Spectroscopy: Spectral Distribution of Radiant Energy

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

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SPECTROSCOPY

Spectral Distribution of Radiant Energy

X-Ray UV Visible IR Microwave

200nm 400nm 800nm

V = Wave Number (cm-1)

The energy of photon:

Polychromatic Ray Monochromatic Ray

1. Spectrophotometer - an instrument which can

Sample Detector Quantitative Analysis

Emit fluorescent light ' Antibonding

as energy decreases ' Antibonding

Electron's molecular energy levels

UV Light Source Detector

Glass cell filled with

As the cell thickness increases, the intensity of I

E differs from K (Specific extinction Coefficient) by a

1. Run the sample for spectrum

Lambert Equation: A = KCL

There is some A vs. C where graph is linear.

Avoid very high or low absorbencies when drawing a standard

Chromophoric Group ---- The groupings of the

Thiamin (365 nm, 435 nm)

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

PPM = 1ug / Cm3

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