THIN LAYER CHROMATOGRAPHY(TLC)

INDEX INTRODUCTION PRINCIPLE ADVANTAGES TECHNIQUE OF TLC PRAPARATION OF TLC PLATE STATIONARY AND MOBILE PHASE ACTIVATION OF TLC PLATES APPLICATION OF SAMPLE DEVELOPMENT OF CHAMBER EVALUATION PROBLEMS IN TLC APPLICATIONS

INTRODUCTION Thinlayer chromatography is a planar form of chromatography Thin LayerChromatography (TLC) is a sensitive, fast, simple and inexpensive analytical technique. It is a micro technique; as little as 10-9g of material can be detected Qualitative and quantitative analysis

PRINCIPLE Chromatography is a sophisticated method of separating mixtures of two or morecompounds. The separation is accomplished by the distribution of the mixture between two phases: one that is stationary and one that is moving. Chromatography works on the principle that different compounds will have different solubilities and adsorption to the two phases between which they are to be partitioned.

ADSORPTION CHROMATOGRAPHY PARTITION CHROMATOGRAPHY SEPERATION LARGELY OCCUR BY

ADVANTAGES Simple Equipment Short development Time Wide choice of stationary phase Quick recovery of separated constituents Separation effects Easy visualization of separated compounds Trace analysis

TECHNIQUE OF THIN LAYER CHROMATOGRAPHY

PREPARATION OF TLC PLATES POURING DIPPING SPRAYING SPREADING PRE-COATED PLATES

STATIONARY PHASE CHOICE OF ADSORBENT Solubility of the substance Nature of the compound Reactivity of the compound Chemical reactivity of compound

ADSORBENTS Silica N No binder, no fluorescence Silica G Gypsum , without indicator Silica G-HR Gypsum without indicator Silica p Preparative grade with indicator Aluminium oxide G Aluminium oxide N No binder Cellulose MN 301 Standard grade Cellulose MN 301 HR High purity grade

Inorganic adsorbent Seperation Aluminium silicate Sterols Bentonite Vit D Calicium carbonate xanthophylls Fullers earth aminoacids Silica gel fattyacids Calicium hydroxide Carotenoids

ORGANIC ADSORBENTS CELLULOSE CHARCOAL ION EXCHANGE RESINS

MOBILE PHASE CHOICE OF MOBILE PHASE Nature of the constituent Nature of the process Rate of migration

Least Eluting Power (alumina as adsorbent) Petroleum ether (hexane; pentane) Cyclohexane Carbon tetrachloride Benzene DichIoromethane Chloroform Methanol Water Pyridine Greatest Eluting Power (alumina as adsorbent) Organic acids

ACTIVATION OF TLC PLATES AIR DRYING HOT- AIR OVEN

PURIFICATION OF ADSORBENTS The iron present as an impurity in silicagel-G cause distortion of the plate. Iron free adsorbent can be achieved by using pre-coated and air dried plates. Use of methanol and conc. HCl. Purified plates are dried and activated at 110 degree.

APPLICATION OF SAMPLE ,

Sample is applied as solution in a non-polar solvent , if we use polar solvent spreading out of startingspot Affect Rf value volatile solvent Area of application should be small Size of the spot should be small

DEVELOPMENT OF TLC PLATES

DEVELOPMENT TECHNIQUES ASCENDING-TECHNIQUE MULTIPLE DEVELOPMENT TWO- DIMENSIONAL CENTRIFUGAL CHROMATOGRAPHY HORIZONTAL TLC PREPARATIVE TLC

ASCENDING TECHNIQUE TLC after development chamber drying e.g.beaker with a lid or a closed jar

CENTRIFUGAL CHROMATOGRAPHY

DETECTION REAGENTS REAGENT DETECTION COMPOUND RHODAMINE 6 B LIPIDS Silver nitrate and H2O2 Halogenated hydrocarbons Thymol sugars Uv-light fluorescence FLORESCENCE CONTANING COMPOUNDS Bromocresol green Organic acids Chloranil reagent phenols Emerson reagent Ninhydrin amino acids Bials reagent glycosides

EVALUATION OF TLC PLATES Qualitative evaluation Rf value = distance traveled by substance distance traveled by solvent front

The Rf (=retardation factor) depends on the following parameters: solvent system absorbent (grain size, water content, thickness) amount of material spotted temperature Ideal 0.2 ≤Rf ≤0.8 Rf’s increase with increasing polarity Methanol >ethyl acetate>Methylene chloride (aka dichloromethane)> hexane

QUANTITATIVE ANALYSIS Direct method measurement of spot area Densitometry spectrophotometry Indirect methods UV-spectrophotometry Colorimetry

PROBLEMS IN TLC Over-large spots Uneven advance of solvent front Streaking

APPLICATIONS Purity of sample Examination of reactions Identification of compounds Biochemical analysis Quality testing Pharmaceutical industry

. SHABANA 08171S0410 M.PHARM ANALYSIS BHARAT COLLEGE OF PHARMACY