Principles of Spectrometry

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)
l = Wave Length
C = Velocity of Radiation (constant) = 3 x 1010 cm/sec.
u = Frequency of Radiation (cycles/sec)
V = uC
1
=
The energy of photon:
h (Planck's constant) = 6.62 x 10-27 (Erg´sec)
l
E = h = h
C
l
u C
u C = ul
=
l

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

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.
Polychromatic
Ray
Infrared
Red
Orange
Yellow
Green
Blue
Violet
Ultraviolet
monochromatic
Ray
SLIT
PRISM
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 s, p, 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. s *
p*
Energy
n
p
s
s ®s*
p®p*
n ®s*
n ®p*
Antibonding
Antibonding
Nonbonding
Bonding
Bonding

14. Electronic Molecular Energy Levels
The higher energy transitions (s ®s*) occur a
shorter wavelength and the low energy transitions
(p®p*, n ®p*) 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
· ·
· ·
s and s* orbitals p and p* orbitals

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

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

18. Food Compound
H3C 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
· ·
· ·
s and s* orbitals p and p* orbitals

20. Beer – Lambert Law
I I
Glass cell filled with
concentration of solution (C)
Light
0
As the cell thickness increases, the transmitted intensity
of light of I decreases.

21. R- Transmittance
I
I0
R = I0 - Original light intensity
I- Transmitted light intensity
% Transmittance = 100 x
Absorbance (A) = Log
I
I0
I0
I
1
T
= 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

22. A µ 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. E =
Absorbance x Liter
Moles x cm
UNITS
A = ECL
A = No unit (numerical number only)
E =
Liter
Cm x Mole

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

25. Steps in Developing a Spectrometric Analytical Method
1. 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
Wavelength (nm)
Absorbance
2.0
0.0
200 250 300 350 400 450

26. Spectrometer Reading

27. Slope of Standard Curve = D A
DC
x
x
x
1 2 3 4 5
1.0
0.5
Concentration (mg/ml)
A at 280 nm
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 3 4
1.2
0.8
0.4
A at 540 nm
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 105 M 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 10-4 moles/liter and
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 105 M 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?