In physical and analytical chemistry, colorimetry is a technique used to determine the concentration of solution. It is widely used in biochemical estimations. A substance estimated by colorimetry, must be either colored or more commonly, capable of forming chromo genes, through interaction with suitable reagents. The instrument known as colorimeter or photometer is in fact, an absorptiometer, since it is the amount of light absorbed, which is measured. This is also used to test the concentration of a solution by measuring its absorbance of a specific wavelength of light.
The colorimerty is based on the following two laws which may be stated as follows:
According to Beer’s law, when light passes through a colored solution, amount of light transmitted decreases with the increase in concentration of the colored substance.
= Intensity of incident light
= Intensity of transmitted light
This law states that the amount of light transmitted decreases with increase in thickness of the layer of colored solution.
= Thickness of the layer of solution
= Intensity of incident light
= Intensity of transmitted light
The thickness of layer of solution is inversely proportional to the intensity of light transmitted.
The name colorimeter refers to an instrument used in colorimetry. It is a device that measures the absorbance of particular wavelengths of light by a specific solution. This device is most commonly used to determine the concentration of a known solute in a given solution by the application of the Beer-Lambert law, which states that the concentration of a solute is proportional to the absorbance.
Components of Colorimeter:-
- Light source
- An adjustable slit
- Considering lens
- A set of colored filters
- sample chamber (cuvette) to hold the working solution
- detector to measure the transmitted light
- meter to display the output from the detector
- Light Source:
For visible region measurementTungsten lamp380-750 nm
In colorimetry the light with wavelength of visible range is used for the determination of the solution. Wavelength of light is defined as “the distance between two peaks as the light travels in wave-like manner.” This distance is expressed in nanometer (nm). Other units may be used are Angstrom () and mill micron (m),
1 nm = 1 m 10 =
- An adjustable aperture/slit:
Colorimeter includes an adjustable slit through which the beam of selected wavelength passes which prevents stray light.
- Condensing Lens:
Light after passing through slit falls on condenser lens which gives a parallel beam of light.
Common colorimeters contain a set of filters, which allow selection of light of narrow wavelengths. These filters are changeable in order to maximize accuracy.
For example, a green filter absorbs all the component colors of white light, except green light which is allowed to pass through. Light transmitted through a green filter has a wavelength from 500-600 nm. Similarly, other suitable filters can be used to select light of narrow wavelengths in the range of 400-700 nm.
The monochromatic light from the filter passes through the colored solution placed in a cuvette. Cuvette is made up of special glass/plastic/quartz material. Cuvettes may be square, rectangular or round shaped with a fixed diameter usually 1 cm and having a uniform surface. The solution absorbs some light and the residual light falls on a detector.
Detectors are photosensitive elements which converts light into an electrical signal.
Electrical energy from a detector is displayed on some type of meter or galvanometer as transmittance or absorbance.
*Colored solutions have the property of absorbing light of certain wavelength and transmitting others. Color of a solution depends on transmitted light. e.g. the hemoglobin solution absorbs blue-green light and transmits the complementary colors and the solution appears red. In colorimetry, filters are chosen appropriately, so that absorption is maximum at the selected wavelength region.
- In colorimetric measurements, cuvettes of same diameter are used. Ratio of intensity of emergimg light () to that of incident light () is known as transmittance ().
- Absorbance is directly proportional to the concentration of colored substance, over a wide range. i.e. a more concentrated solution gives a higher absorbance reading.
STEPS IN COLORIMETRY:-
In colorimetric estimations, it is necessary to prepare a test, a standard and a blank. The test solution is prepared by treating a specific volume of the specimen with reagents indicated in the procedure. A standard solution is prepared by similarly treating a solution of the pure substance of known concentration. A blank is always run by treating a volume of water equal to the specimen with the reagent. This is to correct for the color given by reagents alone in the procedure.
An appropriate filter is inserted into the photometer. The cuvette is filled with water to about three fourth and placed in position. Light is allowed to pass through the cuvette. Absorbance is adjusted to zero.
Blank solution is taken in another cuvette and is placed in the cuvette compartment. Absorbance is noted (B). Similarly, absorbance of test (T) and standard (S) are measured. Satisfactory results are usually obtained when absorbance values (optical density) of T and S are in the range of 0.1-0.7.
Colorimetric analysis is a method of determining the concentration of a a chemical element or chemical compound in a solution with the aid of a color reagent. It is applicable to both organic compound and inorganic compounds and may be used with or without an enzymatic stage. The method is widely used in medical laboratories and for industrial purposes, e.g. the analysis of water samples in connection with industrial water treatment.
