This document discusses high performance thin layer chromatography (HPTLC). It begins with an introduction and overview of HPTLC principles, instrumentation, differences from TLC, and application steps. The document then provides more details on the HPTLC instrumentation components, factors affecting separation, common stationary and mobile phases used, and application techniques. It concludes by discussing detection methods and some key applications of HPTLC in pharmaceuticals, food analysis, clinical studies, and forensics.

1. Presented by :
Arvind Singh Heer
MSc-I
(Sem-I)
Analytical Chemistry
MITHIBAI COLLEGE

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

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

4.  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.

5.  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.

8.  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.

9.  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.

10.  Glass plates.
 Polyester/polyethylene plates.
 Aluminium plates.

11.  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.

12.  Methanol.
 Chloroform: Methanol (1:1)
 Chloroform: Methanol: Ammonia (90:10:1 )
 Methylene chloride: Methanol ( 1:1 )
 Ammonia solution (1%)

13. The selection of sample application
technique and device to be used
depends primarily on,
• Sample volume
• No. of samples to be applied
• Required precision

14.  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.

15.  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.

16.  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.

17.  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.

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.

19.  Vertical development.
 Vario method development.
 Horizontal development.
 Automatic multiple development.

20.  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.

21.  Layer thickness.
 Mobile phase.
 Solvent purity
 Size of developing chamber
 Sample volume to be spotted
 Size of initial spot
 Solvent level in chamber.

22.  Pharmaceutical industry : quality control,
purity check etc.
 Food analysis : quality control, stability
testing etc.
 Clinical applications : metabolism studies,
drug screening etc.
 Forensic : poisoning investigations.

23.  HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY
Manmohan srivastava
-THANK YOU