SPECTROSCOPY

Spectral Distribution of Radiant Energy

 
Wave Number (cycles/cm)

X-Ray UV Visible IR Microwave

200nm 400nm 800nm

WAVELENGTH(nm)
 SPECTROSCOPY

V = Wave Number (cm-1)

Wave Length
C = Velocity of Radiation (constant) = 3 x 1010 cm/sec.
= Frequency of Radiation (cycles/sec)
   
V = 
C 

The energy of photon:

h (Planck's constant) = 6.62 x 10-27 (Ergsec)

C C
E = h = h  = C = 
 
 DISPERSION OF POLYCHROMATIC LIGHT WITH A PRISM

Prism - spray out the spectrum and choose the certain wavelength
( that you want by slit.

Infrared
monochromatic
Ray

Red
Orange
Yellow SLIT
Polychromatic PRISM
Green
Ray Blue
Violet

Ultraviolet

Polychromatic Ray Monochromatic Ray

 SPECTROSCOPY

1. Spectrophotometer - an instrument which can

measure the optical density of a sample at any
wavelength.
 
Light Lens Slit Monochromator Slits

Sample Detector Quantitative Analysis

 Fluorometer
2. Fluorometer - measures the intensity of fluorescent light emitted by
a sample exposed to UV light under specific conditions.

Emit fluorescent light ' Antibonding

as energy decreases ' Antibonding

n->' n->'
n Nonbonding
 '
Ground state  Bonding
Energy '
 Bonding

Electron's molecular energy levels

UV Light Source Detector

Monochromator Monochromator
90C

Sample
 BEER LAMBERT LAW

Light
I0 I

Glass cell filled with

concentration of solution (C)

As the cell thickness increases, the intensity of I

(transmitted intensity of light ) decreases.
 
R- Transmittance
I
R= I0 - original light intensity
I0
I- transmitted light intensity
 
I
% Transmittance = 100 x
I0
1
Absorbance (A) or optical density (OD) = 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).
A CL = KCL by definition and it is called the
Beer Lambert Law.
A = KCL
K = Specific Extinction Coefficient ---- 1 g of solute
per liter of solution
 
A = ECL
E = Molar Extinction Coefficient ---- Extinction
Coefficient of a solution containing 1g molecule of
solute per 1 liter of solution
 Absorbance x Liter
E =
Moles x cm

E differs from K (Specific extinction Coefficient) by a

factor of molecular weight.

UNITS
  A = ECL
A = No unit (numerical number only)
Liter
E =
Cm x Mole
 L = Cm
C = Moles/Liter
Liter Mole
A = ECL = ( )x x Cm
Cm x Mole Liter

A = KCL
A = No unit C = Gram/Liter L = Cm
Liter
K=
Cm  Gram

Liter Gram
A = KLC = ( )x x Cm
Cm x Gram Liter
STEPS IN DEVELOPING A SPECTROPHOTOMETRIC
ANALYTICAL METHOD

1. Run the sample for spectrum

Absorbance

2. Obtain a monochromatic
wavelength for the maximum 2.0

absorption wavelength.
3. Calculate the concentration 0.0
350 450
200 250 300 400
of your sample using Beer Wavelength (nm)

Lambert Equation: A = KCL

SPECTROPHOTOMETR READINGS
ULTRAVIOLET SPECTRUM
 A
Slope of Standard Curve =
C
Absorbance at 280 nm

1.0

0.5

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

Absorbance at 540 nm
1.2

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
 CELLS

UV Spectrophotometer
Quartz (crystalline silica)

 Visible Spectrophotometer
Glass

 IR Spectrophotometer
NaCl
 LIGHT SOURCES

 UV Spectrophotometer
1. Hydrogen Gas Lamp
2. Mercury Lamp
Visible Spectrophotometer
1. Tungsten Lamp
IR Spectrophotometer
1. Carborundum (SIC)
 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- 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
UV SPECTROMETER APPLICATION

Protein
Amino Acids (aromatic)
Pantothenic Acid
Glucose Determination
Enzyme Activity (Hexokinase)
FLUOROMETER APPLICATION

Thiamin (365 nm, 435 nm)

Riboflavin
Vitamin A
Vitamin C
VISIBLE SPECTROPHOTOMETER APPLICATION

Niacin
Pyridoxine
Vitamin B12
Metal Determination (Fe)
Fat-quality Determination (TBA)
Enzyme Activity (glucose oxidase)
 EXAMPLES
1. A solution of purified DNA isolated from Escherichia coli gives
an absorbance of 0.793 at 260 M in a 1 Cm cell at pH 4.5. If
E1%1Cm is 197, calculate the concentration of the solution in
milligrams per milliliter.
 
2. 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 93.1 27.4
B 1.90 94.2 32.8
 3. The molar extinction coefficient (E) of compound x 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 x in sample?
 
4. 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?
 
5. 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
Question 6.

A = 0.01
E = 10000L / mole x cm
L = 1cm
A = ECL

0.01= 10000L/mole X Cm X C (Concentration) x 1Cm

C = mole / Liter

C = X mole / Liter = X mole (236 g/mole) / Liter (1000 Cm 3) x PPM (10-6 g/Cm3)
= X mole (236 g / mole) / Liter x 1 Liter / 1000 Cm3 x ( PPM) 10-6 g / Cm3)
=x PPM

PPM = 1ug / Cm3

1ug = 10-6 g