This document discusses techniques for high performance thin layer chromatography (HPTLC). HPTLC allows for the manual, semi-automatic, or automatic application of samples in concentrations of 0.1 to 5 μg/μl as spots or bands up to 50 microliters. Chromatographic development then occurs in a twin trough chamber or automatic developing chamber, controlling solvent consumption and vapor equilibrium. Compounds can be detected under UV light at 254 or 366 nm, through derivatization, or by dipping plates in iodine solution. The document also describes devices for chromatography immersion, spraying, heating plates, and scanning plates connected to a computer.

1. HIGH PERFORMANCE THIN
LAYER CHROMATOGRAPHY
(HPTLC)
Presented by;
Aiswarya . A. T,
First year M.Pharm,
Department of Pharmacy Practice,
Grace college of Pharmacy.

4. 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

5. Manual Sample Applicator
easy application
performed with disposable capillary pipettes, guided
by the capillary holder
Semi automatic sample applicator
suitable for routine use
With the linomat , samples are sprayed onto the
chromatographic layer in the form of narrow bands
solvent of the sample evaporates almost entirely
gives a narrow band of selectable length
Automatic Sample Applicator
Samples 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

6. Chromatographic
Development
Start of
development
Low solvent
consumption
Reproducible pre-
equilibrium with
Solvent vapour
1. Twin trough chamber

7. 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

8. Detection, scanning &
documentationDetection 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

9. CHROMATOGRAM IMMERSION
DEVICE
Dipping technique
immersed and withdrawn at a controlled
uniform speed
HPTLC 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

10. TLC SPRAY CABINET
complete removal of reagent mist while
spraying TLC plates
TLC PLATE HEATER
homogenous heating across the plate

11. Scanning
connected 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