HPTLC SEMINARl

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HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY :

Rahul Patil HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

Contents:

Contents Introduction. Principle of HPTLC. Difference between TLC & HPTLC. Steps involved in HPTLC. Material used for plates. Mobile phase. Sample application. HPTLC Plate development. Applications of HPTLC.

Introduction:

Introduction Sophisticated form of TLC. In 1973,Halpaap introduced first “Nano TLC plates’’ In 1977,the first major HPTLC publication is “HPTLC-high”

Principle:

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.

Fig. HPTLC Instrumentation:

Fig. HPTLC Instrumentation

Difference between HPTLC & TLC:

Difference between HPTLC & TLC HPTLC TLC Particle size 4-8 µm 5-20 µm Sorbent layer thickness 100 µm 250 µm Efficiency High Low Separations 3-5cm 10-15cm Analysis time Faster Slower

Slide 7:

HPTLC TLC Development chamber Less amount of mobile phase More amount of mobile phase Sample spotting Auto sample Manual spotting Scanning Visible/Fluoros-scanner scan the entire chromatogram Not possible

Steps in HPTLC :

Steps in HPTLC Selection of chromatographic layer. Layer pre-washing. Preparation of sample. Application of sample. Chromatographic development. Detection of spot. Scanning and documentation of chromatoplate .

Sample Preparation :

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.

Application of sample :

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

Slide 11:

Micro syringes are preferred if automatic application devices are not available. Volume recommended for HPTLC-0.5-5 μ l. 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 :

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.

Sorbents used in HPTLC :

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:

Plates used in HPTLC Glass plates. Polyester/polyethylene plates. Aluminium plates.

Solvents used for pre-washing :

Solvents used for pre-washing Methanol. Chloroform: Methanol (1:1) Chloroform: Methanol: Ammonia (90:10:1 ) Methylene chloride: Methanol ( 1:1 ) Ammonia solution (1%)

Pre-conditioning (Chamber Saturation) :

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 R f values.

Mobile phase:

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.

Slide 18:

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:

Methods of HPTLC development Vertical development. Vario method development. Horizontal development. Automatic multiple development .

Detection and visualization:

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 :

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 :

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.

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