1. HPTLC
( High performance thin
layer chromatography)
VAISHNAVI U.S.D.N
M.Pharmacy(Pharmaceutical Chemistry)
170213884006
UNDER THE GUIDENCE:
CEEMA MATHEW,M.pharmacy
Asst.Professor

2. INDEX
INTRODUCTION
 ADVANTAGES OF HPTLC OVER HPLC &
TLC
 FEATURES OF HPTLC
 STEPS INVOLVED IN HPTLC
 DIFFERENCES BETWEEN TLC AND
HPTLC

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3. INTRODUCTION
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4. CHROMATOGRAPHY is a broad
range of physical methods used
to separate and or to analyze
complex mixtures. The
components to be separated are
distributed between two phases: a
s ta tio na ry p ha s e bed and a
m o bile p ha s e which percolates
through the stationary bed.
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5. High performance thin layer
chromatography (HPTLC) is an
enhanced form of
thin layer chromatography (TLC).
A number of enhancements can be
made to the basic method of thin
layer chromatography to automate
the different steps, to increase the
resolution achieved and to 5allow

6. 
n
Pri
Automation is useful to overcome
the uncertainty in droplet size and
position when the sample is
applied to the TLC plate by hand.
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7. Advantages of HPTLC over HPLC
Sample and standard, both can be
used at a time.
 Evaluation time is less.
 Use of internal standard is not
necessary.
 Simultaneously 2 or 3 persons can
work but not in case of HPLC.
 Solvent degasing and removal of
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

8. Advantages of HPTLC over TLC
Visual chromatogram
 simplicity
 Multiple sample handling
 Low running and maintenance costs
and disposable layer etc
 Detection by TLC scanner
 Spotting by applicator
 Highly precised
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9. FEATURES OF
HPTLC
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10. 
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Simultaneous processing of sample and standard
- better analytical precision and accuracy less
need for Internal Standard
Several analysts work simultaneously
Lower analysis time and less cost per analysis
Low maintenance cost
Simple sample preparation - handle samples of
divergent nature
No prior treatment for solvents like filtration and
degassing
Low mobile phase consumption per sample
No interference from previous analysis - fresh
stationary and mobile phases for each analysis no contamination
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Visual detection possible - open system

11. STEPS
INVOLVED IN
HPTLC
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13. Selection of chromatographic layer
· Precoated plates - different support
materials - different Sorbents available
· 80% of analysis - silica gel GF
· Basic substances, alkaloids and steroids Aluminum oxide
· Amino acids, dipeptides, sugars and
alkaloids - cellulose
. Non-polar substances, fatty acids,
carotenoids, cholesterol - RP2, RP8 and
13
RP18

14. Sample and Standard Preparation
To avoid interference from impurities and
watervapours
Solvents used are
Methanol, Chloroform: Methanol (1:1)
Ethyl acetate: Methanol (1:1)
Chloroform: Methanol: Ammonia
(90:10:1)
Methylene chloride : Methanol (1:1)
1% Ammonia or 1% Acetic acid
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15. Layer pre-washing & pre-conditioning
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If any impurities are present, those
are need to be removed hence pre
washing is done.
Conc.HCl : Methanol(1:9)
Plate is dipped in the soln, allowed for the
solvent to pass throughout the plate. Hence
impurities are washed out
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16. Activation of pre-coated plates
Freshly open box of plates do not
require activation
Plates exposed to high humidity or
kept on hand for long time to be
activated
By placing in an oven at 110-120ºc
for 30’ prior to spotting (pre
conditioning)
Aluminum sheets should be kept in
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between two glass plates and

17. Application of sample and standard
· Usual concentration range is 0.1-1µg /
µl
· Above this causes poor separation
· Linomat IV (automatic applicator) nitrogen gas sprays sample and
standard from syringe on TLC plates
as bands
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18. Selection of Mobile phase
Can be explained by STAHL’S TRIANGLE
Hydrophilic
Non-Polar
E
Polar
Lipophilic
M
Active
E- ELUENT
S- SORBANT
M- MIXTURE
S
Inactive
EG:If
E- hydrophilic
then
M- polar &
S- inactive
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19. 
Trial and error
- one’s own experience and Literature
Normal phase
- Stationary phase is polar
- Mobile phase is non polar
- Non-polar compounds eluted first because of lower affinity with stationary phase
- Polar compounds retained because of higher affinity with the stationary phase
Reversed phase
- Stationary phase is non polar
- Mobile phase is polar
- Polar compounds eluted first because of lower affinity with stationary phase
- Non-Polar compounds retained because of higher affinity with the stationary phase
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20. Chromatographic development and drying
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After development, remove the plate and
mobile phase is removed from the plate to
avoid contamination of lab atmosphere
Dry in vacuum desiccator
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21. Chromatographic Develpment
1. TWIN TROUGH CHAMBER
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22. 22

23. 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
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27. APPLICATIONS
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Pharmaceutical research
Bio medical analysis
Clinical analysis
Environment analysis
food industry
Quantification of herbal extracts
Therapeutic drug monitoring to determine its
concentration and metabolite in blood urine
Analysis of environment pollution level
Characterization of hazards in industrial waste
Quantitative determination of prostaglandins &
thromboxanes in plasma.
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28. DIFFERENCES
BETWEEN
TLC AND HPTLC
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29. PARAMETER
TLC
HPTLC
Type of
Hand made/pre
chromatographic coated
plates
Pre coated
Absorbant layer
200-250mm
100-150mm
Particle size
5-20 micrometer 4-8 micrometer
Application of
sample
Manual/semi
automatic
Semi automatic
Shape of sample
spot
band
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30. Spot size
3-6mm
1-2mm
Sample volume
1-10 microlit
0.1-2microlit
No of sample per 15-20
plate
40-45
Optimal
development
distance
10-15cm
5-7cm
Development
time
Depend upon
mobile phase
40% less than
TLC
Quantization
Manual
Manual/
instrumentation
Reproducibility
of result
difficult
Reproducible
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31. REFERENCES

Ajaz Ahmad, M Mujeeb, Bibhu Prasad Panda (2010) An HPTLC Method
for the Simultaneous Analysis of Compactin and Citrinin in Penicillium
citrinum Fermentation Broth. Journal of Planar Chromatography-modern
TLC 23 (4), 282–285
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1994 Rebecca Carrier and Julie Bordonaro - Intro to Biochemical
Engineering Term Project, merged with the 1997 project by Kevin Yip
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Puri, A., Ahmad, A. and Panda, B. P. (2010), Development of an HPTLCbased diagnostic method for invasive aspergillosis. Biomed. Chromatogr.,
24: 887–892. doi: 10.1002/bmc.1382
Wikipedia
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Pharmainfo.dot
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