# SPECTROSCOPY.

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SPECTROSCOPY

Introduction of Spectrometric Analyses
The study how the chemical compound interacts with different wavelenghts 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.

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.

Wave Number (cycles/cm)
Spectral Distribution of Radiant Energy Wave Number (cycles/cm) X-Ray UV Visible IR Microwave 200nm 400nm 800nm Wavelength (nm)

V = Wave Number (cm-1) l = Wave Length C = Velocity of Radiation (constant) = 3 x 1010 cm/sec. u = Frequency of Radiation (cycles/sec) The energy of photon: h (Planck's constant) = 6.62 x (Ergsec) C = u

Spectral Properties, Application and Interactions of Electromagnetic Radiation
Type Radiation Type spectroscopy Type Quantum Transition Energy Wave Number V Wavelength λ Frequency υ Kcal/mol eV cm-1 cm Hz 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 Gamma ray Gamma ray emission Nuclear X-ray absorption, emission Electronic (inner shell) X-ray Ultra violet UV absorption Electronic (outer shell) Visible Infrared IR absorption Molecular vibration Molecular rotation Micro-wave Microwave absorption Magnetically induced spin states Nuclear magnetic resonance Radio

Dispersion of Polymagnetic Light with a Prism
Prism - Spray out the spectrum and choose the certain wavelength (l) that you want by slit.

Ultra Violet Spectrometry
The absorption of ultraviolet radiation by molecules is dependent upon the electronic structure of the molecule. So the ultraviolet spectrum is called electronic spectrum.

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.

Electronic Molecular Energy Levels
The higher energy transitions ( *) occur a shorter wavelength and the low energy transitions (*, n *) occur at longer wavelength.

Chromophore is a functional group which absorbs a characteristic ultraviolet or visible region.
UV 210 nm Double Bonds 233 nm Conjugated Diene 268 nm Conjugated Triene 315 nm Conjugated Tetraene

Spectrophotometer An instrument which can measure the absorbance of a sample at any wavelength.

Fluorometer Instrument to measures the intensity of fluorescent light emitted by a sample exposed to UV light under specific conditions. Emit fluorescent light Antibonding as energy decreases s ' p ' Antibonding n-> s ' n-> p ' n Nonbonding p ->p Ground state ' p Bonding Energy s ->s ' s Bonding Electron's molecular energy levels UV Light Source Detector Monochromator Monochromator 90 C Sample

Food Compound S C H 2 3

Chromophore is a functional group which absorbs a characteristic ultraviolet or visible region.
UV 210 nm Double Bonds 233 nm Conjugated Diene 268 nm Conjugated Triene 315 nm Conjugated Tetraene

Beer – Lambert Law As the cell thickness increases, the transmitted intensity of light of I decreases.

R- Transmittance R = I0 - Original light intensity I- Transmitted light intensity % Transmittance = 100 x Absorbance (A) = Log = Log = 2 - Log%T Log is proportional to C (concentration of solution) and is also proportional to L (length of light path through the solution). I I0 I I0 1 T I0 I I I0

A  CL = ECL by definition and it is called the Beer - Lambert Law.
A = ECL E = Molar Extinction Coefficient ---- Extinction Coefficient of a solution containing 1g molecule of solute per 1 liter of solution

UNITS   A = ECL A = No unit (numerical number only)

L = Cm C = Moles/Liter

Steps in Developing a Spectrometric Analytical Method
Run the sample for spectrum 2. Obtain a monochromatic wavelength for the maximum absorption wavelength. 3. Calculate the concentration of your sample using Beer Lambert Equation: A = ECL 2.0 Absorbance 0.0 200 250 300 350 400 450 Wavelength (nm)

There is some A vs. C where graph is linear.
x nm 1.0 x A at 280 0.5 x 1 2 3 4 5 Concentration (mg/ml) There is some A vs. C where graph is linear. NEVER extrapolate beyond point known where becomes non-linear.

Spectrometric Analysis Using Standard Curve
1.2 0.8 A at 540 nm 0.4 1 2 3 4 Concentration (g/l) glucose Avoid very high or low absorbencies 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

Sample Cells UV Spectrophotometer Quartz (crystalline silica)
Visible Spectrophotometer Glass

Light Sources UV Spectrophotometer 1. Hydrogen Gas Lamp
2. Mercury Lamp Visible Spectrophotometer 1. Tungsten Lamp

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 Structure nm Carbonyl > C = O 280 Azo -N = N Nitro -N=O Thioketone -C =S Nitrite -NO Conjugated Diene -C=C-C=C Conjugated Triene -C=C-C=C-C=C- 268 Conjugated Tetraene -C=C-C=C-C=C-C=C- 315 Benzene

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

Flurometric Application
Thiamin (365 nm, 435 nm) Riboflavin Vitamin A Vitamin C

Visible Spectrometer Application
Niacin Pyridoxine Vitamin B12 Metal Determination (Fe) Fat-quality Determination (TBA) Enzyme Activity (glucose oxidase)

Practice Examples 1. Calculate the Molar Extinction Coefficient E 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 C x 105 M Io I A B 2. The molar extinction coefficient (E) of compound riboflavin is 3 x 103 Liter/Cm x Mole. If the absorbance reading (A) at 350 nm is 0.9 using a cell of 1 Cm, what is the concentration of compound riboflavin in sample?

3. The concentration of compound Y was 2 x 10-4 moles/liter and
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 What is the molar extinction coefficient of compound Y? 4. Calculate the molar extinction coefficient E 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 C x 105 M I0 I A

Spectroscopy Homework
A substance absorbs at 600 nm and 4000 nm. What type of energy transition most likely accounts for each of these absorption processes? 2. Complete the following table.  [X](M) Absorbance Transmittance(%) E(L/mole-cm) L(cm)   2.5 x 4.0 x 2.0 x   [X](M) = Concentration in Mole/L

3. The molar absorptivity of a pigment (molecular weight 300) is 30,000 at 550 nm. What is the absorptivity in L/g-cm.  4. 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?

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