Biochemistry and metabolism

Experiment 6

Introduction

Chromatography:

Chromatography is by far the most useful general group of techniques available for the separation of closely related compounds in a mixture. Here the separation is effected by differences in the equilibrium distribution of the components between two immiscible phases, viz., the stationary and the mobile phases. These differences in the equilibrium distribution are a result of nature and degree of interaction of the components with these two phases. The stationary phase is a porous medium like silica or alumina, through which the sample mixture percolates under the influence of a moving solvent (the mobile phase). There are a number of interactions between the sample and the stationary phase and these have been well exploited to effect the separation of compounds.

Thin layer chromatography [TLC]:

Thin layer chromatographic (TLC) technique readily provides qualitative information and with careful attention to details, it is possible to obtain quantitative data. Thin layer chromatography is a technique used to separate and identify compounds of interest. A TLC plate is made up of a thin layer of silica adhered to glass or aluminum for support. The silica gel acts as the stationary phase and the solvent mixture acts as the mobile phase. In the ideal solvent system the compounds of interest are soluble to different degrees. Separation results from the partition equilibrium of the components in the mixture.

In the simplest form of the technique, a narrow zone or spot of the sample mixture to be separated is applied near one end of the TLC plate and allowed to dry. The strip or plate is then placed with this end dipping in to the solvent mixture, taking care that the sample spot/zone is not immersed in the solvent. As the solvent moves towards the other end of the strip, the test mixture separates into various components. This is called as the development of TLC plates. The separation depends on several factors; (a) solubility: the more soluble a compound is in a solvent, the faster it will move up the plate. (b) attractions between the compound and the silica, the more the compound interacts with silica, the lesser it moves, (c) size of the compound, the larger the compound the slower it moves up the plate.

The plate is removed after an optimal development time and dried and the spots/zones are detected using a suitable location reagent. An important characteristic used in thin layer chromatography is Rf value.

The plate is removed after an optimal development time and dried and the spots/zones are detected using a suitable location reagent. An important characteristic used in thin layer chromatography is Rf value.

Principle

• Chromatographic Separation of Amino acids:

• The present experiment employs the technique of thin layer chromatography to separate the amino acids in a given mixture.

• All 20 of the common amino acids [standard amino acids] are α-amino acids. They have a carboxyl group and an amino group bonded to the same carbon atom (the α- carbon). They differ from each other in their side chains, or R groups, which vary in structure, size, and electric charge. The interaction of the amino acids with the stationary phase like silica varies depending on their 'R' groups. The amino acid that interacts strongly with silica will be carried by the solvent to a small distance, whereas the one with less interaction will be moved further. By running controls [known compounds] alongside, it is possible to identify the components of the mixture.

• Since amino acids are colourless compounds, ninhydrin is used for detecting them. To identify this, after development, the TLC plate is sprayed with ninhydrin reagent and dried in an oven, at 105°C for about 5 minutes. Ninhydrin reacts with α-amino acids that results in purple coloured spots [due to the formation of the complex - Rheuman's purple]. Rf values can be calculated and compared with the reference values to identify the amino acids. [The Rf value for each known compound should remain the same provided the development of plate is done with the same solvent, type of TLC plates, method of spotting and in exactly the same conditions].

Materials required

Reagents:

1. 2% solution of individual amino acids.

2. Solvent mixture of normal butanol, acetic acid and water in the ratio 12:3:5 by volume.

3. Ninhydrin reagent.

Requirements:

1. TLC plate.

2. TLC chamber.

3. Capillary tubes.

4. Reagent spray bottle.

5. Conical flasks.

6. Beakers.

Procedure:

1. Pour the solvent mixture (butanol, acetic acid and water in the ratio 12:3:5 by volume) in to the TLC chamber and close the chamber.

2. The chamber should not be disturbed for about 30 minutes so that the atmosphere in the jar becomes saturated with the solvent.

3. Cut the plate to the correct size and using a pencil (never ever use a pen) gently draw a straight line across the plate approximately 2 cm from the bottom.

4. Using a capillary tube, a minute drop of amino acid (glycine) is spotted on the line.

5. Allow the spot to dry.

6. Spot the second amino acid (tryptophan) on the plate [enough space should be provided between the spots].

7. Repeat the same process for Proline and Phenylalanine amino acids.

8. Place the plate in the TLC chamber as evenly as possible and lean it against the side (immerse the plate such that the line is above the solvent). Allow capillary action to draw the solvent up the plate until it is approximately 1 cm from the end.

9. Remove the plate and immediately draw a pencil line across the solvent top.

10. Under a hood dry the plate with the aid of a blow dryer.

11. Spray the dry plate with ninhydrin reagent.

12. Dry the plates in hot air oven at 105°C for 5 min. [Ninhydrin will react with the faded spots of amino acids and make them visible as purple coloured spots.]

13. After some time, mark the centre of the spots, then measure the distance of the centre of the spots from the origin and calculate the Rf values.

Calculation

Results

Different coloured spots of different amino acids observed at different location on TLC plate

Conclusion

• By calculating Rf values using formula and comparing them with standards we can conclude that spot A is of Glycine (0.2), spot B is of Tryptophan(0.66), Spot C is of Proline (0.43-yellow colour) and spot D is of Phenylalanine (0.68).

• Slight variation in Rf values of amino acids can be observed.

• The ninhydrin, which is originally yellow, reacts with amino acid and turns ((deep purple)). Ninhydrin will react with a free alpha-amino group, NH2-C-COOH. This group is present in all amino acids including proline, proteins or peptides.

• Ninhydrin degrades amino acids into aldehydes, ammonia, and CO2 through a series of reactions; the net result is ninhydrin in a partially reduced form hydrindantin. Ninhydrin then condenses with ammonia and hydrindantin to produce an intensely blue or purple pigment, sometimes called Ruhemann’s purple. While Proline has aliphatic side chains with a distinctive cyclic sturcture

Reference Material

• Separation of amino acids by TLC method

Questions

1. Give examples of ploar and non ploar amino acids.

2. Calculate the Rf value if a solute travelled 5 cm from the base spot and the solvent front is 10 cm from the origin?

3. In a TLC experiment using a 70:30 mixture of Petroleum ether and ethyl acetate, a student noted the development of spots in the origin also, what can you suggest about this observation?