This document provides an overview of high performance thin layer chromatography (HPTLC). It describes the basic principles, instrumentation, advantages, disadvantages and applications of HPTLC. Key points include that HPTLC is an automated form of thin layer chromatography that allows for better separation efficiency and detection limits compared to traditional TLC. The document discusses sample preparation, development, detection, quantification and various instruments used in HPTLC like sample applicators, developing chambers, cameras for visualization and quantification. Finally, it provides examples of HPTLC applications in quality control of herbal medicines, dietary supplements, forensic analysis and metabolite studies.

1. HPTLC
Unit 2 Chromatography
Introduction, Principle, Instrumentation, Application
III Pharm D
Pharmaceutical Analysis
RVS College of Pharmaceutical Sciences,
Sulur, Coimbatore
Staff incharge – Ms. P Brindha Lakshmi, M.Pharm

2. CONTENTS
• INTRODUCTION
• COMPARISION OF TLC & HPTLC
• PRINCIPLE – HPTLC
• INSTRUMENTATION – HPTLC
• ADVANTAGES
• DISADVANTAGES
• APPLICATION

3. TLC - HPTLC

4. INTRODUCTION
• It is also known as High Pressure Thin Layer Chromatography
Planar chromatography
Flat-bed chromatography
• A sophisticated and automated form of the thin-layer
chromatography (TLC) with better and advanced separation
efficiency and detection limits.
• Principle: Separation may result due to adsorption or partition
or by both, phenomenon’s depending upon the nature of
adsorbents used on plates and solvents system used for
development.

5. PRINCIPLE
• It is a powerful analytical method equally suitable for qualitative
and quantitative analytical tasks.
• Separation may result due to adsorption or partition or by both
phenomenon, depending upon the nature of adsorbents used on
plates and solvents system used for development.
• 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.

6. BASIC STEPS IN HPTLC
 Selection of stationary phase (chromatographic layer)
 Sample and Standard Preparation (Layer washing)
 Activation of pre-coated plates
 Application of sample and standard
 Selection of mobile phase
 Chamber saturation
 Chromatogram development
 Detection
 Derivatization
 Quantification
 Digital Camera-Based Image Documentation
 Software-Induced Scanning

7. Selection of stationary phase
(chromatographic layer)
Smaller plates (10*10 or 10*20 cm) with significantly decreased
development distance (typically 6 cm) and analysis time (7–20
min) are used
Following are stationary phases that are mostly used:
Silica gel GF - with a less polar mobile phase, such as
chloroform– methanol, has been used for more than 90% of
reported analysis
Pre-coated plates

8. • Pre-coated plates - different support materials - different Sorbents
available like;
Aluminum oxide - Basic substances, alkaloids and
steroids
Cellulose - Amino acids, dipeptides, sugars and
alkaloids
• RP2, RP8 and RP18: Chemically bonded octa decyl
silane (ODS), an n-alkane with 18 carbon atoms, is the most
frequently used stationary phase.
• C8 and shorter alkyl chains provide other alternatives.
These are used for separation of non-polar substances like fatty
acids, carotenoids, cholesterol, etc

9. Sample and Standard Preparation
(Layer washing)
• To detect damage and impurities in the adsorbent, plates need to
be inspected under white and UV light before use.
• In order to improve the reproducibility and robustness of the
results, It is advisable to pre wash the plates.
• Dry the plates and store in dust free atmosphere.
• Layer washing is done to avoid interference from impurities and
water vapours

10. Generally, methanol is used as a prewashing solvent; however, a mixture of
Chloroform: Methanol (1:1),
Ethyl acetate: Methanol (1:1),
Chloroform: Methanol: Ammonia (90:0:1),
Methylene chloride: Methanol (1:1),
1% ammonia or 1% acetic acid or even mobile phase of the
method may also be used.

11. Activation of pre-coated plates
• Freshly open box of plates do not require activation
• Plates exposed to high humidity or kept on hand for long time to be
activated placing in an oven at 110-120ºC for 30min prior to spotting.
• Aluminum sheets should be kept in between two glass plates and placing
in oven at 110- 120ºC for 15 minutes.

