I express my heartful gratitude to MADAM MANIKA

DAS KOTOKI, HOD and s/t of DEPT. OF BIOTECHNOLOGY,

DARRANG COLLEGE, TEZPUR for giving me an opportunity

to present this presentation on

“SPECTROPHOTOMETRY” and helping me to

collect and organize the presentation data. I sincerely

hope tat the knowledge provided by her will be

beneficial in my future life.

Quantitative measurement of the reflection or transmission properties of a material as a function of wavelength.

A part of BIO-INSTRUMENTATION Involves the use of SPECTROPHOTOMETER

It is a photometer that can measure intensity as a function of the light source wavelength.

It is commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, or gases.

Also designed to measure the diffusivity on any of the listed light ranges that usually cover around 200nm - 2500nm using different controls and calibrations.

A Spectrophotometer

Works upon the following principles-Beer’s LawLambert’s LawBeer-Lambert’s Law

The intensity of a ray of Monochromatic Light passing through an absorbing medium decreases exponentially as the concentration of absorbing medium increases.

I = I e-klc

AUGUST BEER

The intensity of light or a ray of Monochromatic Light that passes through an absorbing medium decreases exponentially as the length of absorbing medium increases.

I = I e-k2l

JOHANN HEINRICH LAMBERT

The Beer–Lambert law, also known as the Lambert–Beer law or the Beer–Lambert–Bouguer law (named after August Beer, Johann Heinrich Lambert, and Pierre Bouguer) relates the absorption of light to the properties of the material through which the light is travelling.

Two laws have been combined together as Beer – Lambert’s Law, which is stated below-

I = I o e -kcl

0.20

Light Source

Collimator(lens)

Monochromator(Prism or Grating)

Wavelengthselector

(slit)

Samplesolution

(in Cuvette)

Detector(Photo Cell)

DigitalDisplayor Meter

Five types of Spectrophotometer according to the range and type of light used-

UV-Vis absorption spectrophotometer (UV-Vis)FT-IR absorption/transmission spectrophotometer (FTIR)Atomic absorption spectrophotometer (AAS)Atomic fluorescence spectrophotometer (AFS)X-ray fluorescence spectrophotometer (XFS)

Two types according to the number of beams used-Single Beam SpectrophotometerDouble Beam Spectrophotometer

Manual Data Input

Computerised DataInput

The use of spectrophotometers spans various scientific fields, such as-

PhysicsMaterials ScienceChemistryBiochemistryMolecular Biology

Also used in industries including semiconductors, laser and optical manufacturing, printing and forensic examination, as well in laboratories for the study of chemical substances.