Chromatography is the most powerful technique to separate chemically closely related substances into the individual components on the basis of their physicochemical properties. The compounds are separated on the basis of their partition coefficients between two immiscible phases. The static phase may be a solid or liquid while the mobile phase may be a solid, liquid or gas. Depending upon the static and mobile phases, a variety of chromatographic techniques are available. These include chromatography on paper, thin layer gel, ion-exchange resin etc. Although modern instrument facilities such as High Performance Liquid Chromatography (HPLC) are available for the separation of chemical substances, the classical techniques - paper chromatography and thin layer chromatography are still easy, can be set up even in an ordinary laboratory without much expenditure. It may be recalled that Calvin and his associates used paper chromatography to elucidate the pathway of carbon dioxide fixation in photosynthesis. The separation, identification and (semi) quantification of amino acids using paper chromatography is described below. The same methodology can be used to separate other smaller molecules such as sugars, organic acids etc. by changing the mobile phase and detection (spray) agents.
The separation of the solutes (amino acids) is based on the liquid-liquid partitioning of amino acids in paper chromatography. The partitioning takes place between the water molecule (static phase) adsorbed to the cellulosic matter of the paper and the organic (mobile) phase.
» Whatman No.1 filter paper
» Chromatography chamber
» Hair-dryer or spot-lamp
» Microsyringe or micropipette
» Mobile Phase (Solvent System)
Mix n-butanol, glacial acetic acid and water in the ratio 4 : 1 : 5 in a separating funnel and stand to equilibrate for 30 min. Drain off the lower aqueous phase into a beaker and place it inside to saturate the chromatography chamber. Save the upper organic phase and use it for developing the chromatogram.
» Dissolve different individual amino acids in distilled water at a concentration of 1mg/mL. Use very dilute (0.05N) HCl to dissolve the free amino acids tyrosine and phenylalanine. Dissolve tryptophan in very dilute (0.05N) NaOH.
» Extraction of Sample:
Grind a known quantity of the sample material (dry/wet) in a pestle and mortar with 10-fold volume of 70% ethanol. Shake the contents at 55°C for 30 min. Centrifuge the contents at 10,000rpm for 10 min. Collect the supernatant. Repeat the extraction of the pellet at 55°C at least twice. Pool the supernatants (for leaf extracts, treat with equal volume of petroleum ether 40-60°C) and shake vigorously. Discard the petroleum ether layer containing chlorophyll. Evaporate the alcohol fraction to dryness under vacuum using either a water-pump or rotary evaporator at 40-45°C. Dissolve the residue in a known volume of absolute ethanol or water for analysis.
» Ninhydtin Reagent
Dissolve 100mg ninhydrin in 100mL acetone.
» Elution Mixture:
Prepare 1% copper sulphate solution. Mix ethanol and copper sulphate solution in the ratio 80:20 (v/v).
||Cut the chromatography sheet carefully to a convenient size (40 x 24cm). Draw a line with pencil across the sheet about 5cm away from one end. Mark a number of points at intervals of 3cm.
||Apply a small volume (say, 25mL)of each amino acid as a separate small spot using a microsyringe. A stream of hot air from a hair-dryer facilitates fast drying of spot. The spot should be as small as possible for better resolution.
||Similarly spot different known aliquots of sample extract.
||After spotting, place the sheet in a stainless steel trough in the chromatography chamber, firmly hold it by placing a long steel rod over the sheet. The spot-end of the sheet should be in the trough (descending chromatography). Otherwise, the sheet may be rolled as a cylinder, tied together with fine thread and placed upright with the spots as the bottom in a large Petridish for upward movement of solvent (ascending chromatography).
||Add the organic (phase) solvent to the trough/petri dish and close the chamber airtight. Develop the chromatogram, preferably overnight or longer, until the solvent moves almost to the other end.
||Note the solvent front and dry the chromatogram free of solvent in a fume chamber.
||Spray the chromatogram with the ninhydrin reagent using an atomizer. Dry the paper for about 5 min at room temp followed by at 100°C in an oven for 2-3 min.
Amino acids appear as purple spots; hydroxyproline and proline give yellow colored spots.
Mark all the spots and calculate their Rf values by the formula
||Distance (cm) moved by the solute from the origin
|Distance (cm) moved by the solvent from the origin
The amino acids present in the sample are then identified by comparing the Rf values with that of the authentic amino acids, co-chromatographed.
||For quantitative estimation, cut each spot into several small bits and transfer to the bottom of the test tube.
Add 3mL of elution mixture. Shake the tubes vigorously for 15 min. Decant the liquid and elute the pieces with another 2mL of elution mixture. Repeat the elution with small aliquots until the bits are colorless. Combine and clear the eluate by centrifuging at 10,000rpm for 10 min. Read the intensity of purple color at 570nm in a colorimeter. Use the spot of leucine (50mg) run as standard for comparison.
||Handle the chromatography sheet very carefully until developed as otherwise amino acids from fingers will contaminate. Hold the chromatographic paper between a fold of filter paper piece.
||After developing the chromatogram with the first solvent system, it may be developed with second solvent system such as phenol: H2O (80 : 20). This is then called bidimensional chromatography. In such case, the sample alone is applied at near one corner of the paper. Bidimensional chromatography is carried out for fine separation.
||As the Rf value varies from run to run, due to solvent system, paper, room temperature, size of the chromatography chamber etc.,it is advisable to co-chromatograph the standards every time.
||Similarly, analysis of sugars, organic acid, phenolic compounds can also be done by paper chromatography (For solvent systems, spraying agents etc., see under thin layer chromatography).