1. HIGH PERFORMANCE THIN LAYER
CHROMATOGRAPHY
under the guidance of
Mr. Md.Yunoos M.Pharm,(Ph.D)
1
Presented by :
G.Venkateswarlu
M. Pharm (1st year)
REGD.NO:12102S0409

2. CONTENTS
 Introduction
 Principle
 Features of HPTLC
 Main differences of HPTLC and TLC
 Steps involved in HPTLC
 Selection of chromatographic layer
 Precoated layers of HPTLC
 Sample and standard preparation
 Pre washing of precoated plates
 Activation of precoated plates
 Application of sample
 Selection of mobile phase
 Chromatographic development and drying
 Post chromatographic techniques
 Quantification
 Applications of HPTLC
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3. High performance thin layer chromatography (HPTLC) is
a sophisticated and automated form of TLC
HISTORY:
 In 1973 Halpaap introduced first nano-TLC plates.
 In 1977 the first major HPTLC publication appeared
 It is a versatile and most simple separation technique
 It is also fast and in expensive technique.
 It does not require time consuming preparations
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INTRODUCTION

4. The main principle of separation is “adsorption”
 The mobile phase solvent flows through the
capillary action
 The components move according to their
affinities towords adsorbent
PRINCIPLE
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5. Simultaneous processing of sample and standard.
Better analytical precision and accuracy and less need
for Internal Standard
 Lower analysis time and less cost per analysis
 Simple sample preparation
 No prior treatment for solvents like filtration and
degassing
 Low mobile phase consumption
FEATURES of HPTLC
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6. MAIN DIFFERENCES OF TLC AND HPTLC
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Parameters TLC HPTLC
TYPE OF CHROMATOGRAPHIC
PLATE
HAND MADE / PRECOATED PRECOATED
PARTICAL SIZE DISTRIBUTION WIDE NARROW
PARTICAL SIZE RANGE 5 – 20 μm 4 – 8 μm
SHAPE SPOT SPOT / BAND
SPOT SIZE 2 – 4 mm 0.5 – 1 mm
LAYER THIKNESS 250 μm 100 – 200 μm
SOLVENT CONSUMPTION 50 ml 5 – 10 ml
NO. OF SAMPLES MAXIMUM 12 36 – 72
OPTIMUM DEVELOPMENT
DISTANCE
10 - 15 cm 5 – 7cm
SAMPLE VOLUME 1-10 μl 0.1-2μl
NO. OF SAMPLE PER PLATE 15 – 20 40 - 50

7.  Selection of chromatographic layer
 Sample and standard preparation
 Layer pre-washing
 Layer pre-conditioning
 Application of sample and standard
 Chromatographic development
 Drying
 Detection of spots
 Scanning
 Documentation
Steps involved in HPTLC method
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Instrumentation

