HIGH PERFORMANCE THIN LAYER

CHROMATOGRAPHY
 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.
 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.
 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 over Can be applied as bands
loading
No. of samples/plate (20X20) 15-20 40-50

Optimum development 10-15 cm 5-7 cm

distance
Development time Depends on mobile phase 40% Less than TLC
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
Factors influencing separation
and resolution of spots
• Type of stationary phase
• Type of pre-coated plates
• Layer thickness
• Binder in layer
• Mobile phase
• Solvent purity
• Size of developing chamber
• Sample volume to be spotted
• Size of initial spot
• Solvent level in chamber
 Strength
• Detection by chemical reaction with a
visualization reagent can be carried out,
which means that more or less every type of
compound can be detected with a suitable
detection reagent.
• Robust and Cheap
• Batch processing
• many samples handled at once – speed of
analysis
 Limitations
The number of theoretical plates available for
separation is limited in routine TLC systems
although HPTLC plates can offer nearly the same
efficiency in a 10 cm distance as an HPLC column
of the same length.

Sensitivity is often limited

Not suitable for volatile compounds
Requires more operator skill for optimal use than
HPLC
 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.

• Pre coated plates:

The plates with different support materials
and sorbent layers with different format and
thickness are used.
Plates with sorbent thickness of 100-250μm are
used for qualitative and quantitative analysis.
 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 work packaging

Polyester sheets (0.2 1.More economical as 1.Charring reactions if

mm thick) produced even in roll forms
2.Unbreakable
3.Less packing material
4.Spots can be cut and eluted
thus eliminates dust from
scrapping
temperature exceeds 120oc as
the plates are dimensionally
unstable beyond this
temperature

Aluminum 1.Increasesed temperature 1.Eluents containing high

Sheets(0.1mm) resistance concentration of mineral
acids or ammonia can attack
chemically on aluminum
 Some of the sorbents used in
HPTLC
No Examples Applications
1. Silica gel 60F (Unmodified ) 80% of analysis is done on this 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
 HPTLC Plate
• 20X20cm
• 10X20cm
• 5X10 cm
• 5X7.5 cm
• Good cut edges of sheets
is important to obtain
constant Rf values.

Some of the binders used:

• Gypsum (G)
• Starch (S)
• Layer containing fluorescent indicator (F)
 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.
Some common methods involved in pre-
washing:

• Ascending method:
• Dipping method:
• Continuous method:
Solvents used for pre-washing

• 1.Methanol
• 2.Chloroform: methanol ( 1:1 )
• 3.Choloroform: Methanol: Ammonia
(90:10:1 )
• 4.Methylene chloride: Methanol ( 1:1 )
• 5.Ammonia solution (1%)
 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.
 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.
 Application of sample
The selection of sample application
technique and device to be used depends
primarily on:
• Sample volume
• No. of samples to be applied
• Required precision and degree of
automation.
Some applicators used for spotting are:
a) Capillary tubes
b) Micro bulb pipettes
c) Micro syringes,
d)Automatic sample applicator.
– The major criteria is that they shouldn’t
damage the surface while applying sample.
• The sample should be completely transferred to
the layer.
• Micro syringes are preferred if automatic
application devices are not available.
• Volume recommended for HPTLC-0.5-5μl to keep
the starting zone down to minimum of 0.5-1 mm
in concentration range of 0.1-μg/ml
• Sample spotting should not be excess or not low.
• Problem from overloading can be overcome by
applying the sample as band.
 Advantages of application of
sample as band are
• Better separation because of rectangular area.
• Response of densiometer is higher in case of band
than that observed from an equal amount/equal
volume of sample applied as a spot.
• Large quantity
Automatic applicators used
1) CAMAG Nanomat: Samples applied in the
form of spots. The volume is controlled by
disposable platinum iridium of glass capillary
which has volume of 0.1-0.2μl.
 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
• Mobile phase optimization is necessary while
performing HPTLC.
• Various components of MP should be
measured separately and then placed in
mixing vessel. This prevents contamination of
solvents and also error arising from volumes
expansion or contraction on mixing.
• Trough chambers are used in which smaller
volumes of MP usually 10-15 ml is required.
• Different components of MP are mixed first
in mixing vessel and then transferred to
developing chambers.
• Chambers containing multi component MP
are not generally used for re-use for any future
development , due to differential evaporation
and adsorption by layer and also once the
chamber is opened , solvents evaporate
disproportionally depending on their volatilities.
 Development of chambers
1.Twin trough chamber.

2.Rectangular chambers
3. V-shaped chambers
4.Sandwitch chamber
5.Horizontal development chamber
6.Automatic development chamber
 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.
 Development and Drying:
• The different methods used for development of
chambers are like-Ascending , descending .2-
dimentional, horizontal , multiple overrun , gradient
,radial ,anti-radial ,multimodal ,forced flow planar
chromatography.
• Plates are spotted with sample and air dried and
placed in the developing chambers.
• After the development plate is removed from
chamber and mobile phase is removed under fume
cup-board to avoid contamination of laboratory
atmosphere.
• 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
• Gradient
• Relative humidity
• Temperature Flow rate in solvent
• Separation distance
• Mode of Derivatization

Greater the difference between

two spots and smaller the initial spot
diameter of sample and better will be the
resolution
Detection and visualization
One of the characteristic feature of
HPTLC is the possibility to utilize post-
chromatographic off line derivatization

Detection are of two types:

• Qualitative
• Quantitative
• 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.

• Quantitation Evaluation:
Quantitative of the chromatogram by HPTLC
basically involves direct and indirect
methods;
Densiometry
 Documentation:
1. Documentation is important because labeling every single
chromatogram can avoid mistake in respect of order of
application.
2. Type of plate, chamber system, composition of mobile
phase, running time and detection method should be
recorded.
3. TO assist the analysts and researchers E .merck has
introduced HPTLC pre-coated plates with an imprinted
identification codes.
4. Suppliers name, item number, batch no. , individual plate
no. are imprinted near upper edge of pre-coated plates. This
will not only help in traceability of analytical data, but will
also avoid manipulation of data at any stage as coding will
automatically get recorded using photo-documentation.
 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
aflotoxins etc
• Clinical Applications: Metabolism studies , drug
screening ,stability testing etc
• Industrial Applications; Process development and
optimization, In-process check ,validation etc.
• Forensic : Poisoning investigations