Spectrophotometer helps in the measurement of how light interacts with materials

1. SPECTROPHOTOMETER
(COLORIMETER)

2. COLORIMETER/ SPECTROPHOTOMETER
• measurement of how light interacts with materials
• spectrophotometer, a device that measures the intensity of light as a
beam of light passes through a sample solution.
• Light can be reflected, scattered, transmitted, and absorbed.
• The key difference between colorimetry and spectrophotometry is that
the colorimetry uses fixed wavelengths that are only in the visible range
while spectrophotometry can use wavelengths in a wider range.

3. Laws pertaining to COLORIMETRY
• Lambert’s Law: If a beam of monochromatic light is allowed to pass
through a homogeneous absorbing medium, each layer of medium
decreases the intensity of light by a constant fraction. 1729
• Beer’s Law: Absorption of radiant energy is proportional to the total
number of molecules in the light path. 1852

4. Principle
• If a beam of light is allowed to pass through a solution, a part of the
radiation gets reflected by the walls of the container and the solution,
a part gets absorbed and the rest gets transmitted.
• According to beer- Lambert’s Law, “the absorbance of a
homogeneous medium is equal to the product of the absorptivity,
optical path length and the concentration of the medium”.
• Hence the mathematical expression of Beer-Lambert Law is
described.

5. Beer-Lambert’s Law
•Mathematical expression of Beer-Lambert Law is described as
•A= Kcd
•Where A= Absorbance(extinction co-efficient} of the medium
•K- absorptivity(depends upon the nature of the substance)-
constant for a substance.
•d = thickness of absorbing medium(cm)
•c = concentration of substance(gr/litre) or(mole/litre)
•When d is 1 cm and c is in mole/litre then the constant is
termed as molar extinction co-efficient (E%/1 cm)

6. spectrophotometer
• A spectrophotometer is a refined version of a colorimeter.
• It functions the same way as a colorimeter but with added features. A colorimeter uses
a filter which enables a broad range of wave lengths to pass through.
• The light source can be visible 400-700 UV 200-400 IR 700-2000 nm.
• 1. The light is made parallel by passing through a collimating lens/mirror before it is
passed to the monochromator.
• 2. Monochromator unit is made up of a rotating prism and a filter with a slit.
• In spectrophotometer :
• a prism or grating is used for the incident beam to split into different wavelengths.
• Filter permits the waves of the particular wavelengths adjusted to fall on the test
solution.

7. Components of a spectrophotometer

8. Spectrophotometer:
• measurement of how light interacts with materials
• spectrophotometer, a device that measures the intensity of light
as a beam of light passes through a sample solution.
• Light can be reflected, scattered, transmitted, and absorbed.
• The key difference between colorimetry and spectrophotometry is
that the colorimetry uses fixed wavelengths that are only in the
visible range while spectrophotometry can use wavelengths in a
wider range.

10. USE OF THE SPECTRONIC 20 SPECTROPHOTOMETER
• The on/off knob (at the left on the front of the instrument);
• 2. The "light control" knob (at the right on the front of the instrument);
• this knob is rotated to set the reading to Absorbance = zero (l00 % transmittance)
when a reference solution is in the light path.
• 3. The cuvette holder (at the left on the horizontal table of the instrument);
• a cuvette is a glass tube of known diameter that will be used to insert samples into the
instrument;
• there is a cover on the cuvette holder that MUST be closed during all readings and
adjustments of the instrument, to prevent entry of light from the room into the
photometer.
• 4. The wavelength control knob and its scale (at the right on the horizontal table of the
instrument); rotating the knob rotates the prism of the spectrometer so that light of
the wavelength indicated on the scale passes through the cuvette.
• 5. The meter (on the upper face of the instrument); the upper scale displays
%Transmittance, and the lower scale displays Absorbance; the light control knob (see 2)
controls the movement of the needle across the meter when a cuvette is in place.

11. Operation
• l. Turn on the Spectronic 20 and allow l5 min. for the instrument to warm up.
• 2. Set the dark control (light control knob) so that the needle reads 0 % transmittance.
• 3. Set the wavelength with the wavelength control knob.
• 4. Prepare a cuvette containing a liquid that contains all the components of the sample solution
• except the substance whose absorbance is to be measured. This is the reference cuvette or
• blank.
• Place the reference cuvette in the sample holder (close the cover) and use the light
• control to adjust the needle to read 100 % transmittance.
• 5. Replace the reference cuvette with a sample cuvette containing the sample solution (close the
• cover). Allow the needle to come to rest and read the Absorbance. (Note that the
• Absorbance scale reads from right to left.)
• 6. To read another sample at the same wavelength, replace the first sample cuvette with the next
• one.
• 7. To read the same sample at another wavelength, reset the wavelength, insert the reference
• cuvette, adjust to l00 % transmittance with the light control knob, then insert the sample and
• read Absorbance.
• The instrument must be reset with the reference cuvette for each wavelength

12. Spectrophotometer
• The instrument has a sample holder which is held between the
monochromator unit and the detector.
• Sample holders are made up of glass or fused silica
• Eg. Wall thickness 1mm, Internal diameter 1cm.
• The detector is a photosensitive device. It can be a meter to read
transmittance (light passing through the sample).

13. In biochemical research
• 1. If the absorbency index at a specific wave length is known, the
concentration of the compound (even in microgram) can be
determined by measuring Optical Density(OD) at that wave length.
• 2. Course of reaction can be measured by measuring the rate of
formation or disappearance of a light absorbing substance.
• 3. Compounds can be identified by determining the characteristic
absorbing spectrum in the UV and visible regions of the spectrum.

14. Deviation from Beer Lambert Law
• Incident light may not appear in the transmitted form for other reasons
besides absorption.
• A certain amount of radiation will be reflected from the surface of the
sample holder or absorbed by the material of the cell.
• The solvent used may also absorb or reflect radiation.
• So, Incident radiation( Io)= Absorbed+ Transmitted+ other loses.
• Hence radiation transmitted by a blank sample is measured(considered as
effective incident radiation).
• The Blank is identical to the test sample, but does not contain the test
substance.
• Hence, Blank Reading= Io – other loses.
• So Absorbance= Blank- Transmission.

15. Beer Lambert Law expresses the linear relationship between
concentration of the sample and the absorbance value recorded.