hptlc steps
TRANSCRIPT
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HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY
(HPTLC)
Presented by;Aiswarya . A. T,
First year M.Pharm,Department of
Pharmacy Practice,Grace college of
Pharmacy.
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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
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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
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Chromatographic Development
Start of development
Low solvent consumption
Reproducible pre-equilibrium with Solvent vapour
1. Twin trough chamber
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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
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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
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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
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TLC SPRAY CABINET
complete removal of reagent mist while spraying TLC plates
TLC PLATE HEATER
homogenous heating across the plate
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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
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