HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

(HPTLC)

Presented by;Aiswarya . A. T,

First year M.Pharm,Department of

Pharmacy Practice,Grace college of

Pharmacy.

Usual concentration of applied samples 0.1 to 1 µg / µl for qualitative Analysis and quantity may vary in quantization based on UV absorption 1 to 5 µl for spot and 10 µL for band application.

MANUAL , SEMI-AUTOMATIC , AUTOMATIC APPLICATION Manual with calibrated capillaries Semi and auto-application through applicators

Applicators use spray on or touch and deliver technique for application.

Application of sample and standard

Manual Sample Applicatoreasy applicationperformed with disposable capillary pipettes, guided by the capillary holder

Semi automatic sample applicatorsuitable for routine useWith the linomat , samples are sprayed onto the chromatographic layer in the form of narrow bandssolvent of the sample evaporates almost entirely gives a narrow band of selectable length

Automatic Sample ApplicatorSamples are either applied as spots through contact transfer (0.1-5 micro lit) or as bands or rectangles (0.5->50 micro lit) using the spray on techniques

Chromatographic Development

Start of development

Low solvent consumption

Reproducible pre-equilibrium with Solvent vapour

1. Twin trough chamber

2. Automatic developing chamber (ADC)

fully automatic and independent of environmental effects The activity and pre-conditioning of the layer , chamber saturation, developing distance and final drying can be pre-set and automatically monitored by ADC

Detection, scanning & documentation Detection and visualization

· Detection under UV light is first choice - non destructive · Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length) · Spots of non fluorescent compounds can be seen - fluorescent stationary phase is used - silica gel GF· Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1% iodine solution· When individual component does not respond to UV - derivatisation required for detection

CHROMATOGRAM IMMERSION DEVICEDipping techniqueimmersed and withdrawn at a controlled uniform speedHPTLC SPRAYER a charger and a pump unit

with two kinds of spray heads

type A is for spray solutions of normal viscosity, e.g. lower alcohol solutions

type B is for liquids of higher viscosity, e.g. sulfuric acid reagents

TLC SPRAY CABINET

complete removal of reagent mist while spraying TLC plates

TLC PLATE HEATER

homogenous heating across the plate

Scanningconnected to computer.The scanner features three light sources: a deuterium lamp , a tungsten lamp and a high pressure mercury lamp.The scanning speed is selectable between 1 and 100 mm/s