In physical and analytical chemical science, colorimetric analysis is a technique used to find the concentration of solution. It is widely used in biochemical appraisals. A substance estimated by colorimetric analysis, must be either coloured or more normally, capable of organizing chromo cistrons, through interaction with suited reagents. The instrument known as tintometer or photometer is in fact, an absorptiometer, since it is the sum of light absorbed, which is measured. This is besides used to prove the concentration of a solution by mensurating its optical density of a specific wavelength of visible radiation.
The colorimerty is based on the undermentioned two Torahs which may be stated as follows:
a-?Beer ‘s Law:
Harmonizing to Beer ‘s jurisprudence, when light base on ballss through a coloured solution, sum of visible radiation transmitted lessenings with the addition in concentration of the coloured substance.
=Intensity of incident visible radiation
=Intensity of familial visible radiation
a-?Lambert ‘s Law:
This jurisprudence states that the sum of visible radiation transmitted lessenings with addition in thickness of the bed of coloured solution.
=Thickness of the bed of solution
=Intensity of incident visible radiation
=Intensity of familial visible radiation
The thickness of bed of solution is reciprocally relative to the strength of visible radiation transmitted.
The name tintometer refers to an instrument used in colorimetric analysis. It is a device that measures the optical density of peculiar wavelengths of visible radiation by a specific solution. This device is most normally used to find the concentration of a known solute in a given solution by the application of the Beer-Lambert jurisprudence, which states that the concentration of a solute is relative to the optical density.
Components of Colorimeter: –
a-?An adjustable slit
a-?A set of coloured filters
a-?sample chamber ( cuvette ) to keep the on the job solution
a-?A sensor to mensurate the familial visible radiation
a-?A metre to expose the end product from the sensor
aˆ? Light Beginning:
For seeable part measurementTungsten lamp380-750 nanometer
In colorimetric analysis the visible radiation with wavelength of seeable scope is used for the finding of the solution. Wavelength of visible radiation is defined as “ the distance between two extremums as the light travels in wave-like mode. ” This distance is expressed in nanometre ( nm ) . Other units may be used are Angstrom ( ) and mill micron ( m ) ,
1 nm = 1 m 10 =
aˆ?An adjustable aperture/slit:
Colorimeter includes an adjustable slit through which the beam of selected wavelength base on ballss which prevents isolated visible radiation.
Light after go throughing through slit falls on capacitor lens which gives a parallel beam of visible radiation.
Common tintometers contain a set of filters, which allow choice of visible radiation of narrow wavelengths. These filters are mutable in order to maximise truth.
For illustration, a green filter absorbs all the constituent colourss of white visible radiation, except green visible radiation which is allowed to go through through. Light transmitted through a green filter has a wavelength from 500-600 nanometer. Similarly, other suited filters can be used to choose visible radiation of narrow wavelengths in the scope of 400-700 nanometer.
The monochromatic visible radiation from the filter passes through the coloured solution placed in a cuvette. Cuvette is made up of particular glass/plastic/quartz stuff. Cuvettes may be square, rectangular or unit of ammunition shaped with a fixed diameter normally 1 centimeter and holding a unvarying surface. The solution absorbs some visible radiation and the residuary visible radiation falls on a sensor.
Detectors are light-sensitive elements which converts visible radiation into an electrical signal.
Electrical energy from a sensor is displayed on some type of metre or galvanometer as transmission or optical density.
*Colored solutions have the belongings of absorbing visible radiation of certain wavelength and conveying others. Color of a solution depends on familial visible radiation. e.g. the hemoglobin solution absorbs bluish green visible radiation and transmits the complementary colourss and the solution appears ruddy. In colorimetric analysis, filters are chosen suitably, so that soaking up is maximal at the selected wavelength part.
Color of solution
Wavelength of soaking up ( nanometer )
aˆ?In colorimetric measurings, cuvettes of same diameter are used. Ratio of strength of emergimg visible radiation ( ) to that of incident visible radiation ( ) is known as transmission ( ) .
aˆ?Absorbance is straight relative to the concentration of coloured substance, over a broad scope. i.e. a more concentrated solution gives a higher optical density reading.
Stairss IN COLORIMETRY: –
In colorimetric appraisals, it is necessary to fix a trial, a criterion and a space. The trial solution is prepared by handling a specific volume of the specimen with reagents indicated in the process. A standard solution is prepared by likewise handling a solution of the pure substance of known concentration. A space is ever run by handling a volume of H2O equal to the specimen with the reagent. This is to rectify for the colour given by reagents entirely in the process.
An appropriate filter is inserted into the photometer. The cuvette is filled with H2O to about three 4th and placed in place. Light is allowed to go through through the cuvette. Absorbance is adjusted to zero.
