HIGH PERFORMANCE THIN LAYER

CHROMATOGRAPHY

Name – SADHAN PADHI

Regd No – 170101181002
Branch – Petro Chemical Engineering
College – Centurion University,
Paralakhemundi.
 Thin layer chromatography
(TLC)
• Is a method for identifying substances
and testing the purity of compounds

• TLC is a useful technique because it

is relatively quick and requires small
quantities of material
• Separations in TLC involve distributing a mixture of
two or more substances between a stationary phase
and a mobile phase.

• The stationary phase: is a thin layer of

adsorbent (usually silica gel or alumina) coated on a
plate.
• The mobile phase: is a developing liquid which travels
up the stationary phase, carrying the samples with it.

• Components of the samples will separate on the

stationary phase according to how much they adsorb
on the stationary phase versus how much they
dissolve in the mobile phase.
 HPTLC
• 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
Principle:
• Chromatography is a physical process of separation
in which the components to be separated are
distributed between two immiscible i.e. the principle
of separation is adsorption
• The mobile phase solvent flows through
because of capillary action. The components
move according to their affinities towards the
adsorbent
• The component with more affinity towards the
stationary phase travels slower. The component
with lesser affinity towards the stationary phase
travels faster
• Thus the components are separated on a
chromatographic plate
 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 0.2-5 μl
Application of μl Can be applied as bands
larger volume Spotting which leads to over
No. of samples/plate (20X20) loading 40-50
15-20
Optimum development distance 10-15 cm 5-7 cm

Development time Depends on mobile phase 40% Less than TLC

Reproducibility of results Difficult Reproducible
Steps of HPTLC
Selection of HPTLC plates

• Hand plates were available which are made up

of cellulose and other materials which are not
used much
• Precoated 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 1. Fragility
and chemicals 2.Relatively High wt
2. Easy to handle and 3.Costs more for
offers additional
superior flat surface for work packaging

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

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

Aluminum Sheets(0.1mm) 1.Increasesed temperature Eluents containing high

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. Aluminum 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

a) Amino group ( NH2)
b ) CN COOH ,Phenols ,Nucleotides
Pharmaceutical preservations.
Binders

• Gypsum (G)
• Starch (S)
• Layer containing fluorescent indicator (F)
 Plate size

• 20X20cm
• 10X20cm
• 5X10 cm
• 5X7.5 cm
• Good cut edges of sheets is important to obtain constant
Rf values.
 Pre washing of pre coated plates

To remove impurities which include water vapours

and other volatile substances

Silica gel 60F is most widely used sorbent. The major

disadvantage of this sorbent is that it contain iron
as impurity which is removed by using
Methanol : water in the ratio of 9:1.This is the major
advantage of the step of pre-washing
 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
• 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
• Better separation because of rectangular area.
• Response of densiometer is higher
• Large quantity
 Automatic applicators
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
2. CAMAG Linomat: Automated sample application device. Sample is loaded
in micro syringe (Hamilton Syringe) 1μl capacity. Sample can apply either as
spot or band by programming the instrument with parameters like spotting
volume ,band length etc
3. CAMAG automatic TLC sampler III: Applies sample as spot or bands
automatically from the rack of sample vials
 Mobile phase
• Mobile phase should be of high grade

• 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
• Chambers containing multi component MP are
not generally used for re-use for any future
development , due to differential evaporation
and adsorption by layer
• Once the chamber is opened , solvents
evaporate disproportionally depending on their
volatilities.
Polarity of Solvents
Water
Polar
Acetic Acid
Ethyleneglycol
Methanol
Ethanol
Isopropanol
Acetonitrile
Nitromethane
Aniline
Dimethylsulfoxide
Ethylacetate
Acetone
Dicholoroethane
Tetrahydrofuran
Chloroform
Diethylether
Benzene
Toluene
Non-polar
Carbontetrachloride
Cyclohexane
Petroleum ether
 Development of chambers
1.Twin trough chamber
2.Rectangular chambers
Simulation chamber
The simultion developing chamber is a thick
walled clear glass tank with vertical
grooves and a heavy ground-glass lid.

Round chamber
These cylindrical chambers are ideal for use
with narrower width plates.

Nano chamber
The nano chamber is suitable for the
development of 10x10cm TLC plates and
features a heavy glass lid for
gas-tight seal and optimum vapour
saturation.

HPTLC chamber
Ideal for the development of HPTLC 5x5cm
plates.
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
• Ascending, descending, horizontal

• 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
 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
 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
• Relative humidity
• Temperature
• 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
Detection under UV light is first choice - non destructive
- Spots of fluorescent compounds can be seen at 254 nm (short
wave length) or at 366 nm (long wave length)
- Spots of non fluorescent compounds can be seen - fluorescent
stationary phase is used - silica gel GF
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.
• Quantitative Evaluation:
Quantitative evaluation of the chromatogram by
HPTLC basically involves estimation of chemical
compounds present in the sample
 Densiometry
•Separation of Herbal sample and reference substances, there is a broad array of other detection
modes available.

