Hydrolysis of a Dipeptide: Identification of the Products by TLC

1. Hydrolysis of a Dipeptide: Identification of the Products by TLC

3. Introduction Like proteins, peptides are polypeptide molecules. The distinction is that peptides are short and proteins are long.

4. Introduction Peptide means �DIGESTIBLE� in Greek. A Dipeptide is a molecule consisting of two amino acids joined by a single peptide bond.

5. Introduction A peptide bond is a chemical bond formed between two molecules when the carboxyl group of one molecule reacts with the amino group of the other molecule, releasing a molecule of water (H2O). This is a dehydration synthesis reaction, and usually occurs between amino acids.

6. Introduction The German chemist Emil Fischer obtained the first dipeptide. He made glycylglycine, in 1901 by the partial hydrolysis of the diketopiperazine of glycine.

7. Peptide Hydrolysis Break down of peptide bonds. Requires acid or base, water and heat. Gives smaller peptides and amino acids Similar to digestion of proteins using enzymes. Occurs in cells to provide amino acids to synthesize other proteins and repair tissues.

8. Peptide Hydrolysis

9. Peptide Hydrolysis The sweetener Aspartame, Neotame and others are dipeptides. Both has similar structure and 8000 times sweeter than sugar

10. Chromatography Chromatography is a method for separating the substances in a mixture. Many types of chromatography are known. Gas chromatography Column chromatography Thin Layer Chromatography Paper chromatography All techniques involve a stationary phase and mobile phase.

11. Thin Layer Chromatography Simplest form of chromatography. Neutral Alumina, Silica gel can be the stationary phase. Mobile phase (organic solvents) passes through the sample by CAPILLARY ACTION.

12. Thin Layer Chromatography Different compounds interact differently with the stationary phase and with various mobile phase. The interactions are based on: Solubility of the compound. Polarity of the solvent and compound. Adsorption of the compound on stationary phase. Molecular size of the compounds.

13. Thin Layer Chromatography Components of the sample will separate readily according to how strongly they adsorb on the stationary phase versus how readily they dissolve in the mobile phase.

14. Thin Layer Chromatography

15. Thin Layer Chromatography The mobility of the compounds depends on the nature of their relative attractions to the mobile stationary phase. For a compound, in a specific stationary and mobile phase, we can define the Retention Factor or Retardation Factor ( Rf ) .

16. Thin Layer Chromatography

17. Thin Layer Chromatography Make chromatogram as mentioned in this procedure. Use pencil to make marks.

18. Thin Layer Chromatography Compound spots are applied using a tooth pick or capillary (Keep on the line of Origin).

19. Thin Layer Chromatography Keep the solvent level below the line of origin. Do not allow the solvent level go beyond the solvent front line. Dry the chromatogram using hot plate. Should not touch the sidewall of the champers

20. Thin Layer Chromatography Use ninhydrin solution (0.3 %) to see the spots in your chromatogram. When reacting with free amines, a deep blue or purple color is evolved.

21. Thin Layer Chromatography Once you develop the chromatogram: 1. Find the Rf value for each compound. 2. Find what are all the fragments are present in aspartame. 3. Find out whether diet coke has aspartame or not.

22. Notes You do not need to make any solutions. Use fresh capillary or tooth pick to keep spot. Please Do not touch the ninhydrin solution with bare hand. Work as pair.