12. Application of sample and standard
• The most critical step to obtain good resolution for quantification in
HPTLC.
• Sample application technique depends on factors such as the type of
sample matrix, workload and time constraints.
• Usual concentration range of sample is 0.1-1μg / μl because the
concentration above this range results in poor resolution of samples.
• There are 2 ways in which sample and standard can be applied
• Automated instruments
• Spray-on technique

13. • The sample is typically contained in a syringe, which is emptied by
a motor.
• Delivery speed and volume are electronically controlled.
• A stream of an inert gas such as nitrogen around the tip of the
syringe atomizes the sample and creates a band on the TLC/HPTLC
plate if either the syringe or the plate is moving linearly.
• Band wise application is done in order to achieve better separation
and high response to densitometer.
• Sample application in the form of narrow bands provides the
highest resolution and sensitivity.
• Circular spots have numerous disadvantages while “line” or “band”
application is advantageous

14. Selection of mobile phase
• The selection of mobile phase is based on adsorbent material used as
stationary phase and physical and chemical properties of analyte.
• The elution power of the mobile phase depends on a property called
eluent strength which is related to the polarity of the mobile phase
components.
• Multi component mobile phase once used not recommended for further
use and solvent Composition is expressed by volumes (v/v) and sum of
volumes is usually 100.
• Only 10 -15 ml of mobile phase is required while using twin trough
chambers.

16. Chamber saturation
• HPTLC plates are developed in chambers (flat-bottom chambers, twin-
trough chambers, or horizontal-development chambers) and generally,
saturated twin-trough chambers are used for the best reproducibility.
• Chamber saturation is carried out as follows: Development chamber is
lined on three sides with filter paper.
• Appropriate volume of mobile phase is prepared and carefully poured into
chamber in order to wet the filter paper is thoroughly so that it adheres
to rear wall of chamber.
• Tilt chamber to the side (about 45°) in order to equalize solvent volume in
both troughs. Replace the lid and let chamber equilibrate for 20 min

17. Chromatogram development
• Desired developing distance that is 70 mm from lower edge of plate is
marked on the right edge of the plate with a pencil.
• Lid should not be removed but slide off to the side and plate should be
placed in the front trough in such a way that the layer and filter paper
face each other and the back of the plate is resting against front wall of
chamber.
• Lid is replaced and plates are developed to the mark.
• After development, plates are removed from chamber and dried vertically
in the direction of chromatograph for 5 min in a stream of cold air.
• Remaining mobile phase and filter paper should be discarded after each
development. Vacuum desiccators can also be used for drying

18. Detection
• Detection under UV light is first choice because it is non-destructive.
• Zones with fluorescence or quench fluorescence are viewed in cabinets
that incorporate short-wave (254 nm) and long-wave (366 nm) UV lamps.
• Spots of non-fluorescent compounds are visualized using fluorescent
stationary phase.
• For example, silica gel GF has been used for visualizing non UV
absorbing compounds like ethambutol, dicylomine

19. Derivatization
• Derivatization is necessary for some compounds that do not respond to UV light
and involves reaction with a specific reagent.
• Derivatization is performed either by immersing the plates or by spraying the
plates with a suitable reagent.
• Immersing: Tank of immersion devices is charged with 200 ml of reagent and
plate is placed in the holder of the immersion device, parameters should be set
according to method to start the process. The plate is removed from plate holder
and dried in stream of cold air.
• Spraying: Charge the bottle of the sprayer with up to 50 ml of reagent. Place the
plate in spray cabinet against a filter paper.
• Spray plate with horizontal and vertical motion until it is homogeneously covered
with reagent. The plate is dried with cold air.
• To induce or optimize the derivatization reaction, it may be necessary to heat the
plates

20. Quantification
• Quantitative analysis are performed in situ by using a chromatogram
spectrophotometer (densitometer or scanner) with a fixed sample light
beam in the form of a rectangular slit that measures the zones of samples
and standards.
• Generally, TLC Scanner 3 using win CATS software is used that scans
the chromatogram by reflectance, transmittance, absorbance or by
fluorescent mode.
• Scanning speed is selectable up to 100 mm/s. Spectra recording is fast.
• Concentration of analyte in the sample is calculated by considering the
sample initially taken and dilution factors

21. Digital Camera-Based Image
Documentation
• UV Cabinets are now being replaced with improved design UV
Cabinets which allow digital camera to be fixed for recording images
of the plate.
• Small labs prefer this device although it does not conform to GLP.
• Today, HPTLC is a primary requirement for any laboratory involved in
herbal analysis for establishing the identity of plant extracts by
comparison with Botanical Reference Material (BRM) extracts to detect
substitutes or adulterants, studies of formulations, etc.
• Forensic analysts have long ago stated that their starting points are a
microscope for physical inspection and TLC for chemical inspection

24. INSTRUMENTATION

25. SAMPLE APPLICATION
• The first step in the workflow of TLC/HPTLC and it affects significantly
the quality of the result at the end of the process.
• Methods
Spot application
Spraying – on samples
• Starting zones in the form of narrow bands ensure the best resolution.
• Very large sample volumes or samples with a high matrix content can be
sprayed-on in the form of rectangles
• Spotting samples using a fixed volume capillary is the simplest and
cheapest way of sample application and generally used in conventional
TLC.
• Sample volumes of 0.5 to 5 μL can be applied.

