A colorimeter is used for quantitative analysis of solution on basis of colour intensity.
In a photoelectric colorimeter (or filter photometer), the photoelectric cell is used to measure the intensity of transmitted light. A narrow band of wavelength is obtained by passing visible light through a colour filter & is not strictly monochromatic, which is used as incident radiation.
Photoelectric colorimeter, in general consists of the following components.
i. Source of visible radiation (s): The wavelength of the range of visible light lies between 400-and 750nm. i) In this region, tungsten filament lamp is the most widely used Mercury vapors lamp or hydrogen discharge lamps are also used. The beam of radiation is collimated by a lens (L).
ii. Optical filters: A filter is used to select a narrow band of wavelength to an incident on the sample. Filters are of two types.
a. A glass filter, that consists of the thin filament of gelatin containing different dyes or coloured glass.
b. Interference filters consist of thin sheets of clear dielectric material enclosed within two clean glass sheets.
iii. Sample holder or Cuvette: The solution to be studied absorptiometrically is kept in the sample cell or
holder or cuvette. These cells are made up of glass.
iv. Detector: The transmitted beam is focused on photoemissive cells or photomultiplier tubes or
photovoltaic cells, which act as a detector. The detector converts the transmitted beam emerging from the sample into an electric current.
v. Recorder: Output of detector is displayed as optical density or percentage transmittance. Display
maybe shown on a dial or in digital form.
Types of Photoelectric colorimeters
Photoelectric colorimeters are of two types
- Single beam photoelectric colorimeter
- Double Beam photoelectric colorimeter
Let us learn about Double Beam photoelectric colorimeter
Principle and working of Single-beam photoelectric colorimeter
The beam of light after passing through the lens and filter split into two beams; one passes through the blank and the other through the sample solution. The detector measures the difference in the absorption of light by the two and so gives the optical density of solute directly.
Arrangement and function of components:
i. Light from sources (such as incandescent tungsten lamps), passes through a collimating lens.
ii. Light is then incident on properly selected filters, which allow only a narrow band of
wavelength to pass through.
iii. Light beam emerging from the filter incident on the mirror which split the beam into two.
iv. One beam of light passes through the cuvette containing solvent (or blank) and then to
the photovoltaic cell.
v. The Second beam of light passes through the sample solution and then to another photovoltaic cell.
vi. Photocell produces electric current proportional to transmitted light.
vii. Two electric currents thus produced pass through variable resistance AB and CD.
viii. Depending upon the magnitude of the currents, a potential gradient is set up across AB and CD
ix. A sensitive galvanometer connected across the resistances serves as a null indicator. A null
indicator in the galvanometer indicates that the potential at the two contact points on AB and CD are the same.
Advantages of Double beam colorimeter over single beam colorimeter
- Any change in intensity of incident light due to voltage fluctuation is observed equally by both cells, are canceled out.
- It is not necessary to replace blank with sample or zero adjustments at each wavelength.
- Errors due to solvent or impurities present in the solvent get canceled.
- Changes in sensitivity of photocell do not affect reading since the null method is used.
What is a spectrophotometer?
A spectrometer consists of two units, a spectrometer & photometer. The spectrometer is a device producing light of the desired wavelength in the visible & ultraviolet Region of the spectrum. The photometer is a device for measuring the intensity of light.
Spectrophotometers allow a continuous choice of wavelength range covered in the spectrometer from 200-800nm. It employs prisms of grating, which make light perfectly monochromatic. Spectrophotometer, in general consists of the following components.
- Source of radiation : The source of light for ultraviolet region(i.e200-400nm) is hydrogen or deuterium discharge lamp & tungsten lamp for the visible region(i.e 400-750nm).
- Monochromator: The essential elements of monochromatic are an entrance slit, a dispersing element (prism or grating) & an exit slit. Polychromatic radiation after passing through a prism or grating gets converted into a monochromatic beam of the desired wavelength.
They are of two types
These contain a prism as the dispersing element. The function of the prism is to resolve radiation from the source into its component wavelength & to provide for the isolation of radiation of very narrow bandwidth. Radiation from the source is focused on the entrance slit & then collimated by a collimating lens. The collimated beam falls on the prism, which can be mechanically rotated so as to allow only a particular portion of the spectrum, produced by the prism to fall on a focusing lens & emerge from the exit slit. The exit slit width control bandwidth of emergent radiation, since dispersion is greater for shorter wavelengths & decreased with a longer wavelength.
