Introduction to spectroscopic methods

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An Introduction to

Spectrometric Methods

Bivek [email protected]

mailto:[email protected]

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Quantum Mechanical Properties of Electromagnetic Radiation (EMR)

The amount of energy involved in transitions from its ground state to exited state (following absorption of energy) and from excited to ground state (by emission of radiation) is given by the following equation;

ΔE = E1 – E2 = hV

where, ΔE = change in energy state of the electron or the energy of electromagnetic radiation absorbed or emitted by an atom or molecule.

E1 = energy of electron in original state,E2 = energy of electron in the final state,h = the Plank’s constant

V= frequency of the electromagnetic radiation in hertz (C/ λ), where, C= speed of electromagnetic radiation (3x108 m/s)

λ = wavelength of electromagnetic radiation

(= 6.63 x 10-34 JS)The greater the energy, the higher the frequency and wavenumber and the shorter the wavelength

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Energy level and transition in atom and molecule

E0

E1

E2

E3

E4

Atomic energy level Molecular energy level

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Emission of Radiation

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Absorption of Radiation

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The Beer-Lambert Law

I0

Sample

I Detector

Source

Cuvette

Transmittance, T, is simply defined as “the fraction of light that reaches a detector after passing through a sample”

T = I/I0 …………… (i)

Where, I0 = intensity of Incident radiationI= intensity of transmitted radiation

Percentage Transmission (%T) = % T = I/I0 x 1000 < %T < 100

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Absorbance (A): A = -log T = log (1/T) = log (I o / I)

Absorbance is also called as Optical Density (O.D.)range from 0 (= 100% T) to infinity (=0%T).

The Beer-Lambert law states Absorbance is directly proportional to:1. concentration, c, of absorbing species in the sample (A c)2. path length of light, L, through the sample (A L)

A = € C L ……………… (iii)Where, € = Molar absorbance coefficient of the absorber

C = Concentration of absorbing solution, andb = Path length through the solution (or thickness)

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Absorbance (A):

A = € C L ……………… (iii) Concentration of the analyte is given in unit mol/L (M) The path length, L, in cm , is called the molar absorptivity or molar absorption coefficient “Absorbance of 1 M solution measured in a cell of 1 cm pathlength”

, is characteristic for each substance at a particular wavelength, .

11111 cmMcmmolLcm

Lmolcl

A

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Concentration

Abs

orba

nce,

A

0

0.5

1

Concentration

Tran

smitt

ance

, TA=cLcertain constant LOne analyte

T=10-A =10- bc

Beer’s law is a relation between absorbance and concentration which is a straight line passes by origin at constant pathlength, b, and at certain wavelength, .

Transmittance decreases exponentially as concentration

increases

Beer’s law is obeyed for monochromatic light

Slope = L


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Beer’s Law and Multicomponent samples

For sample containing several absorbing components (say X and Y) given that there are no interactions between the components, the total absorbance is,

Atotal = Ax+ By = €x Cx L + €y Cy L

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Blank

Detector

Source

I0

Use of Blank

I0

Sample

I Detector

Source

Cuvette

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The Beer-Lambert LawA = € C L

Use of Curve

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This relationship is a linear for the most part. However, under certain

circumstances the Beer relationship gives a non-linear relationship.

These deviations from the Beer Lambert law can be classified into three

categories:

Real Deviations - These are fundamental deviations due to the limitations

of the law itself.

Chemical Deviations- These are deviations observed due to specific

chemical species of the sample which is being analyzed.

Instrument Deviations - These are deviations which occur due to how the

absorbance measurements are made.

Derivation of Beer Lambert Law

http://pharmaxchange.info/press/2012/04/ultraviolet-visible-uv-vis-spectroscopy-%E2%80%93-derivation-of-beer-lambert-law/

ReadoutAbsorbance

0.00

Source

Detector


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Path Length Dependence, L

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Path Length Dependence, L ReadoutAbsorbance

0.22

Source

Detector

b

Sample


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ReadoutAbsorbance

0.44

Source

Detector Samples

Of course, we are not introducing two cells in the light pathway, but let us assume that we doubled the path length of light through the absorbing medium


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ReadoutAbsorbance

0.66

Source

Detector Samples



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Concentration Dependence, c ReadoutAbsorbance

0.00

Source

Detector


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Concentration Dependence, c

ReadoutAbsorbance

0.42

Source

Detector

b

Sample


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Concentration Dependence, c ReadoutAbsorbance

0.63

Source

Detector

b

Sample


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Wavelength Dependence,

ReadoutAbsorbance

0.30

Source

Detector

b

The blue solution do absorb the red radiation


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Wavelength Dependence,

ReadoutAbsorbance

0.00

Source

Detector

b

The red solution can not absorb the red radiation but it can absorb radiation that is complimentary to red.


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Any Questions ?

Education

Introduction to spectroscopic methods