Spectroscopy involves using electromagnetic radiation to study chemical compounds and the interactions between radiation wavelengths and compounds. Different regions of the electromagnetic spectrum (e.g. ultraviolet, infrared, nuclear magnetic resonance) cause energy transitions in compounds through absorption. The energy and wavelength of absorbed radiation is directly related and allows compounds to be identified based on their unique absorption spectra. Spectrometers and fluorometers are instruments used to measure absorption and fluorescent emission spectra, revealing information about molecular structure and properties.

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. 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
λ = Wave Length
C = Velocity of Radiation (constant) = 3 x 1010 cm/sec.
υ = Frequency of Radiation (cycles/sec)
υ 1
V = =
C λ
The energy of photon:
h (Planck's constant) = 6.62 x 10- (Erg×sec)
27
C C
E = h υh
= υ= C = υλ
λ λ

7. Spectral Properties, Application and Interactions of
Electromagnetic Radiation
Wave Wavelength Frequency
Energy Number V λ υ
Type Type Type
Radiation spectroscopy Quantum Transition
Kcal/mol eV cm-1 cm Hz
9.4 x 107 4.9 x 106 3.3 x 1010 3 x 10-11 1021 Gamma Gamma ray
ray Nuclear
emission
X-ray Electronic
9.4 x 103 4.9 x 102 3.3 x 106 3 x 10-7 1017 X-ray
absorption, (inner shell)
emission
Ultra
9.4 x 101 4.9 x 100 3.3 x 104 3 x 10-5 1015 violet UV absorption Electronic
Visible (outer shell)
9.4 x 10-1 4.9 x 10-2 3.3 x 102 3 x 10-3 1013 Infrared IR absorption Molecular
vibration Molecular
rotation
9.4 x 10-3 4.9 x 10-4 3.3 x 100 3 x 10-1 1011 Micro- Microwave
wave absorption
Magnetically
Nuclear induced spin
Radio magnetic
9.4 x 10-7 4.9 x 10-8 3.3 x 10-4 3 x 103 107 states
resonance

9. Spectrum of Radiation

10. Dispersion of Polymagnetic Light with a Prism
Prism - Spray out the spectrum and choose the certain wavelength
(λ) that you want by slit.
Infrared
monochromatic
Ray
Red
Orange
Yellow SLIT
Polychromatic PRISM
Green
Ray Blue
Violet
Ultraviolet
Polychromatic Ray Monochromatic Ray

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 σ, π, 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.

13. Antibonding
σ*
π
* Antibonding
σ σ
π
n→
σ
→
ππ
*
→
*
n→*
*
Energy
n Nonbonding
Bonding
π
Bonding
σ

14. Electronic Molecular Energy Levels
The higher energy transitions (σ →σ*) occur a
shorter wavelength and the low energy transitions
(π→π*, n →π*) 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
• •
• •
σ σ
and * orbitals π π
and * orbitals

16. Spectrophotometer
An instrument which can measure the absorbance of a
sample at any wavelength.
Light Lens Slit Monochromator Slits
Sample Detector Quantitative Analysis

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 σ'
π' Antibonding
n->σ n-> '
' π
n Nonbonding
Ground state π −>π '
π Bonding
Energy σ −>σ
'
σ Bonding
Electron's molecular energy levels
UV Light Source Detector
Monochromator Monochromator
90°C
Sample

18. Food Compound
H 3C S CH2 CH2 CH3

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
• •
• •
σ σ
and * orbitals π π
and * orbitals

20. Beer – Lambert Law
Light
I0 I
Glass cell filled with
concentration of solution (C)
As the cell thickness increases, the transmitted intensity
of light of I decreases.

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

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

23. Absorbance x Liter
E =
Moles x cm
UNITS
A = ECL
A = No unit (numerical number only)
Liter
E =
Cm x M ole

24. L = Cm
C = Moles/Liter
Liter Mole
A = ECL = ( )x x Cm
Cm x Mole Liter

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

26. Spectrometer Reading

27. ∆
A
Slope of Standard Curve =
∆
C
x
A at 280 nm
1.0
x
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
A at 540 nm
0.8
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- 262
Nitro -N=O 270
Thioketone -C =S 330
Nitrite -NO2 230
Conjugated Diene -C=C-C=C- 233
Conjugated Triene -C=C-C=C-C=C- 268
Conjugated Tetraene -C=C-C=C-C=C-C=C- 315
Benzene 261

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 10 M
5
Io I
A 2.23 100 27
B 1.90 100 32
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 moles/liter and
-4
the absorption of the solution at 300 nm using 1 Cm quartz cell
was 0.4. 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 10 M 5
I0 I
A 2.0 100 30

38. Spectroscopy Homework
1. 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)
30 2000 1.00
0.5 2500 1.00
2.5 x 10-3 0.2 1.00
4.0 x 10-5 50 5000
2.0 x 10-4 150
[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?