For the visible region, prism & lenses made of glass are used & for the ultraviolet region, quartz prism is used.
The function of the diffraction grating is to get a monochromatic beam of a smaller bandwidth. The grating monochromators are prepared by drawing closely placed equidistant parallel lines on the glass surface or on a metallic surface. In reflection grating, light radiation incident on each groove is diffracted out over a range of angles & in certain direction reinforcement & interference occur. This can be represented by using the grating formula. n=b(Sin i + Sin r)
Where “b” is the distance between adjacent grooves & “i” is the angle of incident, “r” is the angles of diffraction & “n” is an order of the spectrum. The above-mentioned equation can only be satisfied for a single wavelength at a time.
Rotation of grating to change the angle of incidence (i) will bring each wavelength in turn to position to satisfy the equation. As a result of this, a monochromatic beam is obtained.
Grating monochromators have certain distinct advantages over prisms.
- A grating gives a much better dispersion of light than a prism.
- Grating is made of non-corrosive materials like aluminum, which are not easily attacked by moisture.
- The grating can be used over a longer wavelength range as compared to prisms.
Sample holder or cuvette
The solution to be studied absorptiometrically is kept in a sample cell holder or cuvette. These cells are made up of glass for the visible region & are made up of quartz for ultraviolet region study since glass absorbs ultraviolet light.
4. Detector: The transmitted beam is focused on photoemissive cells or photomultiplier tubes or photovoltaic cells, which act as a detector. The detector converts the transmitted beam emerging from the sample into an electrical current
5. Recorder: Output of detector is displayed as optical density or percentage transmittance. Display may be shown on the dial or in digital form.
Spectrophotometers are of two types:
A). Single beam Spectrophotometers. B). Double beam Spectrophotometers.
Please also read….Gas Chromatography
Single beam Spectrophotometer
Principle & working of the spectrometer
Principle:- A polychromatic beam of light from its source is converted into a monochromatic beam of light by using a monochromator, incident on absorbing sample, part of it gets absorbed & rest of it transmitted which is converted into electrical pulses and it is measured as O.D or %T. The same optical path is used for all measurements.
Arrangement & function of components:
i. Light from source ( R ) (such as incandescent tungsten lamp or hydrogen discharge tube) is focused by field lens to an entrance slit and is incident on the objective lens.
ii. Parallel beam of light then focuses on a grating monochromator ( M ) where after reflection produces an interference pattern which results in the dispersion of light into its component wavelength.
iii. To obtain the desired wavelength, the grating can be rotated by means of wavelength cam, thus monochromatic beam of light emerges from the exit slit.
iv. These monochromatic beams of light then passes through cuvette (c) containing solvent or sample solution.
v. The transmitted light fall on a photoelectric cell (P), which converts radiant energy into an electric signal that can be read on a meter calibrated in 0-100% transmittance and absorbance scales.
The drawback of spectrometer:-
Reading fluctuates with variation in source intensity.
Double beam Spectrophotometer
Principle and working of Double beam Spectrophotometer
The beam of light after passing through the lens & grating split into 2 beams; one passes through the sample solution. The detector measures the difference in the absorption of light by the two & so gives the optical density of solute directly.
Arrangement & function of components:
- The light from the source (tungsten lamp for visible region 400 to 800 nm & Hydrogen discharge or deuterium lamp for U.V region 200 to 400 nm) passes through a collimating lens.
- A beam of light falls on the Monochromator, which can be rotated to obtain the desired wavelength of light.
- Mirrors are used to split the light beam into two beams of light.
- One beam of light passes through the sample cell and the other through the blank cell.
- An optical attenuator mounted in the path of reference or blank beam reduces its intensity to that of the sample beam i.e. light transmitted from the sample
- The attenuator is connected to a recorder pen which more across the chart paper wound on a rotating drum
Application of absorption spectrophotometer
a. Absorption spectrum recorded on a spectrophotometer can furnish important information about the chemical constitution of the compound. E.g. Absorption at 254 nm shows the presence of an aromatic ring in the compound.
b. In quantitative analysis, it is used for the estimation of the number of metal ions like Fe & Mn at ppm-level. It is also used for the determination of fluoride ion, chloride ion & phosphate ions.
c. It is commonly used for the estimation of drugs and vitamins. E.g. vitamins B12 are estimated by the spectrophotometer method.
d. Spectrophotometer is effectively used in water and air pollution studies
e. It is used in the determination of the dissociation constant of organic acids & bases.
f. Elucidation of Organic Compound’s structure:
Spectro photometry is of much use in elucidating the structure of organic molecules. A plot of absorbance versus wavelength is called absorption spectrum. From the absorption spectra in ultraviolet radiation, qualitative information about the functional group, such as carbonyl, nitro & amino can be obtained. This information is used to identify organic compounds.