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9. 1) Hand made plates
2) Precoated plates
Hand made plates:
Cellulose (native )
Cellulose with starch as binder
Silica gel with starch
Acetylated cellulose +CaSO41/2H20
SELECTION OF CHROMATOGRAPHIC LAYER
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10.  Different support materials are used
• Glass
• Polyester sheets
• Aluminium
 Silica gel 60F: 80%analysis
 Aluminium oxide: Basic substances, alkaloids and
steroids
 Cellulose: Amino acids, dipeptides, sugars and
alkaloids
 RP2, RP8, RP18: Non-polar substances, fatty
acids, carotenoids, cholesterol
 Hybrid plates RP18WF254S: Preservatives,
barbiturates, analgesic and phenothiazines
PRECOATED LAYERS OF HPTLC
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11.  For normal phase chromatography solvent for
dissolving the sample should be non polar
 For reverse phase chromatography polar solvents are
used
 Sample standard should be dissolved in the same
solvent to ensure comparable distribution at stationary
zones
SAMPLE AND STANDARD PREPARATION
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12. To avoid any possible interference due to impurities it
is recommended to clear the plates is called pre
washing
Methods used for pre washing are ;
Dipping
Ascending
Continuous
Solvents used for washing are;
chloroform in methanol (1:1)
methylene chloride -methanol (1:1)
1%ammonia or 1% acetic acid
PRE WASHING OF PRE COATED PLATES
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13.  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 minutes prior to spotting
 Aluminum sheets should be kept in between two
glass plates and placing in oven at 110-120ºc for 15
minutes.
ACTIVATION OF PRECOATED PLATES
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14.  Selection of sample application and devices used
depends on
• Sample volume
 Number of samples to be applied
 Sample is applied use of automatic devices and
graduated capillaries
 Volume recommended for HPTLC 0.5-5μl
 Sample should not excess or not low
 Over loading can be over come by applying sample as
band .
APPLICATION OF SAMPLE AND STANDARD
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15. The Nanomat serves for easy
application of samples in the form of
spots on HPTLC plates
The Nanomat is suitable for
HPTLC plates 10 x 10 cm and 20 x 10
cm
 Capillaries of 0.5, 1, 2, and 5 µL
volume are available.
NANOMAT AND CAPILLARY DEVICES
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16.  Trial and error
 3 - 4 component mobile phase should be avoided
 Multi component mobile phase once used not
recommended for further use
PRE CONDITIONING (Chamber saturation)
 Un- saturated chamber causes high R f values
Saturated chamber by lining with filter paper for 30
minutes prior to development
SELECTION OF MOBILE PHASE
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17.  After development, remove the plate
 Dry in vacuum dessicators.
Development chambers:
 Twin trough and flat bottom chamber
 Horizontal developing chamber
 HPTLC various system
 Automatic developing chamber (ADC2)
 Automated multiple development (AMD)
CHROMATOGRAPHIC DEVELOPMENT AND DRYING
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18. 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.
For pre-
equilibration, the
TLC plate is placed
in the empty trough
opposite the trough
started only
when
developing
solvent is
introduced into
the trough
Twin Trough Chambers
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19. HPTLC VARIOSYSTEM
• Development with six
different solvents can be
tested side by side
• Six different conditions of
pre-equilibration, including
relative humidity, can be
tested simultaneously.
It is developed from both
opposing sides towards the
middle.
HORIZONTAL DEVELOPING CHAMBER
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20. AUTOMATIC DEVELOPING
CHAMBER (ADC 2)
The Automatic Developing
Chamber offers convenience,
safety and reproducibility for
isocratic developments of
TLC/HPTLC plates.
AUTOMATED MULTIPLE
DEVELOPMENT(AMD)
Employed for reproducible
gradient elution.
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21. DERIVATIZATION
For proper execution of the
dipping technique, the
chromatogram must be
immersed and withdrawn at a
controlled uniform speed
HPTLC Sprayer
The TLC/HPTLC Sprayer
consists of and a pump unit
with two kinds of spray heads.
Spray head type A is for spray
solutions
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22.  Detection and visualization
 Quantification or densitometric measurements
POST CHROMATOGRAPHIC STEPS INCLUDE
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23. Detection under UV light is first
choice - non destructive Spots of
fluorescent compounds can be
seen at 254nm
.
CAMAG UV Lamp
CAMAG UV Cabinet
Detection and visualization
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24. Sample and standard should be chromatographed on
same plate after development chromatogram is
scanned
 Camag TLC scanner III scan the chromatogram in
reflectance or in transmittance mode by absorbance or
by fluorescent mode
 scanning speed is selectable up to 100 mm/s - spectra
recording is fast - 36 tracks with up to 100 peak
windows can be evaluated
 Calibration of single and multiple levels with linear or
non-linear regressions are possible · When target values
are to be verified such as stability testing and
dissolution profile single level calibration is suitable
Quantification
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25. . It can also be used for
densitometric measurements of
other planar objects, such as
electrophoresis gels.
Key features
Scanning speed 1-100 mm/s
Spectrum recording up to 100
nm/s
TLC Scanner 3
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26.  Pharmaceutical industry : Quality control, content
uniformity, identity/purity check
 Food Analysis: Quality control, additives, Pesticides
,stability testing ,
 Clinical Applications: Metabolism studies , drug
screening ,stability testing etc
 Industrial Applications: Process development and
optimization, In-process check ,validation etc.
 Forensic : Poisoning investigations
 Finger print analysis
APPLICATIONS OF HPTLC
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27. 1.Sethi PD HPTLC High performance thin layer
chromatography , First edition , CBS Publishers and
Distributers ,page No 4-68
2.Beckett AH. StenlakeJB Practical pharmaceutical
chemistry ,Fourth edition(1996) part –two CBS
publishers and Distributers New delhi page No 123-
124
3.http:// www.camag.com /products/application/ disposa
ble.html
4.http:// www.bcon-nstruments.nl /products/ hptlc.html
5.Meyyanathan SN Basic Principles of HPTLC
REFERENCES
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