26. CAMAG® Automatic TLC Sampler 4
(ATS 4)
CAMAG® Linomat 5

27. CAMAG® Nanomat 4

28. DEVELOPMENT
• The key step of the TLC/HPTLC process.
• In addition to stationary and mobile phase, TLC/HPTLC features a gas
phase, which can significantly influence the result of the separation.
• A development can occur in an unsaturated, partially saturated,
saturated and/or preconditioned chamber.
• The primary focus of an automatic is to make the development more
reproducible and independent of human interaction.
• The layer is automatically brought in contact with the mobile phase and
the migration is monitored.
• After completion of chromatography, the plate is dried.

29. CAMAG® Automatic Developing Chamber
2 (ADC 2)
CAMAG® AMD 2 System Automated Multiple
Development

30. CAMAG® Twin Trough Chamber CAMAG® Flat Bottom Chamber

31. DERIVATIZATION
• It is an inherent advantage of TLC/HPTLC that all analytes remain stored
on the plate and can be readily derivatized after chromatography.
Analytes that do not respond to visible or UV light can be rendered
detectable. In many cases, analytes or classes of analytes can be
identified by specific reagents, enabling their selective detection.
• Pre-chromatographic derivatization is possible by overspraying the
sample application zones with the CAMAG® Linomat 5, the CAMAG®
Automatic TLC Sampler 4 (ATS 4) or CAMAG® HPTLC PRO Module
APPLICATION.

32. • For the transfer of liquid reagents for postchromatographic derivatization,
one can choose between spraying or dipping. Automated spraying (CAMAG®
HPTLC PRO Module DERIVATIZATION, CAMAG® Derivatizer) or manual
dipping (CAMAG® Chromatogram Immersion Device 3) are the preferred
techniques, particularly when a quantitative evaluation is intended.
• In most cases, the derivatization reaction needs to be completed by heat
treatment. Heating the plate at the desired temperature with a plate heater
(CAMAG® TLC Plate Heater 3) specifically designed for this purpose is highly
recommended. A plate heating unit is an integral part of the CAMAG®
HPTLC PRO Module DERIVATIZATION.

33. CAMAG® Derivatizer CAMAG® TLC Plate Heater 3

34. CAMAG® TLC Sprayer CAMAG® Glass reagent sprayer

35. DETECTION
• Colored substances can be seen directly on the plate; others are
conveniently changed into colored derivatives.
• Compounds that absorb UV light of UV 254 nm are visualized with the
help of a fluorescence indicator (F254) embedded in the stationary phase
of the plate and excited by a UV lamp.
• Substances excited to fluoresce by UV 366 nm either with or without
derivatization can also be visualized.
• For electronic image acquisition a digital camera captures visible
polychromatic light. The obtained data can be edited, archived and
evaluated. For quantitation, either
hyperspectral data
densitograms
image profiles

36. • Hyperspectral data are obtained by using polychromatic light for
excitation in conjunction with an imaging spectrometer (CAMAG® HPTLC
PRO Module DETECTION). Densitograms are obtained by scanning the
plate with monochromatic light and detecting the reflected light with a
photomultiplier (CAMAG® TLC Scanner 4).
• Images are captured with a tri-colored CCD camera (CAMAG® TLC
Visualizer 2).
• The spectral selectivity offers a great advantage over visual inspection.
• Each individual analyte can be evaluated at its absorption/fluorescence
maximum.

37. CAMAG® TLC Scanner 4 CAMAG® TLC Visualizer 3

38. CAMAG® BioLuminizer 2 CAMAG® UV Cabinet 4

39. APPLICATIONS
HPTLC method deals with qualitative and quantitative analytical
applications such as
herbal and dietary supplements, nutraceuticals, and various types
of medicines.
It is used in
quality control
purity check
clinical applications
metabolism studies
drug screening etc
forensic: poisoning investigations
assaying radiochemical impurities of radiopharmaceuticals
detection and identification of pharmaceutical raw materials
drugs and their metabolites in biological media

40. • HPTLC as biomarker in pharmacognostical research HPTLC analysis of
many plants used in Indian Systems of Medicine has been performed for
various pharmacological activities like CNS, hepatoproctative etc.
• The HPTLC may be used as a rapid method by which to control the
quality of raw plant materials and formulations based on the Lawsonia
inermis plant.
• Michelia champaca L. (Magnoliaceae) popularly known as champa is a
reservoir of numerous bio markers.
• HPTLC method has been used for detection, and quantification of
quercetin in Michelia champaca (leaves and stembark) and the estimated
values indicate that the leaves are the richest source of the quercetin.
• HPTLC method for the estimation of curcumin in marketed turmeric
powder can be used routinely with good reliability and reproducibility