According to Hartely, compounds of similar structures have analogous absorption spectra. E.g. for tyrosine, two structures have been proposed.
The absorption spectrum of tyrosine was found to be similar to phenol, confirming structure (2) for the amino acid tyrosine.
g. Structure of inorganic complexes: Spectrophotometry has been used in the study of complexes.
i) Geometrical Isomerism: The geometrical isomers of the transition complex can be easily distinguished by these techniques. For instance, the cis isomers of [Co(en)2F2]NO3 are violet whereas the trans isomers are green and trans-cinnamic acid has λmax 272 nm while cis-cinnamic acid has λmax 268 nm.
ii) Octahedral-Planner Equilibrium: The change in colour of cis-[Ni(triethylenediamine)(H2O2)]+2 from blue to yellow on adding inert salt to the solutions has been shown from absorption spectra by Jorgensen to involve an octahedral planner equilibrium.
h. Job’s method to determine the metal-ligand ratio in complexes
The molar ratio of metal to the ligand in a coordination complex can be found using Job’s method of continuous variation.
Analytical application of colorimetry & spectrophotometry
(i) Quantitative analysis:
The determination of the amount of substance in a solution by a colorimeter or spectrophotometer usually involves.
- Construction of standard curve or calibration curve.
- Multiplication factor or calibration factor.
a) Calibration curve:
The concentration of the solution (or substance) is determined by using a calibration curve as follows:
- Standard solutions of substances of different concentrations are prepared.
- The absorbance of the standard solution is measured by a colorimeter or spectrometer.
- The absorbance is then plotted against the concentration to give a calibration curve.
- The unknown concentration can be determined from the absorbance of an unknown solution.
b) Multiplication factor:
The multiplication factor is the concentration of a solution that gives unit absorbance. First standard
solution of different known concentrations is prepared & absorbance of a solution is measured by
colorimeter or spectrophotometer. From the concentration & absorbance, the multiplication factor is
The concentration of an unknown solution can be found from its absorbance. i.e. unknown concentration = Absorbance X multiplication factor.
(ii) Identification of structural groups in a molecule :
The structural groups can be identified using the characteristic λmax value. Generally, spectral details like λmax molar absorptive etc. of the unknown compounds are compared with the data of the known compound.
To find out λmax for the given material series of standard solutions is prepared. A series of calibration curves are plotted in terms of absorbance versus concentration.
The wavelength corresponding to maximum absorption is called the wavelength of maximum absorption i.e. λmax
Advantages of double beam spectrophotometers over single beam spectrophotometers
- Any change in intensity of incident light due to voltage fluctuation gets compensated
- It is not necessary to replace blank with sample or zero adjustments at each wavelength.
- Error due to solvent or impurities present in solvent gets canceled.
- Changes in sensitivity of photocells do not affect reading since the null method is used.
Distinguish between Colorimeter and Spectrophotometer
|It is an instrument used for measuring absorption in the visible region.||It is an instrument used for measuring absorption in both U.V. & visible regions.|
|To get a monochromatic beam of light, colour filters are used.||To get a monochromatic beam of light, prism or grating are used.|
|Bandpass is Higher (10-15 nm).||Bandpass is Small (1-2 nm).|
|Normally, a photovoltaic cell is used as a detector which has low sensitivity.||Normally, a photomultiplier cell is used as a detector which has low sensitivity.|
|Incandescent tungsten lamp for visible region.||Incandescent tungsten lamp for visible region & hydrogen discharge or deuterium lamp for U.V region|
|Measurements done on a colorimeter are less precise & accurate compared to a spectrophotometer.||Measurements done on a spectrophotometer are more precise & accurate than colorimeters.|
|A colorimeter is generally used for the quantitative analysis of coloured compounds.||A spectrophotometer is used for quantitative & qualitative analysis of colourless as well as coloured compounds.|
|The extinction coefficient cannot be determined by colorimeters.||The extinction coefficient can be determined by a spectrophotometer.|
|They are cheaper than a spectrophotometer||They are much more costly than colorimeters.|