HIGH PERFORMANCE THIN LAYER

CHROMATOGRAPHY

B.V. Rao
Associate Professor
Chebrolu Hanumaiah
Institute of
Pharmaceutical Sciences
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.

➢ The basic difference between conventional TLC & HPTLC

is only in the particle size & pore size of the sorbents.

➢ It is very useful in quantitative & qualitative analysis of

pharmaceuticals.
PRINCIPLE :

 The principle of se paration is

adsorption.
Stationary phase - solid
Mobile phase - liquid
 ADVANTAGES OF HPTLC OVER OTHER CHROMATOGRAPHIC
METHODS
HPTLC is the most simple separation technique today
available to the analyst due to-
 Simultaneously analysis of sample and standard
 Lower analysis time and less cost per analysis.
 It is very simple to learn and the instrumentation is very
easy to operate.
 The sample preparation is very simple.
 It involves very low maintenance cost.
 Solvents used in HPTLC needs no prior treatment like filtration
and degassing.
 HPTLC allows the use of corrosive and UV absorbing mobile
phases.
 In HPTLC ,the mobile phase consumption for sample is extremely
low.
 DIFFERENCES BETWEEN TLC AND HPTLC
Parameters TLC HPTLC
TYPE OF HAND MADE / PRECOATED
CHROMATOGRAPHIC PRECOATED
PLATE
PARTICAL SIZE WIDE NARROW
DISTRIBUTION
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 10 - 15 cm 5 – 7cm
DEVELOPMENT
DISTANCE
SAMPLE VOLUME 1-10 μl 0.1-2μl
NO. OF SAMPLE PER 15 – 20 40 - 50
PLATE
 INSTRUMENTATION :
 Steps involved
1. Selection of the HPTLC plates & sorbents
2. Sample preparation including prewashing & pre
chromatographic derivatization
3. Application of sample
4. Pre-conditioning
5. Development techniques(separation)
6. Detection including post chromatographic derivatization
7. Quantitation
8. Documentation
PROCEDURE: SELECTION OF CHROMATOGRAPHIC
PLATES
SAMPLE AND STANDARD
PREPARATION
PLATES PRE-WASHING

PLATES PRE-CONDITIONING

APPLICATION OF SAMPLE

CHROMATOGRAPIC DEVELOPMENT

DETECTION OF SPOTS

SCANNING AND DOCUMENTATION OF

CHROMOPLATE USING PC CATS
SOFTWARE
1.SELECTION OF HPTLC PLATES:

TYPES OF
binders
PLATES

Gypsum starch
HAND MADE PRE COATED

sorbent layers with

made up of cellulose different format and
thickness are used

support materials
like glass,
sorbent thickness of
alluminium,
plastic 100 – 250 μm are
used for qualitative &
quantitative analysis
 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.

Common methods involved pre washing are as follows

1. Ascending method
2. Dipping method
3. Continuous method
SOLVENTS USED FOR PRE-WASHING

 Methanol

 Chloroform: methanol ( 1:1 )

 Chloroform: Methanol: Ammonia (90:10:1 )

 Methylene chloride: Methanol ( 1:1 )

 Ammonia solution (1%)

ACTIVATION OF PLATES:
 Plates exposed to high humidity.

 Plates are placed in oven at 110o-120oc for 30

min prior to the sample application.

 If the plates are not dried properly, the

moisture is present on the plates & the
principle changes to partition
chromatography. (stationary phase- liquid,
mobile phase- liquid)
 2.SAMPLE PREPARATION:

 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.
3. Application of sample
1.Sample application 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 bulb pipettes
c) Micro syringes,
d)Automatic sample applicator.

The major criteria is that they shouldn’t damage

the surface while applying sample.
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.
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.

Glass: Borosilicate
MOBILE PHASE:

 Mobile phase should be of high graded.

 Chemical properties, analytes and sorbent layer

factors should be considered while selection of mobile
phase.

 Mobile phase optimization is necessary while

performing HPTLC.

