HPTLC
Mr. P.Prachet,
Assistant Professor,
Department of Pharmaceutical Analysis,
Chalapathi Institute of Pharmaceutical Sciences 1
Definition:
Chromatography is a physical process of
separation in which the components to be
separated are distributed between 2 immiscible
phases-a stationary phase which has a large surface
area and mobile phase which is in constant motion
through the stationary phase.
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Introduction:
• HPTLC is the improved method of TLC which
utilizes the conventional technique of TLC in
more optimized way.
• It is also known as planar chromatography or Flat-
bed chromatography.
• HPTLC takes place in high speed capillary flow
range of the mobile phase.
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Principle
Separation may result due to adsorption or
partition phenomenon depending upon the nature
of adsorbents used on plates and solvents system
used for development.
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Differences between TLC and HPTLC:
Parameter TLC HPTLC
Chromatographic plate used Hand made /pre-coated Pre-coated
Sorbent layer thickness 250 mm 100-200mm
Particle size range 5-20 μm 4-8 μm
Pre-washing of the plate Not followed Must
Application of sample Manual/Semi automatic Semi automatic/
Automatic
Shape Spot Spot/Band
Spot size 2-4mm 0.5-1mm
Sample volume 1-10 μl 0.2-5 μl
Application of larger volume Spotting which leads to Can be applied as
over loading bands
No. of samples/plate (20X20) 15-20 40-50
Optimum development distance 7-10 cm 5-7 cm
Development time Depends on mobile phase 40% Less than TLC
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Reproducibility of results Difficult Reproducible
SAMPLE AND SELECTION OF
STANDARD CHROMATOGRAPHIC PLATES
PREPARATION
LAYER PRE-WASHING
LAYER PRE-CONDITIONING
APPLICATION OF SAMPLE
CHROMATOGRAPIC DEVELOPMENT
DETECTION OF SPOTS
SCANNING AND DOCUMENTATION OF
CHROMOPLATE USING PC CATS SOFTWARE 6
Selection of HPTLC plates
Hand plates were available which are made up of cellulose and other materials
which are not used much now-a –days.
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Supports
Materials Advantage Disadvantage
Glass 1.Ressistant to heat and 1. Fragility
chemicals 2.Relatively High wt
2.Easy to handle and offers 3.Costs more for additional
superior flat surface for packaging
work
Polyester sheets (0.2 mm 1.More economical as 1.Charring reactions if
thick) produced even in roll forms temperature exceeds 120°C
2.Unbreakable as the plates are
3.Less packing material dimensionally unstable
4.Spots can be cut and beyond this temperature
eluted thus eliminates dust
from scrapping
Aluminum Sheets(0.1mm) 1.Increasesed temperature 1.Eluents containing high
resistance concentration of mineral
acids or ammonia can attack
chemically on aluminum
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Some of the sorbents used in
HPTLC
No Examples Applications
1. Silica gel 60F 80% of analysis is done on this
(Unmodified ) layer.
2. Alluminium oxide Basic substances ,alkaloids and
steroids
• 3. Cellulose
(microcrystalline )
Amino acids ,peptides ,sugars and
other liable compounds which
cannot be chromatographed on the
active layers of silica gel.
4. Silica gel chemically
modified COOH ,Phenols ,Nucleotides
a) Amino group ( NH2) Pharmaceutical preservations.
b ) CN
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Plate size:
20X20cm
10X20cm
5X10 cm
5X7.5 cm
Good cut edges of sheets is important to obtain constant
Rf values.
1mm SILICA GEL 0.25mm
Pouring
Dipping
Spraying
Spreading
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Pre washing of pre coated plates
The main purpose of the pre-washing is to remove
impurities which include water vapours and other volatile
substances from the atmosphere when they get exposed in
the lab environment.
Silica gel 60F is most widely used sorbent. The major
disadvantage of this sorbent is that it contain iron as
impurity.
This iron is removed by using Methanol : water in the
ratio of 9:1.This is the major advantage of the step of pre-
washing.
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Activation of plates
Freshly opened box of HPTLC plates doesn’t
need activation.
Plates exposed to high humidity or kept in hand
for long time require activation.
Plates are placed in oven at 110o-120oc for 30 min
prior to the sample application.
Activation at higher temperature for longer period
is avoided as it may lead to very active layers and
risk of the samples being decomposed.
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Sample Preparation
• Proper sample preparation is an important pre-
requisite for success of TLC separation.
• For normal chromatography: Solvent should be
non-polar and volatile.
• For reversed chromatography: Polar solvent is
used for dissolving the sample
• Sample and reference substances should be
dissolved in the same solvent to ensure comparable
distribution at starting zones.
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Application of sample
1.It is the most critical step for obtaining good
resolution for quantification by HPTLC.
2.Some applicators used for spotting are:
a) Capillary tubes
b) Micro pipettes
c) Hamilton syringes
d)Automatic sample applicator.
