1. Presented by
Revan V. Hiwale
Guide
Prof. K. B.Gabhane
VidyaBharati College Of Pharmacy, Amravati-444601
2019-2020
HIGH PERFORMANCE THIN LAYER
CHROMATOGRAPHY
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3. INTRODUCTION
HPTLC is a form of thin-layer chromatography (TLC) that provides superior
separation power using optimized coating material, novel procedures for
mobile-phase feeding, layer conditioning, and improved sample application.
• The basic difference between conventional TLC and HPTLC is only in
particle and pore size of the sorbents.
• The principle of separation is similar that of TLC adsorption.
• It is very useful in quantitative and qualitative analysis of pharmaceuticals.
• Detection limit in nanogram range with UVabsorption detection and in
picogram range with fluorimetric detection.
• Large no of theoretical plates in minimum area of plates .
• Analysis time is greatly redused in HPTLC due to shorter migration distant.
• Higher efficiency due to smaller particle size(5 μm).
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4. PRINCIPLE
Same theoretical principle of TLC (Adsorption
chromatography ) i.e. the principle of separation is
adsorption.
• Mobile phase flow by capillary action effect .
• And component move according to their affinities
towards the adsorbent.
• The component with higher affinity toward adsorbent
travels slowly.
• And the component with lesser affinity towards the
stationary phase travels faster.
• Thus the components are separated on a chromatographic
plate according to their affinity and seperation also based
on their solubility in mobile phase.
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6. MANUAL SAMPLE APPLICATOR
The Nanomat serves for easy application of
samples in the form of spots onto TLC and
HPTLC layers .
• The actual sample dosage performed with
disposable capillary pipettes , which are
precisely guided by the capillary holder.
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7. SEMI AUTOMATIC SAMPLE APPLICATOR
The instrument is suitable for routine use for
medium sample throughout . In contrast to the
Automatic TLC sampler , changing the sample
the Linomat requires presence of an operator.
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8. AUTOMATIC SAMPLE APPLICATOR
• Samples are either applied as spots through
contact transfer (0.1-5 micro lit) or as bands or
rectangles (0.5->50 micro lit) using the spray on
techniques.
• ATS allows over spotting.
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9. AUTOMATIC DEVELOPING CHAMBER (ADC)
In the ADC this step is fully
automatic and independent of
environmental effects.
The activity and pre-conditioning of
the layer , chamber saturation
developing distance and final
drying can be pre-set and
automatically monitored by ADC.
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10. STEPS INVOLVED IN HPTLC
Selection of chromatographic plates
Layer pre-washing
Activation of pre-coated plates
Selection of chromatographic plates
Selection of mobile phase
Pre-conditioning
Chromatographic development and drying
Detection and visulization
Documentation
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13. DOCUMENTATION
Documentation is important because 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.
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16. HPTLC Method for the Determination of Paracetamol, Pseudoephedrine
and Loratidine in Tablets and Human Plasma
Paracetamol (PAR) is acetamide, N-(4-hydroxy phenyl) (1, 2),
which is widely used as a minor analgesic,
Pseudoephedrine HCl (PSH) is (1S,2S)-2-(methylamino)-1-
phenylpropan-1-ol hydrochloride
Loratadine (LOR) (ethyl-4-(8-chloro-5,6-dihydro-11H-
benzo[5,6]cyclohep-ta[1,2-b]pyridin-11-ylidine)-1-piperidine
carboxylate) is a second-generation antihistamine.
Case study
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17. EXPERIMENTAL
Samples
Pure samples
Pure samples were kindly supplied by Al Rowad Pharmaceutical Industries Co., with
certified purities of 99.84, 98.50 and 100.07% for PAR, PSH and LOR, respectively.
Pure standard 4-AP was purchased from Sigma-Aldrich Co., Cairo, Egypt, with a
certified purity of 99.56%.
