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General principles Substances are separated according to their differential solubility between the stationary phase, represented by the water bonded to the cellulose molecules of paper, and the moving phase, the solvent. As the solvent front advances it carried the components at different rates.

Substances to be identified are ‘spotted’ near one end of the filter paper As the solvent moves up the paper, different molecules move at different rates with the smallest molecules moving the fastest The technique is used for small molecules such as amino acids, small peptides and sugars Paper chromatography is a technique used for the separation & identification of relatively small chemical substances by a moving solvent on sheets or strips of filter paper Filter paper cylinder Clip Solvent Concentrated spot of chemicals to be separated and identified Direction of solvent movement The tank should be saturated with solvent vapour

Investigating the composition of a protein. The peptide bonds can be readily hydrolysed by heating with 6M HCl at 100 o C for hours in an evacuated tube. The amino acids released can then be analysed by chromatography or electrophoresis.

Choice of solvent The general principle is “like dissolves like”. If a non-polar solvent is used the hydrophobic amino acids will move much further up the paper, as they are more soluble in non-polar solvents. In contrast if a polar solvent is used the hydrophilic amino acids will advance further up the paper as they are now more soluble.

origin Solvent front solvent Amino acid spots Spray the dry paper with locating agent (ninhydrin) to make the colourless spots visible Dry the paper with gentle heat to develop the amino acid spots Purple spots develop located at different distances from the origin line Mark the solvent front & allow paper to dry

amino acid spots on the origin line Chromatography separates small molecules in a mixture on the basis of size As the solvent moves up the paper, molecules move at different rates When the spots are colourless (most amino acids), a locating agent is needed to visualise their positions on the chromatography paper

Y The R f value is always a value less than one as the solvent front always moves further than the solute molecules X1X1 X5X5 X4X4 X3X3 X2X2 Rf values can be compared with published values to identify the amino acids.

The mixture of unknown amino acids is seen to contain four different amino acids Of these four amino acids, two can be positively identified The mixture contains four amino acids; two unknown together with arginine & leucine

Mixture of amino acids on origin line Paper dried and rotated clockwise through 90 o Solvent front First solvent Second solvent Two-way chromatography provides better separation of substances that behave in a similar fashion in the first solvent. A second run in a different solvent resolves two very close spots more clearly Solvent front

Electrophoresis, like chromatography, is a technique for the separation of molecules Both chromatography and electrophoresis may be used for the separation of small molecules Electrophoresis, unlike chromatography, can be used for the separation of relatively large molecules Electrophoresis is used mainly for separating mixtures of proteins, peptides and DNA fragments Electrophoresis separates molecules from a mixture according to their charge and size The technique involves passing a direct current through either filter paper or a gel

- + Electrophoresis gel saturated with buffer solution (to keep the pH constant) Negative electrode Positive electrode CATHODE ANODE GEL Different molecules show as different bands after staining Sample slots in the gel Buffer solution Buffer tank Contact wick making electrical connection between buffer and gel Sample of mixture placed in a slot cut in the gel

Preparation of samples Filter paper wicks are soaked in the test solution. They are then inserted in fine cuts made in the thin layer of silica gel. NB Further separation can be achieved using filter paper in combination with chromatography in another direction.

Separation of Serum Proteins Blood serum contains many different blood proteins A serum sample is placed in a slot in the gel The pH of the buffer is adjusted to make all the proteins negatively charged When the direct current is passed through the gel, all the proteins will move to the anode After staining, the proteins will show up as different bands in different positions from the origin slot Direction of migration albumin alpha 1 globulin alpha 2 globulin beta globulin gamma globulin (mainly antibodies)

Amino acids are AMPHOTERIC The amino group of the amino acid is a basic group The carboxylic group is an acid group Acids behave as proton donors Bases behave as proton acceptors Protons are hydrogen ions (H + ) A proton is the nucleus of a hydrogen atom H+H+ H+H+ Amphoteric molecules behave as both acids and bases

Amino acids possess a carboxylic acid end and an amino end: The amino group behaves as a base The R group may also possess a carboxylic acid OR an amino group In solution the acid group behaves as a proton donor and the amino group (basic) as a proton acceptor In solution, the acid group becomes negatively charged and the amino group becomes positively charged Neutral amino acids with no acid or amino groups in the R group have one negative and one positive charge. Overall they are electrically neutral molecules H+H+ H+H+

The hydrogen atom contains a single positively charged particle in its nucleus This positively charged particle is a proton Spinning around the nucleus in orbit is a single negatively charged electron In solution, the hydrogen atom of the carboxylic acid group of the amino acid releases a proton (H + ) The electron remains with the carboxylic acid group which becomes negatively charged + + +

This is an ACIDIC amino acid as it possesses TWO carboxylic acid groups In solution the acid groups donate a proton and the amino group accepts a proton The negative electrons remain with the carboxylic acid groups The amino acid now has TWO negatively charged carboxylic acid groups and ONE positively charged amino group Overall, this acidic amino acid is negatively charged H+H+ H+H+ H+H+

This is a BASIC amino acid as it possesses TWO amino groups In solution the acid group donates a proton and the amino groups accept a proton The negative electron remains with the carboxylic acid group The amino acid now has TWO positively charged amino acid groups and ONE negatively charged carboxylic acid group Overall, this basic amino acid is positively charged H+H+ H+H+ H+H+

ANODE (+) CATHODE (-) mixture of the three amino acids placed in well moves towards moves towards stays at the origin GEL SOAKED IN BUFFER SOLUTION acidic amino acid basic amino acid neutral amino acid direct current passed through the gel and later sprayed with ninhydrin The gel is examined under uv