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 PARTITION CHROMATOGRAPHY

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

ADSORBENTSSilica 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 Gypsum without indicator

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

development chambere.g.beakerwith a lid or a closed jar

afterdrying

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 phenols

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 polarityMethanol >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

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SHABANA 08171S0410

M.PHARM ANALYSIS BHARAT COLLEGE OF PHARMACY