Pharmaceutical samples
ATSHI® tablets (batch no. 2234), manufactured by Al Rowad Pharmaceutical Industries
Co., 10th of Ramadan City, Egypt, were labeled to contain 500 mg PAR, 120 mg PSH
and 5 mg LOR.
Chemicals and reagents
Acetone, hexane, methanol and ammonia 33% were of analytical grade (El Nasr
Pharmaceutical Chemicals Co., Abu-Zabaal, Cairo, Egypt).
Solutions
Stock standard solution (1 mg/mL) of PAR, 4-AP,PSH and LOR were prepared in
methanol.
Pharmaceutical dosage form solution
Ten tablets were crushed and triturated well in a mortar. The average tablet weight was
determined, and a powder sample equivalent to 500 mg of PAR was transferred into a
100-mL volumetric flask. About 75-mL methanol was added, and the flask was sonicated
for 15 min. The solution was filtered, and the volume was completed to the mark with
methanol.
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18. Procedure
Chromatographic conditions
Samples were applied in the form of bands of 6 mm width with a
100-µL sample syringe on aluminum plates precoated with silica gel
60F254 (20 × 10 cm), using an autosampler. A constant application
rate of 0.1 µL/s was used, and the space between bands was 8.9
mm. The slit dimension was 6.0 × 0.3 µm, and the scanning speed
was 20 mm/s. The mobile phase consisted of acetone–hexane–
ammonia (4:5:0.4, by volume). Linear ascending development was
carried out in a glass chamber saturated with the mobile phase.
Development of the plates was left till the mobile phase migrates 8
cm. Following the development, the plates were air dried, spots
were visualized under a UV lamp at 254 nm and densitometric
scanning was performed using a CAMAG TLC scanner in the
reflectance–absorbance mode at 254 nm for PAR, 4-AP and LOR
and at 208 nm for PSH and operated by WINCATS software. The
radiation source was a deuterium lamp.
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19. CALIBRATION
Volumes equivalent to 0.1–6 mL for PAR, 0.2–3.5
mL for 4-AP, 1.6–12 mL for PSH and 0.1–2 mL LOR
from stock standard solution of each were
accurately transferred into four series of 10-mL
volumetric flasks, and the volumes were completed
to the mark with methanol. Then, 10 µL from each
flask was spotted in replicates on HPTLC plates.
The procedure under chromatographic conditions
was performed. The peak areas were plotted
against concentrations to obtain the calibration
graphs.
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20. APPLICATION TO HUMAN PLASMA
Into a series of 10 mL volumetric flasks, 1 mL of drug-
free human plasma sample was spiked with different
concentrations of PAR, PSH and LOR from their stock
solutions (1 mg/mL). The volumes were completed to
the mark with methanol to provide final concentrations of
0.5–6, 1.6–12 and 0.4–2 mg/mL for PAR, PSH and LOR,
respectively. The flasks were shaken vigorously and
then centrifugated at 3,000 rpm for 15 min. Then, 1 mL
of the protein-free supernatant was transferred into four
series of 10-mL volumetric flasks, and the volume was
completed with methanol. Furthermore, 10 µL of each
solution was applied to the HPTLC plate and the
procedure was followed as described under calibration.
The regression equations were calculated.
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21. RESULTS
The HPTLC method offers a simple way to quantify directly on a
HPTLC plate by measuring the optical density of the separated
bands. The amounts of compounds are determined by comparing
to a standard curve from reference materials chromatographed
simultaneously under the same condition (20).
The HPTLC densitometric method has advantages of low
operating costs and high sample output, and the need for
minimal sample preparation and mobile phase having pH 8 or
more can be used (21).
Although the proportion of the three studied drugs in their
pharmaceutical formulation is complex (500:120:5, for PAR, PSH
and LOR, respectively), the proposed method has offered a
solution to this problem as HPTLC is a separation method.
The calibration curves were constructed by plotting the peak
areas versus the corresponding concentrations, and the
regression equations were calculated for the three drugs and 4-
AP.
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