 Twin trough chambers are used in which smaller

volumes of Mobile phase usually 10-15 ml is required.
o Various components of Mobile phase should be measured
separately and then placed in mixing vessel and then
transferred to developing chambers.. This prevents
contamination of solvents and also error arising from
volumes expansion or contraction on mixing.

Development Chambers:
1. Twin trough chamber
2. Rectangular chambers
3. V-shaped chambers
4.Sandwitch chamber
5.Horizontal development chamber
6.Automatic development chamber (ADC)
DEVELOPMENT CHAMBERS:
1.Twin trough chamber:

Rectangular chambers Automatic development chamber (ADC)

 4.PRE-CONDITIONING (CHAMBER SATURATION):
 Preconditioning of HPTLC chamber is required for efficient
separations.
 It is the process in which the chamber is saturated with the mobile
phase components used for development. This ensures equilibrium is
established in the atmosphere within the chamber with the mobile
phase components.
 The chamber is lined with filter paper, moistened with the mobile
phase before keeping the HPTLC plate. Alternatively pour mobile
phase in the twin trough chamber, set a side for 30 min, place the
HPTLC plate quickly and close with the lid immediately, to avoid
mobile phase vapours leaving the chamber.
 5.DEVELOPMENT TECHNIQUES:
 The different methods used for development of chambers are like-
Ascending, descending, 2-dimentional, horizontal, multiple overrun,
gradient & forced flow planar chromatography.

 Plates are spotted with sample and air dried then 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 ❖Gradient elution

❖ Type of pre-coated plates ❖Relative humidity

❖ Layer thickness
❖Temperature
❖ Binder in the layer
❖Separation distance
❖ Mobile phase composition
❖Mode of development
❖ Solvent purity
❖ Size of developing chamber Greater the difference between
❖ Sample volume to be spotted two spots and smaller the spot
diameter of sample and better will
❖ Size of spot diameter
be the resolution.
❖ Solvent level in chamber
6.DETECTION AND VISUALIZATION
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 from mixture by comparing the Rf values of the
sample components with the standards.

 Quantitative detection:

Quantitative of the chromatogram by HPTLC basically involves

direct and indirect methods.
Indirect method:
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
1) Spectrophotometry
2) Flourimetry
3) Colorimetry
Collection of samples from scrapping will results in the loss of
sample, so vaccum devices and elution chamber are used.
Densitometry

•Visualization of spots/bands done directly on plate.

•Amount of compound unknown is measured by comparing standard
curve from reference chromatogram with same condition.
❖Densitometer basically consists of :
Light source
Visible Radiation
UV Radiation
Wave length selection device.
Condensing and focusing system.
Photo-sensing detectors.
 DETECTION
METHODS

SPRAYING DIPPING GAS PHASE

Derivatization by Spraying:

 It comes with a rubber pump but may also be operated from a

compressed air or nitrogen supply.

 It also consists of two kinds of spray heads.

 Spray head type A is for spray solutions

of normal viscosity , e.g. lower alcohol solution.

 Spray head type B is for liquids of higher viscosity.

Eg: sulphuric acid reagent 28

Derivatization by Dipping
 For proper execution of the dipping technique, the
chromatogram must be immersed in reagent and withdrawn at a
controlled uniform speed.

29
 Derivatization through gas phase:
• It offers rapid and uniform transfer of the reagent.

• Only few reagents are suitable they include I,Br,Cl, as

well as volatile acids, bases and some other gases like
H2S , NO.

• In gas phase derivatization can be easily accomplished

in twin trough chambers where the reagent is placed
or generated in the rear trough , while the plate facing
the inside of the chamber is positioned in the front
trough.
30
 DOCUMENTATION

HPTLC plates have been evaluated quantitatively and

qualitatively should be documented as per guidelines of
GLP.

Common methods of documentation are:-

➢ PHOTO DOCUMENTATION.

