The document provides a comprehensive overview of high-performance thin-layer chromatography (HPTLC), detailing its principles, instrumentation, application processes, advantages, and disadvantages. Key topics include the factors affecting HPTLC, the differences between TLC and HPTLC, and troubleshooting tips for common issues encountered during analysis. Applications of HPTLC span various fields, including pharmaceuticals, food analysis, and environmental testing.

1. High-Performance
Thin-Layer
Chromatography
(HPTLC)
Presented by:-
Kishan singh tomar
Himanshu thakur
Himanshu patel
Amit nishad
Abhishek shelke
Anubhav singh
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2. Content
 Introduction
 Principal
 Factors affecting HPTLC
 Instrumentation
• parts
• types
 Steps involve in sample
• Preparation
• Application
• Evaluation
 Advantages
• disadvantages
• application
 Trouble shooting
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3. Introduction
 High-Performance Thin-Layer
Chromatography (HPTLC) is an advanced
chromatographic technique for separating,
identifying, and quantifying compounds in
complex mixtures.
 It combines the separation power of TLC(Thin-layer
chromatography)with detection and identification
capabilities.
 Improved separation and quantification
capabilities.
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4. PRINCIPLE
• The basic principle here applied is adsorption.
• Separation based on differential migration of
compounds between a stationary phase (thin-
layer) and a mobile phase.
• Thin-Layer: Consists of a uniform layer of
adsorbent material (e.g., silica gel GF) on a glass
plate.
• Mobile Phase: Solvent or mixture of solvents that
travels up the thin-layer via capillary action.
• Separation Mechanism: Compounds move at
different rates, leading to separation based on
their chemical properties.
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5. Different steps of HPTLC
 Automatic Sample Applicator
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6. Detector Chamber (UV Cabinet)
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7. Densitometer (Scanner)
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8. Photo Documentation Unit
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9. Factor affecting
HPTLC
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10. Factors Affecting Hptlc
• Type of stationary phase
• Type of pre-coated plates
• Layer thickness
• Binder in the layer
• Mobile phase composition
• Solvent purity
• Size of developing chamber
• Sample volume to be spotted
• Size of spot diameter
• Solvent level in chamber
• Gredient elution
• Relative Humidity
• Temperature
• Separation Distance
• Mode of Development
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11. 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,
• θ- contact angle.
Related to HPTLC
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12. • 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
 Plate Height :
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13. • 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
 Resolution :
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14. • 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
 Capacity Factor / Retention Factor
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15. • 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
 SPOT CAPACITY:
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16. Instrumentation
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17. Instrumentation of HPTLC-
6 Scanner
Autometic developing chamber
Applicator
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18. 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 loading
to over Can be applied as bands
No. of samples/plate (20X20) 15-20 40-50
Optimum development
distance
10-15 cm 5-7 cm
Development time Depends on mobile
phase
40% Less than TLC
Reproducibility of results Difficult Reproducible
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19. 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.
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20. Plate size-
• 20X20cm
• 10X20cm
• Good cut edges of sheets is important to obtain
constant Rf values.
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21. 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
• 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
• CAMAG automatic TLC sampler III: Applies sample as spot or
bands automatically from the rack of sample vials
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22. 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
• Trough chambers are used in which smaller
volumes of MP usually 10-15 ml is required
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23. Development of chambers
1.Twin trough chamber
2.Rectangular chambers
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24. 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.
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25. 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.
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26. 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 voids manipulation of data at any
stage as coding will automatically get recorded
using photo-documentation

27. Steps involve in
preparation,
application and
evaluation of
sample
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28. 1. Selection and stationary
phase
Selection of chromatographic plate :- it should be not
reactive, economical beneficial, standard grade material
made for quantitative and qualitative analysis.
Selection of chromatographic plate material :-
•Glass
•Polyethylene/ polyester
•Aluminum
Selection of sorbent material:- small size particle with non
reactive tendency towards analyte and easily stick to plate.
