HIGH PERFORMANCE

THIN LAYER CHROMATOGRAPH

YPresented by :

Arvind Singh Heer

MSc-I(Sem-I)

Analytical Chemistry

MITHIBAI COLLEGE

CONTENTS Introduction. Principle of HPTLC. Instrumentation of HPTLC. Difference between TLC & HPTLC. Steps involved in HPTLC. Applications of HPTLC.

INTRODUCTION Sophisticated form of TLC. In 1973,Halpaap introduced first

“Nano TLC plates.’’ In 1977,the first major HPTLC

publication is “HPTLC-high”

PRINCIPLE Separation may result due to

adsorption or partition or by both phenomenon depending upon the nature of adsorbents used on plates and solvents system used for development.

INSTRUMENTATION OF HPTLC CONSISTS OF FOLLOWING Lamp selector Entrance lens slit Monochromator entry slit Grating Mirror Slit aperture disc Mirror Beam splitter Reference photo multiplier Measuring photo multiplier Photo diode for transmission measurements.

INSTRUMENTATION

DIFFERENCE BETWEEN HPTLC & TLC

STEPS IN HPTLC Selection of chromatographic layer. Plates. Activation of pre-coated material. Preparation of sample. Layer pre-washing. Application of sample. Pre-conditioning. Mobile phase. Chromatographic development. Detection of spot. Scanning and documentation of

chromatoplate.

SORBENTS USED IN HPTLC Silica gel 60F, it analyses 80% of

drugs.

Aluminium oxide, it analyses the basic substances and steroids.

Cellulose.

Silica gel chemically modified in amino group and CN.

PLATES USED IN HPTLC Glass plates.

Polyester/polyethylene plates.

Aluminium plates.

SAMPLE PREPARATION For normal chromatography , solvent

should be non-polar and volatile.

For reversed chromatography , polar solvent is used for dissolving the sample.

Sample and reference substances should be dissolved in the same solvent to ensure comparable distribution at starting zones.

SOLVENTS USED FOR PRE-WASHING Methanol.

Chloroform: Methanol (1:1)

Chloroform: Methanol: Ammonia (90:10:1 )

Methylene chloride: Methanol ( 1:1 )

Ammonia solution (1%)

APPLICATION OF SAMPLE The selection of sample

application technique and device to be used depends primarily on,

• Sample volume• No. of samples to be applied• Required precision

Micro syringes are preferred if automatic application devices are not available.

Volume recommended for HPTLC-0.5-5μl.

Sample spotting should not be excess or not low.

Problem from overloading can be overcome by applying the sample as band.

SOME APPLICATORS USED FOR APPLICATION OF SAMPLE By capillary tube,0.1-0.2μl volume

sample spot is applied.

By micro syringes, 1μl sample can apply either as spot or band.

By automatic sample applicator.

By micro bulb pipette.

PRE-CONDITIONING (CHAMBER SATURATION)

Time required for the saturation depends on the mobile phase.

If unsaturated chamber used for development, the solvent evaporates from the plate mainly at the solvent front and it results in increased Rf values.

MOBILE PHASE Solvent composition expressed in

v/v. Mobile phase should be of high

graded. Chemical properties , analyses and

sorbent layer factors should be considered while selection of mobile phase.

If possible mobile phase containing more than 3 or 4 components should be avoided.

Prevents contamination of solvents.

Multi-component of mobile phase once used is not recommended for re-use.

Chemical reaction avoided between SP & MP. e.g. Acetic acid, Ammonia.

METHODS OF HPTLC DEVELOPMENT Vertical development.

Vario method development.

Horizontal development.

Automatic multiple development.

DETECTION AND VISUALIZATION First spots detects under UV light

because it is non destructive. Fluorescent compound spots can

be seen at 254nm or 366nm. For non fluorescent compound

spots, fluorescent stationary phase (silica gel GF) is used.

Non UV absorbing compounds detects by dipping the plates in 0.1% iodine solution.

FACTORS INFLUENCING SEPARATION AND RESOLUTION OF SPOTS Layer thickness. Mobile phase. Solvent purity Size of developing chamber Sample volume to be spotted Size of initial spot Solvent level in chamber.

APPLICATIONS OF HPTLC Pharmaceutical industry : quality

control, purity check etc.

Food analysis : quality control, stability testing etc.

Clinical applications : metabolism studies, drug screening etc.

Forensic : poisoning investigations.

REFRENCE HIGH PERFORMANCE THIN LAYER

CHROMATOGRAPHY Manmohan srivastava

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