Thin Layer Chromatography-Lecture Slides, Slides of Chemistry

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Dr. S.D. SARASWATHY Assistant Professor Department of Biomedical Science Bharathidasan University Tiruchirappalli

Thin Layer

Chromatography (TLC)

Biotechniques

KEY CONCEPTS

• Introduction
• General Principle
• TLC Technique
• Applications

• TLC technique involves the distribution of

components of a mixture to be separated between

two phases.

• The components of the mixture are partitioned

between an adsorbent (stationary phase), and a

solvent (mobile phase).

• Different compounds will have different solubility

and adsorption to the two phases between which

they are to be partitioned.

• In TLC separation of the individual substances is

based on their relative affinities towards stationary

and mobile phases.

PRINCIPLE

• The stationary phase: is a thin layer of adsorbent

(usually silica gel or alumina) coated on a plate.

• The mobile phase: is a developing liquid which

flows through the stationary phase, carrying the

samples with it.

• Components with more affinity towards stationary

phase travels slower.

• Components with less affinity towards stationary

phase travels faster.

PRINCIPLE

• R f

value indicates the position of migrated spots on

chromatogram.

• In TLC the results are represented by R f

value which

represents the migration of solute relative to the

solvent front.

• The R f

value is calculated as:-

Distance travelled by the solute R f Value = Distance travelled by the solvent front

R

f

VALUE

• A plastic, glass or aluminum sheet is coated with a thin layer of silica gel (adsorbent).
• Plates must be dried, activated and stored in desiccator until used.

TLC - TECHNIQUE

STEP 1: Preparation of Slurry STEP 2: Preparation of Tank

• Solvent mixtures should be freshly prepared for analysis.
• Solvent is poured down side of the tank (1.5cm depth).
• Tank is covered with the glass lid and kept for saturation.
• A very small amount of sample (solution) to be analyzed is applied in a small spot with a capillary tube, ~1cm from the bottom of the TLC plate. STEP 3: Application of Sample (Spot)

TLC - TECHNIQUE

• Individual components in the sample move up at different rates.
• More polar analytes interact more strongly with the stationary phase move very s lowly up.
• More nonpolar analytes interact less strongly with the polar silica gel and more strongly with the less polar mobile phase m ove higher up.
• Once the solvent reaches the top (below ~1-2 cm) of the TLC sheet the plate is removed from the developing chamber and position of solvent front is marked. http://classes.kvcc.edu/chm220/TLC% Lab/lab/procedures1.htm

TLC - TECHNIQUE

• The solvent is allowed to evaporate from the TLC sheet.
• As the compound is colorless, it can be visualized by suitable methods. - Lipids - Iodine vapors - Amino acids - Ninhydrin reagent.
• Also, manganese-activated zinc silicate (fluorescent compound) , is added to the adsorbent that allows the visualization of spots under a black light (UV 254 lamp).
• Once visible, the R f value of each spot can be determined. http://orgchemboulder.com/Technique/Procedures /TLC/TLC.shtml

• Qualitative results of TLC
  • expressed as fractions of 1.
  • can be expressed from Rf values (Ex: Rf x 100)
  • no more than two decimal places
• R f values can be used to aid in the identification of a substance by comparison to standards.
• Comparison should be made only between spots on the same sheet, run at the same time.
• Identical substances will have the same R f value, whereas non- identical compounds will differ in their R f values.

RESULTS OF TLC - R

f

VALUE

• TLC is used in qualitative and quantitative analysis to separate organic compounds and to test the purity of compounds.
• This technique is useful for separation of lipids, amino acids and sugars etc.
• It is useful in:
  • Identification of components of a mixture.
  • Following the course of a reaction,
  • Analyzing fractions collected during purification,
  • Analyzing the purity of a compound. APPLICATIONS