A complete Detail on HPTLC which gives a broad idea about the topic

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