SPECTROSCOPY

Spectral Distribution of Radiant Energy

 Wave Number (cycles/cm)

X-Ray UV Visible IR Microwave

200nm 400nm 800nm

WAVELENGTH(nm)

V = Wave Number (cm-1)

Wave Length

C = Velocity of Radiation (constant) = 3 x 1010 cm/sec.

= Frequency of Radiation (cycles/sec)

 

The energy of photon:

h (Planck's constant) = 6.62 x 10-27 (Ergsec)

V =C

E = h = hC

C=

C =

SPECTROSCOPY

DISPERSION OF POLYCHROMATIC LIGHT WITH A PRISM

Polychromatic Ray

Infrared

RedOrange

YellowGreen

Blue

Violet

Ultraviolet

monochromatic Ray

SLITPRISM

Polychromatic Ray Monochromatic Ray

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

SPECTROSCOPY

1. Spectrophotometer - an instrument which can measure the optical density of a sample at any wavelength.

 

Light Lens Slit Monochromator

Sample Detector Quantitative Analysis

Slits

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

Emit fluorescent lightas energy decreases

Ground state

Sample

90C

DetectorUV Light Source

Monochromator Monochromator

Antibonding

Antibonding

Nonbonding

Bonding

BondingEnergy

'

'

'

''

n->n

n->'

Electron's molecular energy levels

Fluorometer

BEER LAMBERT LAW

Glass cell filled with concentration of solution (C)

IILight

0

As the cell thickness increases, the intensity of I (transmitted intensity of light ) decreases.

 

R- Transmittance

R = I0 - original light intensity

I- transmitted light intensity

 

% Transmittance = 100 x

Absorbance (A) or optical density (OD) = 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

I0

I

1 T

I I0

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

E =Absorbance x Liter

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)

E = LiterCm x Mole

L = Cm

C = Moles/Liter

A = KCL

A = No unit C = Gram/Liter L = Cm

A = ECL = (Liter

Cm x Mole) x

Mole

Literx Cm

K=Liter

Cm Gram

A = KLC = (Liter

Cm x GramGramLiter x Cm) x

STEPS IN DEVELOPING A SPECTROPHOTOMETRIC 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 = KCL

Wavelength (nm)

Absorbance

0.0

2.0

200 250 300 350 400 450

SPECTROPHOTOMETR READINGS

ULTRAVIOLET SPECTRUM

Slope of Standard Curve = AC

1 2 3 4 5

1.0

0.5

Concentration (mg/ml)

Absorbance at 280 nm

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

NEVER extrapolate beyond point known where becomes non-linear.

SPECTROMETRIC ANALYSIS USING STANDARD CURVE

1 2 3 4

0.4

0.8

1.2Absorbance 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

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 nmCarbonyl > 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

A 2.0 100 30

Question 6.Question 6.

A = 0.01A = 0.01E = 10000L / mole x cmE = 10000L / mole x cmL = 1cmL = 1cmA = ECLA = ECL

0.01= 10000L/mole X Cm X C (Concentration) x 1Cm0.01= 10000L/mole X Cm X C (Concentration) x 1Cm

C = mole / LiterC = mole / Liter

C = X mole / Liter = X mole (236 g/mole) / Liter (1000 CmC = X mole / Liter = X mole (236 g/mole) / Liter (1000 Cm33) x PPM (10) x PPM (10-6-6 g/Cm g/Cm33))= = XX mole mole (236 (236 gg / / molemole) / ) / LiterLiter x 1 x 1 LiterLiter / 1000 / 1000 CmCm33 x ( PPM) 10 x ( PPM) 10-6-6 gg / / CmCm33))=x PPM=x PPM

PPM = 1ug / PPM = 1ug / CmCm33

1ug = 1ug = 1010-6-6 g g