Criteria of Colorimetric Analysis:-
Specificity of the color reaction: Very few reactions are specific for a particular substance, but many give colors for a small group of related substances only, i.e. they are selective. By introducing other complex-forming compounds, by alternating the oxidation states, and by controlling the pH, close approximation to specificity may often be obtained.
Proportionality between color and concentration: For visual colorimeters it is important that the color intensity should increase linearly with the concentration of the substance to be determined. This is not essential for photoelectric instruments, since a calibration curve may be constructed to relate the instrumental reading of the color with the concentration of the solution. Put another way, it is desirable that the system should follow the Beer’s law even when the photoelectric colorimeters are used.
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Stability of the color: The color produced should be sufficiently stable o permit an accurate reading to be taken. This also applies to those reactions in which colors tend to reach maximum after a time; the period of maximum color must be long enough for precise measurements to be made. The influence of other substances must be known, and the influence of experimental conditions (temperature, ph, stability in air, etc.)
Reproducibility: the colorimetric procedure must give reproducible results under specific experimental conditions. The reaction need not necessarily represent a stoichiometrically quantitative chemical range.
Clarity of the solution: The solution must be free from precipitate if comparison to be made with a clear standard. Turbidity scatters light as well as absorbing it.
High Sensitivity: The color reaction should be highly sensitive, particularly when minute amounts of substances are to be determined. It is also desirable that the reaction product absorbs strongly in the visible rather than the ultraviolet; the interfering effect of other substances in the ultraviolet is usually more pronounced.
In enzymatic analysis (which is widely used in medical laboratories the color reaction is preceded by a reaction catalyzed by an enzyme. As the enzyme is specific to a particular substrate, more accurate results can be obtained. Enzymatic analysis is always carried out in a buffer solution at a specified temperature (usually 37) to provide the optimum conditions for the enzymes to act.
Cholesterol (CHOD-PAP method)
1. Cholesterol + oxygen — (enzyme cholesterol oxidase) — cholestenone + hydrogen peroxide
2. Hydrogen peroxide + 4-aminophenazone + phenol — (enzyme peroxidase)–colored complex.
Glucose (God-Perid method)
1. Glucose + oxygen + water — (enzyme glucose oxidase) —gluconate + hydrogen peroxide.
2. Hydrogen peroxide + ABTS — (enzyme peroxidase) — colored complex.
In this case, both stages of the reaction are catalyzed by enzymes.
Triglycerides (GPO-PAP method)
1. Triglycerides + water — (enzyme esterase) — glycerol + carboxylic acid.
2. Glycerol + ATP – (enzyme glycerol kinase) — glycerol-3-phosphate + ADP
3. Glycerol-3-phosphate + oxygen — (enzyme glycerol-3- phosphate oxidase) — dihyroxyacetone phosphate + hydrogen peroxide.
4. Hydrogen peroxide + 4-aminophenazone + 4-chlorophenol — (enzyme peroxidase) — colored complex.
1. Urea + water – (enzyme urease)–ammonium carbonate.
2. Ammonium carbonate + phenol + hypochlorite —- colored complex.
In this case, only the first stage of the reaction is catalyzed by an enzyme. The second stage is non-enzymatic.
CHOD = cholesterol oxidase
GOD = glucose oxidase
GPO = glycerol-3- phosphate oxidase
PAP = phenol + aminophenazone (in some methods the phenol is replaced by 4-chlorophenol, which is less toxic)
Perid = peroxidase
Calcium + o-cresothaline complexone —– colored complex.
Copper + bathocuproin disulfonate —- colored complex.
Creatine + picrate —- colored complex.
Iron + bathophenanthroline disulphonate — colored complex.
Phosphate + ammonium molybdate + ammonium metavandate —- colored complex.
In ultraviolet (UV)methods there is no visible color change but the principle is exactly the same, i.e. the measurement of a change in the absorbance of the solution .UV methods usually measure the difference in absorbance at 340 nm wavelength between nicotinamide adenine dinucletide (NAD)and its reduced form (NADH).
Pyruvate + NADH (enzyme lactate dehydrogenase) —- L-lactate + NAD
Principle and practice of analytical chemistry by F.W.Fifield and David Kealey.
Vogel’s Textbook of Quantitative chemical Analysis (Sixth Addition)
By J.Mendham, R.C. Denney, J.D. Barnes and M.J.K. Thomas.
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