– The major criteria is that they shouldn’t
damage the surface while applying sample. 14
Sample Applicator – Spraylin software
Automated sample application device. Sample
is loaded in micro syringe (Hamilton Syringe)
100μl capacity. Sample can apply either as spot
or band by programming the instrument with
parameters like spotting volume ,band length
etc.
Glass: Borosilicate
Needle: Especially developed for the need of sample
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applicator in HPTLC
Application of sample by Spraylin software:
1 2
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3 4
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Mobile phase
• Mobile phase should be of high graded.
• Chemical properties, analytes and sorbent layer
factors should be considered while selection of
mobile phase.
• Use of mobile phase containing more than three or
four components should normally be avoided as it
is often difficult to get reproducible ratios of
different components
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Petroleum ether
Carbon tetrachloride
Cyclohexane
Carbon disulphide increasing polarity
Ether
Acetone
Benzene
Toluene
Ethyl acetate
Chloroform
Alcohol
Water
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Development of chambers
1.Twin trough chamber.
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Pre-conditioning : (Chamber Saturation)
•Chamber saturation has a pronounced influence on the
separation profile.
•Time required for the saturation depends on the mobile
phase.
•If plates are introduced into the unsaturated chamber,
during the course of development, the solvent evaporates
from the plate mainly at the solvent front and it results in
increased Rf values.
•If tank is saturated prior to the development, solvent
vapors soon get uniformly distributed through out the
chamber. As soon as the plate is kept in such a saturated
chamber, it soon gets pre-loaded with solvent vapors
thus less solvent is required to travel a particular
distance, resulting in lower Rf values.
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Development
o The different methods used for development of
chambers are like-Ascending, descending. 2-
dimentional, horizontal, multiple over-run, gradient,
radial, anti-radial, multimodal, forced flow planar
chromatography.
o Plates are spotted with sample and air dried and placed
in the developing chambers.
o After the development plate is removed from chamber
and mobile phase is removed under fume cup-board to
avoid contamination of laboratory atmosphere.
o The plates should be always laid horizontally because
when mobile phase evaporates the separated components
will migrate evenly to the surface where it can be easily
detected
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Two Dimensional Technique
Drying
• Drying of chromatogram should 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, evaporation of
essential volatile oils if any present in the spot or
compounds sensitive to oxygen may get
destroyed due to the rise in temperature.
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Detection
One of the characteristic feature of HPTLC is the
possibility to utilize post-chromatographic off-line
derivatization
Detection are of two types:
• Qualitative
• Quantitative
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• Qualitative detection:
HPTLC is routinely used for qualitative analysis of
raw materials, finished products, plant extracts etc. It
involves the identification of unknown sample
mixture by comparing the Rf values of the sample
components with the standards.
Retardation factor (Rf) :
distance travelled by solute
distance travelled by solvent
• Quantitative detection:
Quantitative of the chromatogram by HPTLC basically
involves direct and indirect methods;
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Direct method (Destructive):
Ferric chloride -Phenolic comp. & tannins
Ninhydrin in acetone - Amino acids
Dragendroff’s reagents - Alkaloids
Indirect method (Non-destructive): It involves removal of
analyte from the plate followed by quantitation. Eg;
Scrapping and elution which is followed by analysis of
eluant by convenient methods like Spectrophotometry and
Flourimetry
Collection of samples from scrapping will results in the
loss of sample, so vacuum devices and elution chamber are
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used.
Advantages of densitometer
/Scanner/ JUSTTLC
• The purpose of scanner is to convert the spot /band on
the layer in to densitogarm consisting of peaks similar in
appearance to HPLC.
• The position of the scanned peaks on recorder chart is
related to Rf values.
• Peak height/area is related to the concentration of the
substance in the spot.
• Quantitation is faster, reliable, accurate and reproducible
• Photodocumentation of HPTLC plates is possible.
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Capturing the TLC plates using EOS utility
software
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Applications of HPTLC
• Pharmaceutical industry: Quality control,
content uniformity, uniformity test, identity/purity
check.
• Food Analysis: Quality control, additives,
pesticides, stability testing, analysis of sub-
micron levels of aflatoxins.
• Clinical Applications: Metabolism studies, drug
screening, stability testing etc
• Industrial Applications; Process development
and optimization, In-process check, validation
etc.
• Forensic : Poisoning investigations
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References
1. HPTLC- Quantitative analysis of pharmaceutical Formulations
by P.D. Sethi
2. Principles of instrumental analysis skoog, Holler, Nieman
3. Instrumental methods of analysis Willard ,Merrit, Dean
4. Pharmaceutical analysis Munson
5. Sharma J.friedB.Handbook of TLC
6. www.pharmainfo.net
7. http://images.google.co.in/images?q=hptlc+plates&ie=ISO-
8859-1&hl=en
8. http://images.google.co.in/images?svnum=10&hl=en&lr=&ie=IS
O-8859-1&q=linomat
9. www.camag.com
10.http://www.infoexpo.ch/abstract 36
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