Thin layer chromatography

Chromatography

1. Chromatography Amandeep Singh Assistant Professor Department of Biotechnology GSSDGS Khalsa College Patiala Thin Layer Chromatography

2. Partition/Distribution Coefficient The way in which a given compound partition/distribute itself between 2 immiscible phases (Stationary phase & Mobile phase) K= Cs Cm Where K= Partition/distribution Coefficient Cs= Concentration of substance in stationary phase Cm= Concentration of substance in mobile phase If K= 0.2 between cellulose (Stationary phase) and CCL4 (mobile phase), it means concentration of substance in CCL4 is 5 times higher that in cellulose. As 1 = 0.2 5

3. Techniques Plane Chromatography Stationary phase is coated on plane surface. Examples: Paper Chromatography Thin Layer Chromatography (TLC) Column Chromatography Stationary phase is Packed in a column . Examples: Gas Chromatography Ion-Exchange Chromatography High Performance Liquid Chromatography (HPLC) Affinity Chromatography Exclusion Chromatography

4. Thin Layer Chromatography Preparation of Layer For Preparation of stationary phase for thin layer chromatography, silica gel or alumina gel is mixed with water to make slurry. The thickness of the slurry on plane should be 0.25 mm for analytical preparation and 5 mm for preparative preparations. Silica gel or Alumina gel + Water = Thick Suspension (Stationary Phase) ( Slurry) For Analytical Separation For Preparative Separation 0.25 mm 5 mm Reason for covering with watch glass is to make atmosphere in beaker saturated with solvent vapour .

5. Rf Value Rf value in chromatography is known as Retention value or Retention factor or Retardation factor. The  Rf values  indicate how soluble the particular pigment is in the solvent by how high the pigment moves on the paper. Two pigments with the same  Rf value  are likely to be identical molecules. Small  Rf values  tend to indicate larger, less soluble pigments while the highly soluble pigments have an  Rf value  near to one . Rf = distance traveled by substance/distance traveled by solvent front OR Rf = distance traveled by solute/distance traveled by solvent A high Rf (i.e. 0.92) would refer to a substance that is very non-polar. It means that substance moved a 92% of the entire distance the solvent traveled . A low Rf value (0.10) would refer to a substance that is very polar.It means that substance was only able to move 10% of the entire distance the solvent traveled. Rf = Distance travelled by solute Distance travelled by solvent

6. Calculating the Rf Value

7. Detection By Chemicals : By spraying Ninhydrin (reacts with amino acids to give purple or brown colour spots) Iodine Crystals (Iodine vapours react with spots of the sample to give brown colour ) By Radiation : By using Ultra-Violet Light on the spots which will show Fluorescence . The spots that appeared after the chromatography is over are colourless . To make them visible, any of the detection techniques listed below can be used:

8. How does TLC works? Silica gel is a form of Silicon dioxide (SiO2 ). At surface of silica gel, silicon atoms are attached to –OH groups. Surface of silica gel is very polar because of –OH groups and can form H-bond with suitable compound around it. Main Body of Silica - O – Si – O – Si – O – Si – O – Si – O - OH OH OH OH O O O O

9. If a compound/solute to be separated is polar , it can interact with polar –OH groups of silica. It can either make H-bonds (strong) or it can interact with VanderWall Interactions (weak) with the silica. If a compound make H bond with the silica then it will travel less with the solvent (non-polar). On the other hand if the compound make Vanderwall interactions with silica, then it can travel far with the solvent. If the compound/solute is non-polar , then it can not make any bond with the silica and it will move along with the solvent. Because of –OH group on surface of silica, solute can make either these two types of interactions H-bonding Vander Wall Interactions Stick Strongly Stick Loosely Travel Less distance Travel More distance How does TLC works?