Venki hptlc ppt

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

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
2

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
3
INTRODUCTION

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
4

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
5

MAIN DIFFERENCES OF TLC AND HPTLC
6
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

 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
7
Instrumentation

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
9

 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
10

 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
11

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
12

 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
13

 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
14

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
15

 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
16

 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
17

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
18

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
19

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.
20

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
21

 Detection and visualization
 Quantification or densitometric measurements
POST CHROMATOGRAPHIC STEPS INCLUDE
22

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
23

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
24

. 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
25

 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
26

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
27

Venki hptlc ppt

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