HPTLC

1. HIGH PERFORMANCE THIN
LAYER
CHROMATOGRAPHY
BY: DHARATI JOSHI
M.Sc. Biotechnology
1

2. CONTENTS
Introduction about TLC & HPTLC
About TLC
Difference between TLC & HPTLC
About HPTLC
Principle of HPTLC
Instrumentation
Steps involve in HPTLC
Factors affecting HPTLC
Applications of HPTLC
2

3. HPTLC is a modified version of TLC.
HPTLC also comes under planer chromatography or flat-
bed chromatography.
Planer chromatography is one type of chromatography
technique in which the stationary phase is on a flat plate &
the mobile phase moves through stationary phase by
capillary action.
HPTLC also a type of liquid chromatography in which mobile
phase is in liquid state while stationary phase in solid state.
3

4. Liquid
chromatography
Paper
chromatography
TLC
(10-12 µm)
HPTLC
(5-6 µm)
Planer
chromatography
4

5. Thin Layer Chromatography
Thin Layer chromatography is a method used to
separate non-volatile mixture.
TLC is performed on a sheet of glass, aluminium foil,
plastic which is coated with a thin layer of adsorbent
material such as silica gel, aluminium oxide or cellulose.
It is a type of planer chromatography.
5

6. Principle:
The separation depends on the relative affinity of
compounds towards stationary & mobile phase.
The molecules which has higher affinity for stationary
phase are move slower than others. That’s way
separation is occur.
After the separation spots are obtained on a plate. These
are visualized by simply projecting UV light onto sheet.
6

7. 7

8. Difference between TLC & HPTLC:
In both process steps are same. The main difference
between them is in the characteristics of separation.
HPTLC plates are based on optimized silica gel60 with
smaller particle size than used for TLC.
8

9. COMPARISON OF TLC & HPTLC
9

10. HPTLC
HPTLC is a more sophisticated & enhanced version of
TLC.
It is automated version of TLC to increase more
accuracy & resolution.
Automation in sample loading is useful to overcome the
variability in droplet size & position. Where in TLC the
sample is applied to TLC plate by hand.
10

11. Principle
It follows same principle as TLC(Adsorption
chromatography) i.e. the principle of separation is
adsorption.
Mobile phase flow by capillary action effect.
And component move according to their affinities
towards the adsorbent.
11

12. The molecule with higher affinity toward adsorbent
travels slowly.
And the component with lesser affinity towards the
stationary phase travels faster.
Thus the components are separated on a
chromatographic plate according to their affinity and
separation also based on their solubility in mobile phase.
12

13. Instrumentation of HPTLC
13
Applicator Automatic developing chamber

14. Instrumentation of HPTLC
14ScannerDigital camera for photodetection

15. 15

16. 1. Selection of chromatographic plates:
Hand made plates which are made up of cellulose and
other materials are not much use now a day.
Pre-coated plates are used which are available with
different support(matrix) & different sorbent(stationary
phase).
As a support materials: Glass, Polyester, Aluminium.
As a sorbent: Silica gel60F, Aluminium oxide, cellulose.
Smaller particle size of silica helps in greater resolution
and sensitivity. 16

17. 17
HPTLC Plates

18. 2. Layer pre-washing:
It is a purification step.
The main purpose of the pre-washing is to remove
impurities such as water vapour, other volatile
substances which are comes from the lab environment.
The major disadvantage of silica gel60F is, it contain
iron as impurity.
This iron is removed by using Methanol : water (9:1),this
is the major advantage of pre- washing step.
18

19. 3. Activation of pre-coated plates:
Freshly opened box of plates does not need activation.
If plates exposed to high humidity or kept in hand for a
longer time at that time activation of plate is require.
Plates are activated by placing in an oven at 110-120°C
for 30 min, this step will removes water that has been
physically absorbed on surface of plate.
Activation at higher temperature for longer time is
avoided which leads to very active layer and there is risk
of sample decomposition.
19

20. 4. Sample preparation:
It is important to prepare proper sample for successful
separation.
Sample and reference substances should be dissolved in
the same solvent to ensure comparable distribution at
starting zones.
Solvents used are : Methanol, Chloroform: Methanol
(1:1), Ethyl acetate: Methanol (1:1) etc.
20

21. 5. Sample application:
Usual concentration range is 0.1-1µg/µl.
Above this causes poor separation.
Volume recommended for HPTLC is 0.5-5µl.
The size of sample spot must be not large than 1mm in
diameter.
The problem of overloading can be overcome by applying
the sample as band.
The major criteria is that it should not damage the
surface while applying sample. 21

22. 6. Selection of mobile phase:
Chemical properties of analytes & sorbent layer should
be considered while selection of mobile phase.
The less amount of mobile phase is required then TLC.
Due to small amount of mobile phase, it prevents errors
in result which is occur due to mobile phase.
22

23. 7. Pre-conditioning (chamber saturation):
Chamber is saturated by lining with filter paper for 30
min prior to development of chromatograph.
For low polarity mobile phase there is no need of
saturation.
However saturation is needed for highly polar mobile
phase.
Chamber saturation influence separation profile.
23

24. 8. Chromatographic development and drying:
 Plates are spotted with sample and air dried & placed in
the developing chambers.
The different methods used for development of
chromatographs are like: Ascending, Descending,
Horizontal.
After development, remove the plate and mobile phase is
removed from the plate to avoid contamination of lab
atmosphere.
Dry in vacuum desiccators with protection from heat
and light. 24

25. 9. Detection and visualization:
Detection under UV light is first choice.
Fluorescent compounds can be seen at 254 nm (short
wavelength) or at 336 nm (long wavelength).
Spots of non fluorescent compounds can be seen by
using fluorescent stationary phase.eg silica gel Gf.
Non UV absorbing compounds are visualized by dipping
the plates in 0.1% iodine solution.
25

26. 26

27. 10. Scanning & documentation:
The scanner converts band into peak and peak area
which is use to determine the concentration of
substance on spot/band.
This is measured by instrument and recorded.
This result is also get by hard copy or we can get print
form of our result.
27

28. Factors affecting HPTLC:
Types of stationary phase
Types of mobile phase
Layer thickness
Temperature
Mode of development
Amount of sample
Dipping zone
28

29. Applications of HPTLC:
Pharmaceutical industry: Quality control, purity test.
Food Analysis: QC, stability testing.
Clinical applications: Metabolism studies, drug
screening etc.
Biomedical analysis: Separation of gangliosides
Forensic: Poisoning investigation
Environment analysis: Pesticides in drinking water etc.
Analysis of drug in blood etc.
29

30. REFERENCES:
www.camag.com
HPTLC by Manmohan Shrivastava
www.slideshare.net
www.pharmatutor.org
www.hptlc.com
www.ncbi.nlm.nih.gov
www.sciencedirect.com
www.researchgate.net 30

31. 31
HAVE YOU ANY QUESTIONS?

32. 32