

When blank is the sample Io is determined

otherwise I is measured

Separates white lightinto various colors

detects light &measures intensity

- white light of constant intensity


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Uses of visible spectrophotometry

Analysis of unknowns using Beer’s Law calibration curve

Absorbance vs. time graphs for kineticsSingle-point calibration for an

equilibrium constant determinationSpectrophotometric titrations – a way

to follow a reaction if at least one substance is colored – sudden or sharp change in absorbance at equivalence point, a piece-wise function

(Been there, done that!)


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Practical Applications

• Medicinal Chemistry– compound ID (steroids, nucleosides)– monitoring isomerization, chirality

• Pharmaceutical Biotechnology– concentration/purity measurements– monitoring conformation of protein drugs

• Pharmacokinetics/Med. Chem.– HPLC monitoring and purification


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Quantitative Analysis (Beer’s Law):

1) Widely used for Quantitative Analysis Characterization- wide range of applications (organic & inorganic)- limit of detection 10-4 to 10-5 M (10-6 to 10-7M; current)- moderate to high selectivity- typical accuracy of 1-3% ( can be ~0.1%)- easy to perform, cheap

2) Strategies

a) absorbing species- detect both organic and inorganic compounds

containing any of these species (all the previous examples)

Chromophore Example Excitation max, nm Solvent

C=C Ethene __> * 171 15,000 hexane

CC 1-Hexyne __> * 180 10,000 hexane

C=O Ethanaln __> * __> *

290180

1510,000

hexanehexane

N=O Nitromethanen __> * __> *

275200

175,000

ethanolethanol

C-X X=Br X=I

Methyl bromideMethyl Iodide

n __> *n __> *

205255

200360

hexanehexane


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b) non- absorbing species- react with reagent that forms colored product- can also use for absorbing species to lower limit

of detection- items to consider:

, pH, temperature, ionic strength- prepare standard curve (match standards and

samples as much as possible)

Standard Addition Method (spiking the sample)

- used for analytes in a complex matrix where interferences in the UV/Vis for the analyte will occur: i.e. blood, sediment, human serum, etc..

- Method:(1) Prepare several identical aliquots, Vx, of the unknown sample.(2) Add a variable volume, Vs, of a standard solution of known

concentration, cs, to each unknown aliquot.(3) Dilute each solution to an equal volume, Vt.(4) Make instrumental measurements of each sample to get an

instrument response, IR.(5) Calculate unknown concentration, cx, from the following equation.

Note: This method assumes a linear relationship between instrument response and sample concentration.

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