Selection of sorbent material:- plates-Silica gel 60F,
Aluminum oxide, Cellulose, silica gel chemically modified -
a)amino group(NH2), b) CN group
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29. 2.Pre-washing of plate
What is necessity of washing:- it is done to remove impurity
such as volatile impurity and water vapors.
e.g. Silica 60 majorly have iron impurities which is washed by
methanol (9:1).
Method of washing :-
•Ascending
•Dipping
•Continuous
Solvent used for washing:-
•Methanol
•Chloroform: Methanol (1:1)
•Chloroform: methanol: Ammonia(90:10:1)
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30. 3. Activation of precoated
plates
Activation condition: -
• 110-120 ̊C for 30 min in hot air oven.
• Activation of plate is only necessary for hand made plate or
highly moisture absorbed plate.
• Higher temperature may leads to decomposition of
compound.
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31. 4. Sample preparation
Standard sample:- sample taken for reference proposes
and only main sample is present.
Test sample:- This are generally in mixture of compounds.
Both of the solution are made in high concentration dissolve
in optimum amount of solvent because small amount of
substance is going to be tested.
Solvent use for sample preparation:-
• Methanol
• chloroform: methanol (1:1)
• ethyl acetate: methanol (1:1)
•Chloroform: methanol: ammonia (90:10:1)
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32. 5. Selectionofsolvent
Ideal requirement for solvent:- solvent should be non
reactive to HPTLC environment and have maximum
solubility of test substance.
Types of solvent :-
• diethyl ether
• Methylene chloride
• chloroform
• Hexane
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33. 6. Sampleapplication
Range:- Normally 0.1-1 µg/ µl is use because above it poor
separation is observe and for HPTLC 0.5-5µl volume is
used to place 1mm spot.
Size:- sample size diameter should be in 1mm
Types of applicator:-
• capillary tube
• micro bulb pipettes
• micro syringes
• automatic sample applicator (auto sampler)
•Micro pipette
Drying of spot after correct placement.
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34. 7. preparationofchamber
• Obtain a twin trough chamber for 20x10 cm plates.
• Fit the rear trough of chamber with a filter paper of
corresponding size.
• Pour 20 ml of developing solvent over the filter paper into
the rear trough ensuring complete wetting.
• Pour a sufficient amount of developing solvent into the front
trough to have a level of 5 mm.
• Close the lid of the chamber and allow 20 min for saturation.
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35. 8.Development ofcompound
After that stationary phase is put into the chamber and wait
to move solvent 80% of total surface.
Different method used for development of chamber:-
• Ascending
• Descending
• Horizontal
Advance development device :-
• Automatic multiple development
• Circular device
• anti-circular device
• multiple developments
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36. 9.Detectionandvisualization
Types of detector: -
•Uv detector (widely use)
For fluorescent compound can be detected on 254nm
(short wavelength).
For non fluorescent compound can be detected by using
fluorescent stationary phase( silica gel Gf).
For non uv absorbing compound like ethanbutol,
dicylomine dipping the plate in 0.1% iodine solution.
•Fluorescence detector
•Mass spectroscopy
•Fourier-transform infrared.
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37. 10.scanning and
documentation of result
The scanner converts band into peak and peak height or
area is related to the concentration of
substance on spot/band.
The different type of results can be interpreted by total
area cover and the height of peak for each compound.
The documentation requirement is according to GLP and
record proposes which include name and sign person
perform experiment, date and time, observation graph and
result document with speciation of lot number, batch
number, etc.
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38. Advantages &
disadvantages
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39. Some Advantages of HPTLC are:
1. HPTLC has high resolution and sensitivity in
detection.
2. High separation efficiency of components.
3. Can detect trace amounts of compounds.
4. Visualization can be performed without staining.
5. Can analyze multiple samples simultaneously.
6. High resolution of complex mixtures.
7. Minimal sample preparation required.
8. Rapid analysis compared to traditional
chromatography.
