1. SPECTROSCOPY

2. 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.

3. 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.

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

5. Electromagnetic Radiation
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

7. 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

9. Spectrum of Radiation

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

11. 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.

12. 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.

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

15. 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

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

17. 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

18. Food Compound S C H 2 3

19. 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

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

21. 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

22. 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

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

24. L = Cm
C = Moles/Liter

25. 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)

26. Spectrometer Reading

27. 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.

28. 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

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

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

31. 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.

32. 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

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

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

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

36. 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?

37. 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

38. 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

39. 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?