HPTLC: INTRODUCTION, PRINCIPLE AND APPLICATION

1. HPTLC
( High Performance Thin Layer Chormatography)
By: Poonam Aher

2. Contents:
 Introduction.
 Introduction to H.P.T.L.C.
 principle.
 Selection of HPTLC plates.
 Activation of pre coated plates.
 Sample preparation.
 Evaluation of spot or band.
 Application of HPTLC.
 Difference between TLC and HPTLC.

3.  Introduction:
chromatography is a physical process of separation in
which the component is to be separated are distributed
between two immiscible a stationary phase with has large
surface area and a mobile phase which is in constant
motion through the stationary phase.

4. •Introduction to H.P.T.L.C
H.P.T.L.C is the improved method of T.L.C which utilize
the conventional technique of TLC in more optimize way.
It is also known as a planer chromatography or Flat- bad
chromatography.

5.  Principle:
HPTLC take place in high speed capillary flow range of
the mobile phase, There are three main step in HPTLC
1] sample to analyzed to chromatogram layer volume
precision and suitable position are achieved by use of
suitable instrument.
2] solvent (mobile phase) migrates the planned distance in
layer (stationary) by capillary action in this process sample
separated in its components.
3] separation tracks are scanned in densitometer with light
beam in visible or uv region

7. Selection of HPTLC plates:
Previously hand made plate is used in TLC for both
qualitative and quantitative work, certain draw back
with that is non uniformly layer , formation of thick layer
paved for advent pre coated plates.
Now a days pre coated plates are available in different
format and thickness by different manufactures. these
plates are used for both qualitative and quantitative
purpose in HPTLC.
 glass plates .
Polyester /polyethylene.
Aluminium plates.

8. GLASS PLATES:
 Resistance to heat
 Easy to handle Thickness 1.3mm
 Offer superior plane and smooth surface.
Fragile
High weight
High production cost

9.  POLY ESTER POLYETHYLENE:
 Thickness of plate 0.2mm
 It can be produce in Roll form.
 Unbreakable.
 Less packing material required.
Development of plate is not above temp. 1200losses of its shape.

10.  Aluminium plates:
 Thickness of plate 0.1mm
 It can be produce in Roll form.
 Unbreakable.
 Less packing material required.
Development of plate is not above temp. 1200losses of its shape.

11. 1.Plate coater ,
a.Hand operated
 The manual plate coater
functions in the same manner
as the automatic coater , except
with this model the plates are
pushed through by hand , one
after the other and lifted off on
the other slide.
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12. b.AUTOMATIC PLATE COATER
 The glass plates to be coated are
conveyed underneath a hopper filled with
the adsorbent suspension.
 The plates are moved by a motorized
conveying system at a uniform feeding
rate of 10cm/s, to ensure a uniform speed.
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13. 2.Drying Rack
 The Drying Rack consists of ten
individual aluminum trays.
 A tin box for storing the trays and two
wire handles , to move the stack while
hot ,are supplied.
 The drying rack is convenient to use ,
particularly when TLC plates are prepared
with the automatic plate coater in large runs.
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14. 3.PLATE CUTTER
 Used to cut HPTLC plates easily and
more precisely.
 Cuts plates with a thickness up to
3mm.
 Does not damage the sensitive layer.
 Easy to handle .Read the required
size from the scale directly.
 Helps saving costs on pre-coated plates
of high quality by preventing off cuts.
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15. Sorbents used in HPTLC Plates:
Sorbent used in conventional
TLC can be used in HPTLC with or
with out modification.
Silica gel 65F(modified)
Highly purified silica gel60.
Aluminium oxide.
Microcrystalline.
Silica gel G particle size of sorbent
Reversed stationary phase. HPTLC 6 m
Hybrid plates. TLC 10 m
Layer thickness in HPTLC -100-200 m
in TLC -250 m

16.  Layer prewashing :
 Ascending method .
 continuous method.
 Deeping method.
 solvent used for washing
 Methanol
 Chloroform: methanol:amonia(90:10:1)
 Chloroform: methanol(1:1)
 Ammonia solution (1%)

17. Activation of precoated plates:
The plates are activated by placing in oven at 110-1200c for
30 minutes, this step will remove the water that has been
physical absorbed on the surface at solvent layer.
Freshly open box of HPTLC plates usually not required
activation.
Activation at higher temperature and long time is avoided
which may tends to vary active layer and sample
decomposition .

18. SAMPLE PREPARATION:
Proper sample preparation is pre requisite for the success
HPTLC separation.
Beside maximizing the yield of analyte in the selected
solvent ,stability of the analyte during extraction and
analysis must consider. there for choice of suitable solvent
for given analysis is very important .
Solvent for dissolving the sample should be non polar and
non volatile as far as possible since polar solvent are likely
to induce circular chromatogram at the origin.