9. Quantitative and qualitative analysis can be
performed.
10.Minimal use of solvents and reagents.
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40. Disadvantages of HPTLC are:
1. HPTLC is relatively expensive compared to
traditional TLC.
2. Requires specialized equipment and materials.
3. Higher cost of HPTLC plates.
4. High purity solvents are needed.
5. More complex stationary phase.
6. Quality control and testing add to costs.
7. Skilled technicians are required for optimal results.
8. Maintenance and calibration of equipment add to
costs.
9. Costly software and data analysis tools are needed.
10.Need for high-quality standards and reference
materials.
11.Cost of training and education for technicians.
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41. APPLICATION
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42. 1. Pharmaceutical Analysis:-
 Quantitative analysis of drugs and pharmaceuticals
in various formulations.
 Determination of drug purity and content.
2. Botanical and Herbal Analysis:-
 Identification and quantification of active
compounds in plant extracts.
 Authentication of herbal products.
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43. 3. Food and Beverage Analysis:-
 Detection and quantification of food
additives, preservatives, and
contaminants.
 Analysis of natural products and flavors.
4. Cosmetics and Personal Care
Products:-
 Analysis of ingredients and contaminants
in cosmetics and personal care products.
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44. 5. Environmental Analysis:-
 Detection and quantification of pollutants
and contaminants in environmental
samples.
6. Forensic Science:-
 Drug analysis in biological samples.
 Analysis of trace evidence.
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45. TROUBLESHOOTING
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46. 1. Poor Separation or Resolution:-
 Ensure proper sample application and spot size.
 Optimize mobile phase composition, development
distance, and saturation time.
 Check the condition of the stationary phase and
replace if necessary.
2. Streaking or Tailings:-
 Ensure even application of the sample, avoiding
overloaded spots.
 Optimize the sample concentration to prevent
overloading.
 Modify the mobile phase composition and saturation
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47. 3. Uneven Spot Shapes:-
 Ensure the cleanliness of the TLC plate
and avoid contamination.
 Optimize the sample application technique
to achieve a round and well-defined spot.
4. Faint or Weak Spots:-
 Increase the sample concentration or
volume for better visualization.
 Optimize the development conditions (e.g.,
mobile phase composition, development
distance).
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48. 5. Irregular Migration Patterns:-
 Check for impurities in the solvent or
contamination of the TLC plate.
 Ensure consistent saturation of the TLC chamber
and equilibration before use.
6. Peak Tailing:-
 Optimize mobile phase composition and pH.
 Ensure proper plate activation before use.
 Use a clean and properly conditioned TLC plate.
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49. 7. Non-Reproducible Results:-
 Standardize all experimental conditions,
including temperature, humidity, and equipment
calibration.
 Use high-quality TLC plates and solvents from
reliable sources.
8. Low Sensitivity:-
 Optimize sample concentration and application
volume.
 Consider derivatization techniques to enhance
sensitivity.
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50. References:-
1. Kovachev BB. Exploration of Feature Selection Techniques in
Machine Learning Models on HPTLC Images for Rule
Extraction.
2. Attimarad M, Ahmed KK, Aldhubaib BE, Harsha S. High-performance
thin layer chromatography: A powerful analytical technique in
pharmaceutical drug discovery. Pharm Methods. 2011 Apr;2(2):71-5.
doi: 10.4103/2229-4708.84436. PMID: 23781433; PMCID:
PMC3658041.
3. Shivatare RS, Nagore DH, Nipanikar SU. HPTLC’an important tool in
standardization of herbal medical product: A review. Journal of
scientific and innovative research. 2013;2(6):1086-96.
4. Srivastava MM. An overview of HPTLC: A modern analytical
technique with excellent potential for automation, optimization,
hyphenation, and multidimensional applications. High-performance
thin-layer chromatography (HPTLC). 2010 Sep 20:3-24.
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51. 21-03-2024 51
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