INTRODUCTION TO SPECTRAL METHODS OF ANALYSIS

Maam Glenn Medina De La Salle University

Absorption and emission of radiation by molecules

An isolated molecule possesses a certain amount of energy, exclusive of that associated with its motion

Absorption and emission of radiation by molecules

Most of this energy is in the form of kinetic energy and electrostatic potential energy of electrons in the nuclei Forms
vibration of the individual atoms about their mean positions in the molecule rotation of the molecule about its center of mass

Molecular Transitions
Absorption of electromagnetic (EM) radiaton: Transition to a higher energy level
Emission of EM radiation: Transition to a lower energy level

Absorption and emission of radiation by molecules

Absorption lines associated with

orbital changes
X-ray, ultraviolet (UV), and visible radiation

vibrational changes
Infrared (IR) radiation

rotational changes
Microwave radiation

Sample Problems
1. When O2 absorbs UV radiation in the wavelength 147 nm, by how many kJ per mole is the Energy increased?
2. How much is the energy of CO2 increased when it absorbs IR radiation with a wavenumber 2300 cm-?
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Answers to Sample Problems

1.814 kJ/mol 2.28 kJ/mol

The Line Spectrum

An isolated molecule can only absorb and emit energy in discrete amounts corresponding to the allowable changes in its energy level
It can only interact with radiation having certain discrete wavelengths Thus the absorption and emission properties of an isolated molecule can be described in terms of a line spectrum

The Line Spectrum

Line spectrum consists of a finite number of extremely narrow absorption or emission lines, separated by gaps in which the absorption and emission of radiation are not possible

Petty 2004

Continuous Spectrum
Possible for molecules [solid/liquid] Strong interaction between the force fields of individual molecules Absorption and emission take place throughout a continuous spectrum of wavelengths

Spectrophotometer

The monochromatic light, with irradiance Po, strikes the sample of length b. Some of the Po may be absorbed by the sample. Thus, P Po

T- Transmittance

T=

P P0

P0 - Original light intensity

P- Transmitted light intensity [Irradiance] P P0 1 T Irradiance energy per second per unit area of light beam

% Transmittance = 100 x

Absorbance (A) = Log

= Log Log

P0 P

P is proportional to c and b (length of light path) P0

Beer-Lambert law
For molecules that absorb ultraviolet or visible light, the absorbance of a solution increases as attenuation of the beam increases. Beer's Law states that

A = abc
where a is a constant of proportionality, called the absorptivity.

When c is expressed as Molarity, then a becomes e, which is the molar absorptivity.

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Sample Problem
1. Find the absorbance and transmittance of a 0.0002040 M solution with a molar absorptivity of 313 M-cm- in a cell with 2.0 cm path length

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Spectroscopic Instruments
1. 2. 3. Spectroscope an instrument that is used to measure the amount of radiation absorbed or emitted by matter Spectrograph consists of a device that disperses the wavelength so that multichannel detection is possible Spectrometer a spectroscopic instrument that uses a monochromator or polychromator with a transducer to convert radiant intensities into electrical signals
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A very simple spectrometer based on a prism

Spectroscopic Instruments
4. Spectrophotometer a spectrometer that allows measurement of the ratio of the radiant powers of two beams to give the absorbance. The wavelength can be varied continuously, making it possible to obtain an absorption spectrum Photometer uses a filter for wavelength selection in conjunction with a suitable radiation transducer. It is simpler and less expensive than spectrophotometers. It is also often used as detectors for HPLC, FIA, Electrophoresis and others
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5.

Single-Beam Spectrophotometer

Single-Beam Spectrophotometer

Double-Beam Spectrophotometer

Baseline Spectrum

Steps in Developing a Spectrometric Analytical Method

1. Run the sample for spectrum

Absorbance

2. Obtain a monochromatic wavelength for the maximum absorption wavelength.

2.0

3. Calculate the concentration of your sample using Beer Lambert Equation: A = abc

0.0 200 250 300 350 400 450

Wavelength (nm)

Slope of Standard Curve =

A C

1.0
x

0.5

4 2 3 Concentration (mg/ml)

There is some A vs. C where graph is linear.

NEVER extrapolate beyond point known where plot becomes non-linear.