•Detection is a very flexible and independent step. Multiple detection is possible without
repeating the chromatography.

•Allows measuring the absorption and/or fluorescence of underivatized or derivative substances

at wavelengths between 200 and 800 nm. Up to 31 wavelengths can be evaluated and spectra
of any peak can be recorded.

•Following integration densitometric data can be quantitatively evaluated.

 Documentation

• Labeling every single chromatogram can avoid mistake in

respect of order of application

• Type of plate, chamber system, composition of mobile

phase, running time and detection method should be
recorded

• Suppliers name, item number, batch no., individual plate

no. are imprinted near upper edge of pre-coated plates.
Helps in traceability of analytical data
• Avoids manipulation of data at any stage as coding will
automatically get recorded using photo-documentation
 POST CHROMATOGRAPHIC
DERIVATIZATION
• Chromatogram Immersion
Device.
• TLC Sprayer.
• Reagent Spray.
• TLC Spray Cabinet.
• TLC Plate Heater
 Detecting Agents
• p-Anisaldehyde – sulfuric acid: phenols,
sugars, steroids, and terpenes
• Aluminium chloride: flavonoids
• Antimony (III) chloride: vitamins A & D,
carotenoids, steroids, sapogenins, steroid
glycosides, terpenes
• Bromthymol blue: lipids and phospholipids
• Diphenylamine: glycosides, glycolipids
• Dragendorff reagent: nitrogen compounds,
alkaloids, antiarrhythmic drugs,
• surfactants
• Gentian Violet – Bromine: lipids
Iodine vapor: Relatively unspecific universal
reagent for many organic compounds
 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
 Conclusion
1. CHROMATOGRAPHY IS A SCIENCE OF SEPERATION USED EITHER FOR IDENTIFICATION OR
QUANTITATION OF CHEMICAL SUBSTANCES

2. VARIOUS MODES OF CHROMATOGRAPHIC TECHNIQUES WERE DEVELOPED BASED ON

INITIAL DISCOVERY BY MICHAEL T.SWETT

3. HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY IS MOST ADVANCED TECHNIQUE

NOW A DAYS

4. IT IS THE MODERN SEPARATION TECHNIQUE WHICH IS ACCEPTRD AS AN EXTREMLY

FESIBLE,RELIABLE & COST EFFICIENT METHOD

5. BY THIS TECHNIQUE ACCURATE SEPARATION OF SAMPLE TAKES PLACE

6. IT CAN SPEED UP OUR WORK & SO MANY THINGS AT A TIME USUALLY NOT POSSIBLE WITH
OTHER ANALYTICAL TECHNIQUE

7. HPTLC CAN SIMULTANEOUSLY HANDLE SEVERAL SAMPLES EVEN OF DIFFERENT NATURE &
COMPOSITION SUPPORTING SEVERAL ANALYTE AT A GIVEN TIME
 References:
• HPTLC- Quantitative analysis of pharmaceutical Formulations by P.D. Sethi
• Dr. Harish Chandra Andola, “High Performance Thin Layer Chromatography (HPTLC): A Modern
Analytical tool for Biological Analysis”, Nature and Science , 2010;8(10)

• CAMAG, 2010-2011. BASIC EQUIPMENT FOR MODERN THIN LAYER CHROMATOGRAPHY.

SWITZERLAND: CAMAG.AVAILABLE FROM:
CAMAG.COM/DOWNLOADS/FREE/BROCHURES/CAMAG-BASIC-EQUIPMENT-08.PDF

• http://www.pharmainfo.net/files/images/stories/article_images/INTRODUCTION_ANALYTICALME

• THODFig10.JPG

PATEL, R.B. AND PATEL, M.R. AND PATEL, B.G. (2011) EXPERIMENTAL ASPECTS AND
IMPLEMENTATION OF HPTLC. IN: SHRIVASTAVA, M.M. HPTLC. NEW YORK: SPRINGER, PP. 41-

• 54.

SHRIVASTAVA, M.M. (2011) AN OVERVIEW OF HPTLC: A MODERN ANALYTICAL TECHNIQUE

WITH EXCELLENT POTENTIAL FOR AUTOMATION, OPTIMIZATION, HYPHENATION, AND
MULTIDIMENSIONAL APPLICATIONS. IN: SHRIVASTAVA, M.M. HPTLC. NEW YORK: SPRINGER,
PP. 3- 24.
•
http://www.infoexpo.ch/abstract
 Thank You
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