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Basic Principles of Colorimetry

The document discusses the principles and applications of photometry and spectrophotometry, focusing on their roles in quantitative and qualitative analysis in biochemistry. It explains the functioning of colorimeters and spectrophotometers, detailing their components, measurement principles, and the laws governing absorbance. Key concepts include Beer’s Law and Lambert’s Law, which relate light absorption to concentration and path length, respectively.

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13 views

Basic Principles of Colorimetry

The document discusses the principles and applications of photometry and spectrophotometry, focusing on their roles in quantitative and qualitative analysis in biochemistry. It explains the functioning of colorimeters and spectrophotometers, detailing their components, measurement principles, and the laws governing absorbance. Key concepts include Beer’s Law and Lambert’s Law, which relate light absorption to concentration and path length, respectively.

Uploaded by

vbunny2004v
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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Photometry and Spectrophotometry

Competency: BI 11.18 Discuss the principles of Spectrophotometry


BI 11.19 Outline the basic principles involved in the functioning of instruments
commonly used in a biochemistry laboratory and their applications

Spectrophotometric analysis is the widely used method of quantitative and qualitative


analysis in the laboratory.

Photometry is defined as the measurement of light; Spectrophotometry is defined as


the measurement of the intensity of light at selected wavelengths.

The method depends on the light absorbing properties of the substance or its
derivative of the substance being analyzed.

The intensity of the transmitted light passing through the solution containing an
absorbing substance (chromogen) is decreased by the absorbed fraction. The fraction
is detected, measured and used to relate the light transmitted or absorbed to the
concentration of analyte in question.

The instruments used to read absorbance:

1. Colorimeter ( Filter Photometer or Filter Absorptiion spectrometer)


2. Spectrophotometer (Absorption Spectrometer)

Colorimeter Spectrophotometer

Absorbance can be measured only Absorbance can be measured at Specific


within certain Wavelength ranges with wavelengths using a Diffraction
Filters being used to obtain the required grating Prism
wavelength.
Filters employed correspond to Illford
spectrum filters with wavelength band
width of 40 nm.
More expensive compared to colorimeter
and requires greater technical skill.
Also reduce interference from unwanted
chromogens unlike that in colorimeters.
Basic Principles of Colorimetry

A Colorimeter is used to measure the concentration of a substance in a


patient’s sample by comparing the amount of light it absorbs with that absorbed by a
standard preparation that contains a known amount of substance being tested. The
absorbance of light occurs at given wavelengths in the visible spectrum (360 to 700
nm).

When a beam of monochromatic light is passed through a colored solution,


some amount of light is absorbed and the rest is transmitted. The concentration of
the substance in the solution is directly proportional to the amount of light absorbed
and inversely proportional to the logarithm of transmitted light. The colorimeter
function is based on the principles of Beer’s Law and Lambert’s Law.

Beers’ Law states that the amount of light absorbed, Absorbance, is directly
proportional to concentration of the substance (analyte) (1)

Lambert’s Law states that the amount of light absorbed is proportional to the path
length of the solution, l. (2)

AαC 1

Aαl 2

Combining equations (1) and (2)

AαCXl

A = kCl

K, is constant called as Absorptivity

For a given colorimeter, path length is constant

Comparing an unknown with a known concentration:

A (unknown) C (unknown)
------------------ = -----------------
A (Standard) C (Standard)
A (unknown)
Concentration of unknown = ---------------- X C (standard)
A (standard)

Application of Beer’s Law:

A calibration relationship between absorbance and concentration can be established


for a given instrument and under specified conditions using a series of reference
solutions with increasing concentrations of the analyte.

Frequently, a linear relationship exists up to a certain concentration or absorbance.

When this linear relationship exists, the solution is said to obey Beer’s law.

SPECTROPHOTOMETER

The basic components of spectrophotometer include:

1. A light source
2. A device to isolate light of a desired wavelength
3. A cuvette
4. A photodetector
5. A readout device
6. A data system

Light sources:

Types of light sources used in spectrophotometers include

a) Incandescent lamps: used to produce visible portion of the spectrum.


b) Xenon discharge lamps: used to provide continous spectra in the UV region.
c) Light emitting diodes
d) Lasers: provide intense light of a narrow wavelength.
Spectral Isolation:

Radiant energy of desired wavelength can be isolated using

a) Filters ( interference or dichromatic filters)


b) Prisms
c) Diffraction gratings

Combination of lenses or slits may be inserted before or after the monochromatic


device to render light rays parallel or to isolate narrow portions of the light beam.

Filters are the simplest type made of a thin layer of colored glass. The spectral purity
is given in terms of its spectral bandwidth.

A prism separates white light into a continuous spectrum.

Diffraction gratings are prepared by depositing a thin layer of aluminum copper alloy
on the surface of a thin glass plate and then ruling many parallel grooves over it.

Cuvette

Most commonly used cuvettes have a 1.0 cm light path and are made of borosilicate
glass or plastic for visible region.

For readings below 340 nm, quartz cuvettes are required. Cuvettes must be clean and
optically clear because etching or deposits on the surface affect absorbance values.

Photodetectors

A photodetector is a device that converts a light into an electric signal that is


proportional to the number of photons striking its surface.

Ex: Photomultiplier tube


Photodiodes

A photomultiplier tube is most commonly used for measuring light intensity both in
UV region and the visible region.

Read out devices: Digital read out devices provide visual numeric display of
absorbance or converted values of the concentration

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