Spectrometric Analysis Using Standard Curve

1.2

0.8

0.4

3 1 2 Concentration (g/l) glucose

Avoid very high or low absorbance when drawing a standard curve. The best results are obtained with 0.1 < A < 1. Plot the Absorbance vs. Concentration to get a straight line

CUVETS/ SAMPLE CELLS

UV-Vis fused silica (SiO2)
Vis Glass

IR NaCl or KBr plates

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Light Sources
UV Spectrophotometer 1. Hydrogen Gas Lamp

2.

Mercury Lamp

Visible Spectrophotometer 1. Tungsten Lamp

Graph showing how absorbance varies with wavelength

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BEERS LAW
Applies to monochromatic radiation
Works well for dilute solutions (0.01 M)

In concentrated solutions, e changes

*Concentration-dependent chemical equilibrium (e varies with concentration)
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Choosing the lmax

1. Maximum sensitivity at maximum absorbance 2. Little variation in the absorbance
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Absorbance
1. Ideal range: 0.4 0.9

2. Abs, light passing thru, P hard to measure

3. Abs, light passing thru, hard to distinguish between S and Ref
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Sample Problems
1. Pure hexane has negligible UV absorbance above l = 200 nm. A solution prepared by dissolving 25.8 mg benzene (78.11) in hexane and diluting to 250.0 mL had an absorption peak at 256 nm and an absorbance of 0.266 in a 1.000-cm cell. Find the molar absorptivity of benzene. A sample of hexane contaminated with benzene had an absorbance of 0.070 at 256 nm in a cuvet with a 5.000-cm path length. Find the concentration of benzene in mg/L.
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2.

Answers
1. 201 M-cm-

2. 5.4 mg/L

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Chemical Structure & UV Absorption

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

Chromophoric Structure
Group
Carbonyl

Structure
>C=O

nm
280

Azo
Nitro Thioketone Nitrite Conjugated Diene Conjugated Triene Conjugated Tetraene Benzene

-N = N-N=O -C =S -NO2 -C=C-C=C-C=C-C=C-C=C-C=C-C=C-C=C-C=C-

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270 330 230 233 268 315 261

UV Spectrometer Application
Protein Amino Acids (aromatic) Pantothenic Acid Glucose Determination Enzyme Activity (Hexokinase)

Visible Spectrometer Application

Niacin
Pyridoxine Vitamin B12 Metal Determination (Fe) Fat-quality Determination (TBA)

Enzyme Activity (glucose oxidase)

Assignment
1. Calculate the Molar Extinction Coefficient at 351 nm for aquocobalamin in 0.1 M phosphate buffer. pH = 7.0 from the following data which were obtained in 1 cm cell. Solution A C (M) 2.23 x 10-5 Po 100 P 27

1.90 x 10-5

100

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2. The molar extinction coefficient of compound riboflavin is 3 x 103 L/cm mole. If the absorbance reading at 350 nm is 0.90 using a cell of 1.00 cm, what is the concentration of riboflavin in the sample?

3. The concentration of compound Y was 2 x 10-4 moles/liter and the absorption of the solution at 300 nm using 1 cm quartz cell was 0.40. What is the molar extinction coefficient of compound Y?
4. Calculate the molar extinction coefficient at 351 nm for aquocobalamin in 0.1 M phosphate buffer pH =7.0 from the following data which were obtained in 1 cm cell. Solution A C (M) 2.0 x 10-5 Po 100 P 30

5. A substance absorbs at 600 nm and 4000 nm. What type of energy transition most likely accounts for each of these absorption processes? 6. Complete the following table.

[X](M)
2.5 x 10-3 4.0 x 10-5 2.0 x 10-4

Absorbance Transmittance(%) e(L/mole-cm)

L(cm)

0.5 0.2

30

2000 2500

50

5000 150

1.00 1.00 1.00

7. The molar absorptivity of a pigment (molecular weight 300) is 30,000 at 550 nm. What is the absorptivity in L/g-cm? 8. The iron complex of o-phenanthroline (Molecular weight 236) has molar absorptivity of 10,000 at 525 nm. If the absorbance of 0.01 is the lowest detectable signal, what concentration in part per million can be detected in a 1-cm cell?

9. Describe (and differentiate) the following terms:

a. Fluorescence b. Phosphorescence c. Luminescence