BACKGROUND
Chromatography
Chromatography is one of the most important techniques for separating closely related compounds present in a mixture. The separation becomes effective because of the difference present in the components distribution of equilibrium between two immiscible phases.
The variation in the equilibrium distribution occurs because of the nature and the degree of interaction of different components in these two phases. The stationary phase is a porous medium like silica in which the sample mixture percolates under a moving solvent influence.
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There are several interactions in between sample and the stationary phase and these all have been well oppressed to affect the compound separation. Thin layer chromatography or TLC offers qualitative information and in details it provides good data. A TLC plate is made from thin layer of silica that is applied in a glass.
The silica gel acts as a stationary phase and the solvent mixture acts as mobile phase. In a solvent system various compounds are soluble to variety degrees. Separation occurs because of the partition equilibrium of the components in the mixture. Rf value is one of the important characteristics that are used in TLC.
All common amino acids are a-amino acids. They have a carboxyl group and a amino group that is bonded to the same carbon atom. They are different from each other through their side chains that vary in their structure, electric charge and size. The amino acids interact with the stationary phase like silica depending on their R groups.
Those amino acids that interact very strongly with silica will be transferred by the solvent to a small distance whereas the one with very less interaction will move further. By running some controls it becomes possible to identify the mixture components.
So, the basic objective of the test is to separate and identify the amino acids in a mixture by thin layer chromatography.
REQUIREMENTS
Reagents:
- Individual amino acids2% solution
- Solvent mixture of butanol, acetic acid and water in the ratio 12:3:5.
- Ninhydrin reagent.
Miscellaneous:
- TLC plate
- TLC chamber
- Capillary tubes
- Reagent spray bottle
- Conical flasks
- Beakers
PROCEDURE
At first put the solvent mixture into TLC chamber and then close the chamber. Then keep the chamber undisturbed for about 30 min so that the jar atmosphere becomes saturated with the solvent.1
Now cut the plate into perfect size and with the help of a pencil draw straight line across the plate from about 2 cm from the bottom. Now use a capillary tube and small drop of amino acid is spotted on the line.
Then leave few minutes so that spot becomes dry. Now put second spot of amino acid on the plate. Repeat the whole process for spotting the unknown acid. Now keep the plate in the TLC chamber evenly and lean it against the side.2
Leave for few minutes for allowing the capillary action to bring the solvent up the plate till it reaches 1 cm from the end.3 Now remove the plate and at once draw a pencil across the solvent top. Now dry the plate under the hood with the help of blow dryer.
Now take ninhydrin reagent to spray the dry plate. In a hot air oven set at 105 ˚C dry the plates for about 5 min. After this mark the centre of the rising spots and measure the distance between the center and origin and calculate the Rf values.4
CONCLUSION
It is one of the more efficient methods for separation of amino acid, vitamins, sugars from multicomponent mixtures.
REFERENCES
- H. R. Mahler and E. H. Cordes, “Basic Biological Chemistry,” Harper and Row, New York, 1968.
- C. Haworth and J. G. Heathcote, “The Direct Determination of Amino Acids on Thin-Layer Chromatograms by Densitometry,” Biochemistry Journal, Vol. 114, No. 3, 1969, pp. 667-668.
- K. Lorentz and B. Flatter, “Staining of Amino Acids with Benzoquinone in Paper Chromatography,” Analalytical Biochemistry, Vol. 38, No. 2, 1970, pp. 557-559.
- B. Basak and S. Laskar, “Spray Reagents for the Detection of Amino-Acids on Thin-Layer Plates,” Talanta, Vol. 37, No. 11, 1990, pp. 1105-1106.
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- Test for Proteins
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- Estimation of protein by Folin Lowry method
- Determination of saponification value of the given oil/fat
- Determination of saponification value of fat (coconut oil)
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