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).
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