SPECTROPHOTOMETER

SPECTROSCOPY

1. UV/Vis refers to absorption spectroscopy in the ultraviolet-visible

spectral region.

2. It uses light in the visible and adjacent (near-UV and near-infrared

(NIR)) ranges.

3. Absorption measures transitions from the ground state to the excited

state.

4. Fluorescence deals with transitions from the excited state to the

ground state.

The Beer-Lambert Law

The Absorbance (or optical density) and Transmission (or Transmittance) of

light through a sample can be calculated by measuring light intensity
entering and exiting the sample,

Light energy of Intensity ‘IO ' passes through a sample with

concentration 'C'. Some light energy is absorbed by the sample. The amount
of

light energy exiting the sample has Intensity 'I'

The following terms are defined:

• · Light Intensity entering a sample is " IO “

• · Light Intensity exiting a sample is "I"

• · The concentration of analyte in sample is "C"

• · The length of the light path in glass sample cuvette is "L" .

• · "K" is a constant for a particular solution and wave length.

The Beer-Lambert Law is given by the following equations:

Light Absorbance (A) = log (IO / I)= KCL

Light Transmission (T) = I/ IO = 10-KCL

The Spectrophotometer Instrument

All spectrophotometer instruments designed to measure the absorption of

radiant

energy have the basic components as follows:

1. A stable source of radiant energy (Light): Tungsten, xenon flash lamp,

deuterium

2. A wavelength selector to isolate a desired wavelength from the source

(filter or

Monochromator);

3. A transparent container (cuvette) for the sample and the blank;

4. A radiation detector (phototube) to convert the radiant energy received to

a

measurable signal; and a readout device that displays the signal from the
detector.
 APPLICATIONS
1. Quantitative Determination of Nucleic Acid.

• DNA(DS,SS)

• RNA

2. Quantitative Determination of Protein.

3. Calorimetric Assays:

• Protein Lowry

• Protein Barford

• Protein Pierce.

• Etc.

4. Microbial Assays.

5. Enzyme Assays.

6. Intracellular Ca2+.

7. ATP.

8. Immunology Assays.

9. Reporter Gene Assays.

AMBUJA KU SAHOO.

Ambuja.sahoo@gmail.com

MPHIL(BOT)

MP10BT006