Hydrolysis of a Dipeptide: Identification of the Products by TLC

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Hydrolysis of a Dipeptide Identification of the
Products by TLC
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Things to Learn

• Learn about Thin Layer Chromatography.
• Using TLC to identify the products.

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Introduction

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

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Introduction

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

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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.

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Introduction

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

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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.

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Peptide Hydrolysis
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Peptide Hydrolysis

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

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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.

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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.

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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.

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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.

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Thin Layer Chromatography
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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 ) .

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Thin Layer Chromatography
Distance moved by a compound Rf Distance
moved by the solvent front The maximum Rf
values can be 1.0 and the minimum is 0.
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Thin Layer Chromatography

• Make chromatogram as mentioned in this procedure.
• Use pencil to make marks.

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Thin Layer Chromatography

• Compound spots are applied using a tooth pick or
  capillary (Keep on the line of Origin).

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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

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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.

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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.

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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.