Blank solution is taken in another cuvette and is placed in the cuvette compartment. Optical density is noted ( B ) . Similarly, optical density of trial ( T ) and standard ( S ) are measured. Satisfactory consequences are normally obtained when optical density values ( optical denseness ) of T and S are in the scope of 0.1-0.7.
COLORIMERTIC Analysis: –
Colorimetric analysis is a method of finding the concentration of a a chemical component or chemical compound in a solution with the assistance of a colour reagent. It is applicable to both organic compound and inorganic compounds and may be used with or without an enzymatic phase. The method is widely used in medical research labs and for industrial intents, e.g. the analysis of H2O samples in connexion with industrial H2O intervention.
Standards of Colorimetric Analysis: –
Specificity of the colour reaction: Very few reactions are specific for a peculiar substance, but many give colourss for a little group of related substances merely, i.e. they are selective. By presenting other complex-forming compounds, by jumping the oxidization provinces, and by commanding the pH, close estimate to specificity may frequently be obtained.
Proportionality between colour and concentration: For ocular tintometers it is of import that the colour strength should increase linearly with the concentration of the substance to be determined. This is non indispensable for photoelectric instruments, since a standardization curve may be constructed to associate the instrumental reading of the colour with the concentration of the solution. Put another manner, it is desirable that the system should follow the Beer ‘s jurisprudence even when the photoelectric tintometers are used.
Stability of the colour: The colour produced should be sufficiently stable o license an accurate reading to be taken. This besides applies to those reactions in which colourss tend to make upper limit after a clip ; the period of maximal colour must be long plenty for precise measurings to be made. The influence of other substances must be known, and the influence of experimental conditions ( temperature, pH, stableness in air, etc. )
Reproducibility: the colorimetric process must give consistent consequences under specific experimental conditions. The reaction need non needfully stand for a stoichiometrically quantitative chemical scope.
Clarity of the solution: The solution must be free from precipitate if comparing to be made with a clear criterion. Turbidity spreads light every bit good as absorbing it.
High Sensitivity: The colour reaction should be extremely sensitive, peculiarly when minute sums of substances are to be determined. It is besides desirable that the reaction merchandise absorbs strongly in the seeable instead than the UV ; the interfering consequence of other substances in the UV is normally more marked.
Enzymatic Methods: –
In enzymatic analysis ( which is widely used in medical research labs the colour reaction is preceded by a reaction catalyzed by an enzyme. As the enzyme is specific to a peculiar substrate, more accurate consequences can be obtained. Enzymatic analysis is ever carried out in a buffer solution at a specified temperature ( normally 37 ) to supply the optimal conditions for the enzymes to move.
Cholesterol ( CHOD-PAP method )
1. Cholesterol + O — ( enzyme cholesterin oxidase ) — cholestenone + H peroxide
2. Hydrogen peroxide + 4-aminophenazone + phenol — ( enzyme peroxidase ) — colored complex.
Glucose ( God-Perid method )
1. Glucose + O + H2O — ( enzyme glucose oxidase ) — -gluconate + H peroxide.
2. Hydrogen peroxide + ABTS — ( enzyme peroxidase ) — colored complex.
In this instance, both phases of the reaction are catalyzed by enzymes.
Triglycerides ( GPO-PAP method )
1. Triglycerides + H2O — ( enzyme esterase ) — glycerin + carboxylic acid.
2. Glycerol + ATP – ( enzyme glycerin kinase ) — glycerol-3-phosphate + ADP
3. Glycerol-3-phosphate + O — ( enzyme glycerol-3- phosphate oxidase ) — dihyroxyacetone phosphate + H peroxide.
4. Hydrogen peroxide + 4-aminophenazone + 4-chlorophenol — ( enzyme peroxidase ) — colored complex.
1. Urea + H2O – ( enzyme urease ) — ammonium carbonate.
2. Ammonium carbonate + phenol + hypochlorite — — colored complex.
In this instance, merely the first phase of the reaction is catalyzed by an enzyme. The 2nd phase is non-enzymatic.
CHOD = cholesterin 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
Non-enzymatic methods: –
Calcium + o-cresothaline complexone — — – colored complex.
Copper + bathocuproin disulfonate — — colored complex.
Creatine + picrate — — colored complex.
Iron + bathophenanthroline disulphonate — – colored complex.
Phosphate ( Inorganic )
Phosphate + ammonium molybdate + ammonium metavandate — — colored complex.
Ultraviolet methods: –
In UV ( UV ) methods there is no seeable colour alteration but the rule is precisely the same, i.e. the measuring of a alteration in the optical density of the solution.UV methods normally measure the difference in optical density at 340 nm wavelength between nicotinamide A dinucletide ( NAD ) and its decreased signifier ( NADH ) .
Pyruvate + NADH ( enzyme lactate dehydrogenase ) — — L-lactate + NAD
Principle and pattern of analytical chemical science 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.