Hptlc Naveen Balaji

1. HPTLC
High Performance Thin Layer
Chromatography
Presented by
NAVEEN BALAJI
1st M Pharm
Department of Pharmaceutics
Sree siddaganga college of pharmacy
Tumakuru
Under the guidance of
Dr. Veeresh.P.Veerapur,M.Pharm,Ph.D,
Professor
Department of Quality Assurance
Sree siddaganga college of pharmacy
Tumakuru

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3.  Chromatography
 Introduction
 Principle
 Steps involved in HPTLC
 Difference b/w TLC & HPTLC
 Applications of HPTLC
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4. Chromatography is a technique for separating mixtures into
individual components in order to analyze, identify, purify, and/or
quantify the components.
Separate
• Analyze
• Identify
• Purify
• Quantify
ComponentsMixture
CHROMATOGRAPHY
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5.  HPTLC is Automated & Sophisticated form of
TLC.
 HPTLC is a improved method of TLC which
utilizes the conventional technique of TLC in
more optimized way.
 It is also known as Planar (or) Flat bed
chromatography
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6.  ADSORPTION CHROMATOGRAPHY
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7. 2) Selection of
chromatographic plates
3) Layer pre-washing
4) Activation of Plates
6) Application of Sample
7) Pre- Conditioning
8) Chromatograpic Development & Drying
10) Scanning and Documentation of
Chromoplate Using PC CATS SOFTWARE
1) Selection & optimization
of MP
7
5) Sample Preparation
Steps involved in HPTLC
9) Detection & visualization

8. Hydrophilic
Hydrophobic
Inactive
Active
Polar
Non-polar
.S
M
A
Stahl’s Triangle
Mixture
Solvent
Adsorbent activity
1) Selection of Mobile phase :-
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9.  Optimization of MP [Solvent optimization]
 First Level: Neat solvents (diethyl ether,
ethanol, Ethyl Acetate, acetonitrile, etc.,.)
 Second level:
 If Rf value are too high – Solvent strength
should be decreased – non-polar solvent
 If Rf value are too low – Solvent strength
should be increased – polar solvent
 Usual ratio: 9:1, 8:2, 7:3, 6:4, 5:5
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10.  Third level:
 Mixtures of solvents from different
selectivity group are chosen
 Solvent mixture can be binary, tertiary or
quaternary
 Usual ratio: 1:1, 9:1, 1:9
1:1:1, 8:1:1, 1:1:8
 Small amount of modifiers such as acetic
acid, formic acid.
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11.  Fourth level:-
 Small variation in the proportion of solvent
mixture Is done.
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12.  PRE-COATED PLATES:-
The pre-coated plates with different support materials
and sorbent layers are used for Qualitative &
Quantitative analysis.
 SUPPORT MATERIALS USED ARE :-
1) Polyethylene/polyester
2) Aluminium
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13.  SORBENTS USED IN PLATES :-
Silica gel 60F
Aluminium oxide
SOME OF THE BINDERS USED ARE :-
Gypsum, Starch.
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14. HPTLC plates
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15.  It is a purification step.
 The main purpose of pre-washing is to
remove impurities which include Iron from
Silica gel, water vapours and other
volatile substances from the atmosphere
when they get exposed in the lab
environment.
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16.  Freshly opened box of HPTLC plates doesn’t
need activation.
 If plates exposed to high humidity or kept in the
hand for longer time then activation results by
removing moisture.
 The plates are activated by placing in an oven at
110º-120ºC for 30 min, this step will remove
water on surface.
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17.  Sample & reference substance should be
dissolved in the same solvent to ensure
comparable distribution at starting zones. It
needs a high concentrated solution, as very
less amount of sample need to be applied.
 Solvents used are :-
methanol
chloroform : methanol (1:1)
Ethyl acetate : methanol (1:1)
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18.  Major criteria is that they shouldn’t damage
the surface while applying sample.
 volume of sample recommended is 0.5-5µl.
 The sample should be completely
transferred to the layer.
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19.  Problem from overloading can be
Overcome by applying the sample as band.
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20. GLASS SYRINGE :-
CAMAG Nanomat:
 It is semi- automatic applicator.
 Samples are applied in the form of spots.
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22.  It is a Automatic Sample Applicator.
 Sample can be applied either as spot or
band.
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24.  Chamber saturation has an influence on
Separation profile.
 When the HPTLC plate is introduced into an
unsaturated chamber, during the course of
development, The solvent evaporates from
the plate mainly at solvent front. hence
resulting in increase in Rf values.
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25.  If tank is saturated prior to the development, solvent
vapors soon gets uniformly distributed throughout
the chamber. As soon as the plate is kept in such a
saturated chamber, less solvent is required to travel
the distance, resulting in lower Rf values.
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26. A) Flat Bottom Chamber-
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8) Chromatographic Development
and Drying :-

27. B) Twin Trough Chamber-
 Less solvent is required for each
chromatographic run because it has got a
twin surface at its base.
 It is economical.
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28. C) AUTOMATIC
DEVELOPING
CHAMBER
 controls & maintains the
Temperature & humidity.
 All relevant parameters
are entered via
Keyboard.
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Ascending
Descending
Two-dimensional
Horizontal
Radial
Plates are spotted with sample, air dried and placed in the
developing chambers.
After development, plate is removed from the chamber.
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30.  Drying of chromatogram can be done in
vacuum desiccators with protection from
heat and light.
 If hand dryer is used there may be
chances of getting contamination of plates.
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31. 31

32. Convert the spot/band into chromatogram
consisting of peaks.
It is a instrumental measurement of visible,
UV absorbance, fluorescence quenching
directly on the layer without resorting to
scrapping and elution.
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33. Theory
The transmission of light in a translucent material can be
described by:
Io = Ia + It + Ir + Ix
Where
Io = Intensity of incident light
Ia = Intensity of absorbed light
It = Intensity of transmitted light
Ir = Intensity of reflected light
Ix = Intensity of light lost due to
scattering 33

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35.  Quantitation is faster , reliable ,accurate
and reproducible.
 Photo documentation of HPTLC plates is
possible.
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36.  Different scanner system are available
 Mono wavelength scanner - introduced by
H. Jork in 1966 – uses monochromatic light
with variable wavelength for illumination
purpose
 Diode-array scanner – introduced in 2000 by
Spangenberg – uses white light for
illumination purpose. We can scan at
different wavelength simultaneously.
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37. Diode array TLC scanne
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38.  1. Documentation is important because labeling
every single chromatogram can avoid mistake in
respect to order of application.
 2. Type of plate, chamber system, composition of
mobile phase, running time and detection method
should be recorded.
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39.  3) Video scan software Digistore captures
the images of evaluation.
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40. Parameter TLC HPTLC
1.Chromatographic
plate used
Hand made /pre-coated Pre-coated
2.Sorbent layer
thickness
250 mm 100-200 µm
3.Particle size range 5-20 μm 4-8 μm
4.Pre-washing of the
plate
Not followed Must
5.Application of sample Manual/Semi
automatic
Semi
automatic/Automatic
6.Shape Spot Spot (or) Band
7.Reproducibility results Difficult Reproducible

41.  Pharmaceutical industry: Quality control, content
uniformity, identity/purity check.
 Food Analysis: Quality control, additives, stability
testing, etc.,
 Forensic: Poisoning investigations.
 Environmental analysis: pesticides in drinking
water, selenium in water.

42.  HPTLC- Quantitative analysis of
pharmaceutical formulations, by
Dr.P.D.Sethi.
 Textbook of pharmaceutical analysis, third
edition by S. Ravishankar, Rx publications .
 Google.in
 www.camag.com
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