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Spectrophotometer: Principle

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26 views3 pages

Spectrophotometer: Principle

Uploaded by

Sai sowmya botta Sai sowmya botta
Copyright
© © All Rights Reserved
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Spectrophotometer

Introduction
Spectrophotometry is a method to measure how much a chemical substance absorbs light
by measuring the intensity of light as a beam of light passes through sample solution.

Principle
The basic principle is that each compound absorbs or transmits light over a certain range of
wavelength. Spectrophotometry is widely used for quantitative analysis in various areas
(e.g., chemistry, physics, biology, biochemistry, material and chemical engineering, clinical
applications, industrial applications, etc).

A spectrophotometer is an instrument that measures the amount of photons (the intensity


of light) absorbed after it passes through sample solution. Depending on the range of
wavelength of light source, it can be classified into two different types:

• UV-visible spectrophotometer: uses light over the ultraviolet range (185 - 400 nm) and
visible range (400 - 700 nm) of electromagnetic radiation spectrum.
• IR spectrophotometer: uses light over the infrared range (700 - 15000 nm) of
electromagnetic radiation spectrum.
Devices
A spectrophotometer, in general, consists of two devices; a spectrometer and a photometer.
A spectrometer is a device that produces, typically disperses and
measures light. A photometer indicates the photoelectric detector that measures the
intensity of light.
*Spectrometer: It produces a desired range of wavelength of light. First a collimator (lens)
transmits a straight beam of light (photons) that passes through a monochromator (prism)
to split it into several component wavelengths (spectrum). Then a wavelength selector (slit)
transmits only the desired wavelengths

*Photometer: After the desired range of wavelength of light passes through the solution
of a sample in cuvette, the photometer detects the amount of photons that is absorbed and
the n sends a signal to a galvanometer or a digital display.

Mechanism

the amount of photons that goes through the cuvette and into the detector is dependent on the
length of the cuvette and the concentration of the sample. Once you know the intensity of light after
it passes through the cuvette, you can relate it to transmittance (T). Transmittance is the fraction
of light that passes through the sample. This can be calculated using the equation:

Transmittance(T)=It/Io

Where It is the light intensity after the beam of light passes through the cuvette and Io is the light
intensity before the beam of light passes through the cuvette. Transmittance is related to absorption
by the expression:

Absorbance(A)=−log(T)=−log(It/Io)

Where absorbance stands for the amount of photons that is absorbed. With the amount of
absorbance known from the above equation, you can determine the unknown concentration of the
sample by using Beer-Lambert Law.

Beer-Lambert Law
Beer-Lambert Law (also known as Beer's Law) states that there is a linear relationship between the
absorbance and the concentration of a sample.

Beer's Law is written as: A=ϵlc

where

• AA is the measure of absorbance (no units),


2
• ϵϵ is the molar extinction coefficient or molar absorptivity (or absorption coefficient),
• ll is the path length, and
• cc is the concentration.

Applications

Some of the major applications of spectrophotometers include the following:

Detection of concentration of substances


Detection of impurities
Structure elucidation of organic compounds
Monitoring dissolved oxygen content in freshwater and marine ecosystems
Characterization of proteins
Detection of functional groups
Respiratory gas analysis in hospitals
Molecular weight determination of compounds
The visible and UV spectrophotometer may be used to identify classes of compounds in both the
pure state and in biological preparations.

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