19. Application of sample and standard solution.
Sample application is one imp and critical step for obtaining the
good resolution for quantification by HPTLC. sample/std are
applied as sport or band depending upon the analysis spot
application is done by using
1)Capillary tubes.
2)Micro bulb pipettes.
3)Micro syringe.
4)Automatic sample applicator.
compare sample/ std applications
FIG:Automatic HPTLC sampler

20. Sample Application
Usual concentration of applied samples 0.1 to 1 µg / µl for
qualitative Analysis and quantity may vary in quantitation 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.
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21. a. Manual Sample Applicator
 The Nanomat serves for easy application of
samples in the form of spots onto TLC and
HPTLC layers .
 The actual sample dosage performed with
disposable capillary pipettes , which are
precisely guided by the capillary holder.
The nanomat is suitable for
 Conventional TLC plates including self-coated
Plates up to 20 × 20cm
 HPTLC plates 10 × 10 cm and 20 × 10 cm
 TLC and HPTLC sheets up to 20 × 20 cm
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22. B .Semi automatic sample applicator
 The instrument is suitable for routine use for
medium sample throughout . In contrast to the
Automatic TLC sampler , changing the sample
the Linomat requires presence of an operator.
 With the linomat , samples are sprayed onto
the chromatographic layer in the form of
narrow bands.
 During the spraying the solvent of the sample
evaporates almost entirely concentrating the sample
Into a narrow band of selectable length.
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23. C .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.
 Application in the form of rectangles
allow precise applications of large volume
with out damaging the layer.
 ATS allows over spotting.
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24. 7. DEVELOPING CHAMBER
1. Flat chamber
2.Twin trough chamber
3.Automatic developing chamber
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25. 1.TWIN TROUGH CHAMBER
• Low solvent consumption:
20 mL of solvent is sufficient for the
development of a 20x20cm plate.
This not only saves solvent , but also reduces
the waste disposal problem
• Reproducible pre –equilibrium with
Solvent vapor: For pre-equilibration, the
TLC plate is placed in the empty trough
opposite the trough which contains
the pre-conditioning solvent. Equilibration
can be performed with any liquid and
for any period of time.
• Start of development : It is started
only when developing solvent is introduced
into the trough with the plate.
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26. Automatic developing chamber (ADC)
 In the ADC this step is
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.
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27.  HPTLC can develop by
Ascending .
Descending .
Circular.
Anti circular.
FIG: HPTLC of Ginseng.

28. DERIVATIZATION DEVICE
a) Spraying
b) Dipping
c) Derivatization through gas
phase
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29. Reasons for Derivatization:
 Changing non-absorbing substance into
detectable
derivatives.
 Improving the detectability.
 Detecting all sample components.
 Selectivity detecting certain substance.
 Inducing fluorescence.
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30. a) Derivatization by spraying
 It comes with a rubber pump but may
also be operated from a compressed air
or nitrogen supply.
 It also consists of a charger and a pump
unit with two kinds of spray heads.
 Spray head type A is for spray solutions
of normal viscosity , e.g. lower alcohol
solution.
 Spray head type B is for liquids of higher
viscosity , e.g. sulphuric acid reagent
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31. b) Derivatization by Dipping
 For proper execution of the dipping
technique the chromatogram must be
immersed and withdrawn at a controlled
uniform speed.
 By maintaining a well defined vertical
Speed and immersion time , derivatization
Conditions can be standardized and tide
Marks , which can interfere with densitometry
evaluation , are avoided.
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32. c)Derivatization through gas phase
 It offers rapid and uniform transfer of the reagent.
 It is unfortunate that only few reagents are suitable they
include I,Br,Cl, as well as volatile acids, bases and some other
gases like H2S , NO.
 In gas phase derivatization can be easily accomplished in twin
trough chambers where the reagent is placed or generated in
the rear trough , while the plate facing the inside of the chamber
is positioned in the front trough.
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33. DETECTION OF SPOTS
Detection can be done by iodine vapor in
iodine chamber. Visual inspection at
254nm of UV region in UV cabinet

34. Scanning &
Documentation
1.HPTLC plates are scanned at
selected UV regions WL by the
instrument & the detected spots are
seen on computer in the form of
peaks.
2.The scanner converts band into
peaks & peak height or area is
related to the concentration of the
substance on the spot.

37.  Evaluation of spot or band:
After detection of spot /band upon objective of experiment
chromatogram is used for several purpose.
Quality Evaluation .
Quantitative Evaluation .

38.  Application of HPTLC:
• Pharmaceutical research.
• Biomedical Analysis.
• Clinical Analysis.
• Environment Analysis.
• Food industry.
 Therapeutic drug monitoring to determine its concentration
and metabolites in blood urine etc.
 Analysis of environment pollution level.
 Quantitative determination of prostaglandin s and
thromboxanes in plasma.
 Determination of mercury in water.
 Characterization of hazard in industrial waste.

39. Comparison between HPTLC and TLC on the basis of parameters

40. Features of HPLC & HPTLC

41.  References.
 Willard H.H., Merrritt L.L., Dean J,A., Settle F.A.,
Instrumental methods of analysis, seventh edn., CBS
publishers and distribution pvt. Ltd.,new delhi,
 Chatwal G.R., Anand S.K.,Instrumental methods of
chemical analysis, Himalaya publishing house, Delhi,
 Mahajan S.S., Instrumental methods of analysis, First
edition, popular prakashan, New delhi, 274-280.

42. *Thank you*