Analysis of amino acids by paper chromatography- Theory

Chromatography is a technique for analyzing or separating mixtures of substances into their components. There are various forms of chromatography techniques. Generally all of these forms involve two distinct phases; the Stationary phase and the mobile phase.
In paper chromatography the stationary phase:- Absorbent paper, Mobile phase:- A suitable liquid or a mixture of solvents.
Mobile phase flows through the stationary phase and carries the components of the mixture with it.
The stationary phase retards the passage of components of the sample.
When components pass through the system at different rates they become separated in time.
This depend on their solubility in the solvent and how well the chromatography paper absorbs.
More soluble the component is in the solvent and the less it absorb into the paper, the faster it would move with the solvent on the paper and hence the spot appears further up the paper.
There are three types of paper chromatography.

• Ascending paper chromatography
• Descending chromatography
• 2-D paper chromatography

In paper chromatography cellulose in the form of paper sheets makes an identical support medium as it has the ability to absorb water molecules between cellulose fibers and forms a stationary hydropholic phase.

In analysis of amino acids by paper chromatography, the location of the amino acids can be determined by spraying the paper with Ninhydrin. The Ninhydrin forms a blue violet complex with an amino acid except for proline/hydroxy proline which gives a yellow color complex.

Basis of biochemical tests; Biuret test, Benedict test, Iodine/potassium iodide test, Emulsion test

Biuret test
A test for peptide bonds to identify proteins.

In the presence of dil.copper sulphate in alkaline solution, nitrogen atoms in the peptide chain form a purple complex with copper(ll) ions. Biuret is a compound derived from urea which also contains the –CONH- group and  gives a positive result.

Benedict test

A test for sugar

Reducing sugars- Benedict’s solution contains copper sulphate. Reducing sugars reduce soluble blue copper sulphate. Here the containing copper(ll) ions will change to insoluble red- brown copper oxide containing copper(l). This is seen as the precipitate.

Non-reducing sugars- The disaccharide will hydrolysed to its monosaccharide elements by boiling with dil. HCl. Such as Sucrose will be hydrolysed to glucose and fructose which are both reducing sugars. That will give the reducing sugar results with the Benedict’s test.

Iodine/potassium iodide test

A test for starch.

What is given at last is a poly-iodide complex formed with starch.

Emulsion test

A test for Lipids

Lipids are iinsoluble with water. Adding water to a solution of the lipid in alcohol results in an emulsion of lipid droplets or micelles in the water. These reflect light and give a white, opalescent appearance.

Test for phenols:- Phenol, 2-napthol, Catechol, Resorcinol

Azo-dye formation

Procedure	Observation
2drops of aniline were dissolved in 1ml of con.HCl and 3ml of water were shaken in a test tube. It was kept in ice. Also 20% sodium nitrite solution and phenol dissolved in NaOH were kept in ice. Few drops of sodium nitrite were added to the cold aniline. Maintaining the temperature around 0-4°C this prepared diazonium solution was added to the cold phenol.	Phenol: intense orange colour dye 2-napthol: red colour dye Catechol: dark brownish dye Resorcinol: red colour dye

Test for primary aromatic amines (Benzoylation):- Anilene, O.toluidene, P.toluidene, M.toluidene

Benzoylation

Procedure	Observation
1ml of the compound, 20ml of 10% NaOH solution and 1.5ml of Benzoyl chloride were mixed together in Stoppered conical flask. Mixture was vigorously shaken.	Aniline: White semi-solid O.toluidene: Pale pink semi-solid M.toluidene: Pale brown semi-solid P.toluidene: Dark brown semi-solid

Life of Albert Einstein in pictures

Sorry that I couldn't post anything from a long time. So here I am, back again. Couldn't think of anything to post..may be some pictures of Einstein will cheer you.. :P