➢ VIDEO DOCUMENTATION.
APPLICATIONS
* Food analysis

* Pharmaceutical industry

* Clinical applications

* Industrial applications

* Forensic applications

* Cosmetics analysis
Food analysis:
Estimation of vitamins, pesticides ,food substances.
Curcumin-curcuminoids in turmeric powder.
Sudan-1,2,3 etc in chilli powder.
Cholesterol in edible oil.
Saffron in food.

Forensic applications:
Detection and estimation of traces of drugs or
poisonous compounds.

Cosmetic analysis:
Presence of colouring agents, preservatives,
trace materials and prohibited substances
Features HPLC HPTLC
Stationary phase Liquid/solid Solid
Mobile phase Liquid Liquid
Conditioning phase None Gas
Sample should be Non volatile
Results By detector Detector + eyes
Analysis On-line Off-line
Resolution Very high Moderate to high
Chromatographic Closed Open
sys
Seperating medium Tubular column Planar layer(plate)
Analysis in parallel No Yes
High pressure Yes Up to 100 samples
Time per sample 2-60min 1-3min
Data obtained Little to high Very high
Post- Limited possibilities Simple,possible for
chromatography every sample
Fraction collection Requires prep.scale Simple no special
PARAMETERS:
 Separation Efficiency:
 Position of the mobile phase at time t as it moves through a
sorbent layer

 Zf - distance moved by the mobile phase from the sample origin

 k - velocity constant.

k is dependent on:

 Ko - permeability constant, dp -average particle diameter,

 g -surface tension of the mobile phase,
 η- viscosity of the mobile phase, and
 θ- contact angle.
Plate Height:
 Broadening of a chromatographic spot can be expressed in terms of
the theoretical plate number ‘N’ of the given chromatographic
system:

 Zf and Zs are the migration lengths of the mobile phase and solute,
and Ws is the chromatographic spot width in the
direction of the mobile phase migration.

 N is proportional to the migration length of the

mobile phase Zf, when Zs /Ws ratio remains
constant.
 Increase in Zf -increase of N and better separation
 Resolution:
 The separation between two spots is measured by the quantity Rs,
and is called resolution.
 The Rs of two adjacent chromatographic spots 1 and 2 is defined
as the distance between the two spot centers divided by the mean
spot widths.

 When Rs=1, the two spots are reasonably well separated.

 Rs values larger than 1 indicates better separation
and smaller than 1 suggest a poor separation.
 Rs is also given by the equation

Rf(1) and Rf(2) are the Rf values

of chromatographic spots 1 and 2
 CAPACITY FACTOR/ RETENTION FACTOR
 The ratio of its retention time in the stationary phase to that in the
mobile phase.

 Measures the degree of retention

 Ratio of total number of moles of analytes in each phase.

 If a substance doesn't migrate, its Rf =0 and k=1
 If a substance migrates with solvent front, its Rf=1 and k=0
 If k< 1, more faster elution ( lesser rt)
 If k > 5, Lesser elution ( Longer rt)
 Ideally k for an analyte between 1 & 5.
 SPOT CAPACITY
 Separation number or spot capacity is defined as the maximum
number of substances, which are completely separated between
Rf 0 and 1, provided that the separation conditions are isocratic

 measures the efficiency of separation.

 Separation number is given by the expression

 Zf - migration distance of the front

 bo - width of the spot at Rf =0

 bl - width of the spot with Rf = 1

REFERENCES:
1. www.spinger.com/978-3-642-1404-2
2. www.camagproducts.com
3. P.D Sethi, HPTLC- quantitative analysis of pharmaceutical
formulations, published by CBS publishers and distributors, pg
no 3-23.
4. Snyder and Kirkland, introduction to modern liquid
chromatography, 2nd edition, published by Wiley and
intersceince, pg no 247.
5. G. vidyasagar, instrumental method of drug analysis, published
by pharmamed press, pg no 137-149.
6. www.camag.com
7. Ashutoshkar, pharmaceutical analysis volume 2, published by
CBS publishers and distributors, pg no 299-314.
8. B.k.sharma, chromatography, GOEL publishing house, Meerut
pg no 5-16.