HPTLC is an improved form of TLC that provides higher resolution, shorter development times, and less solvent consumption compared to conventional TLC. It utilizes pre-coated plates with smaller adsorbent particles and pore sizes. HPTLC is useful for both qualitative and quantitative analysis in applications such as pharmaceutical quality control, food analysis, and clinical and forensic studies. The basic steps in HPTLC include sample preparation and application, chromatographic development and separation, and detection and visualization of spots.

1. HIGH PERFORMANCE THIN LAYER
CHROMATOGRAPHY
Presented by-
Harsh Wardhan Billore
M. Pharm 1st semester
Subject:-Modern Analytical Techniques
(Branch :-Pharmaceutics)
Guided by-
Dr. Tahir Nizami
( Associate Professor )
Smriti College of Pharmaceutical Education ,Indore

2. INTRODUCTIO
N
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.
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.

3. .
• HPTLC is a well known and versatile separation method which shows a lot of
advantages and options in comparison to other separation techniques.
• The method is fast and inexpensive. It does not require time consuming
pretreatments.
• The basic difference between conventional TLC and HPTLC is only in particle and pore size of
the sorbents.
• It is very useful in quantitative and qualitative analysis of pharmaceuticals.
• HPTLC (High performance thin layer chromatography) is the automated, sophisticated form
and improved method of TLC.

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.

5. PRINCIPL
E
.

6. ADVANTAGES OF
HPTLC
.
 High resolution of zones due to higher number of
theoretical plates.
 Shorter developing times
 Less solvent consumption
 Enormous flexibility
 Parallel separation of many samples with
minimal time requirement
 Simplified sample preparation due to single use
of the stationary phase.

7. DIFFERENCES BETWEEN TLC &
HPTLC
PARAMETERS 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

8. PARAMETERS TLC HPTLC
Sample volume 1-10 μl 0.2-5 μl
Application of larger volume Spotting which leads to over
loading
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

9. INSTRUMENTATION OF
HPTLC
1. Plate coaters
2. Drying racks
3. Plate cutters
4. Immersion device
5. Plate heater
6. Sample application
7. Development chamber
8. Derivatization
devices
9. Scanning densitometer

10. 1.PLATE
COATER
1.Hand Operated 2.Automatic Plate Coater
Work same as automatic
coater, except with, plates
are pushed by hand, one
after the other & lifted off
on the other side.
The glass plates to be coated are
conveyed underneath a hopper
filled with a adsorbent
suspension with a uniform
feeding rate 10cm/s, for uniform
speed.

11. 2.DRYING
RACK
 The Drying Rack consists of ten individual aluminum trays.
 A tin box for storing the trays and two wire handles , to move the stack whilehot
,are supplied.
 The drying rack is convenient to use ,particularly when TLC plates areprepared with the
automatic plate coater in large runs.

12. 3.PLATE
CUTTER
 Used to cut HPTLC plates easily and more precisely.
 Cuts plates with a thickness up to 3mm.
 Does not damage the sensitive layer.
 Easy to handle .Read the required size from the scale directly.
 Helps saving costs on pre-coated plates of high quality by preventing offcuts.

13. 4.IMMERSION
DEVICE
 For proper execution of the dipping technique , the chromatogram must be immersed
and withdrawn at a controlled uniform speed.
 Key features
•Uniform vertical speed
•Immersion time selectable between 1 and 8 seconds.
•The device can be set to accommodate 10cm and 20cm plate height.
•Battery operated , independent of power supply.

14. 5. PLATE
HEATER
 The TLC plate heater is designed for heating TLC plates to a given temperature,
while ensuring homogenous heating across the plate.
 The TLC plate heater has a heating surface which is resistant to all common
reagents and is easily cleaned.
 Programmed and actual temperature are digitally displayed.
 The temperature is selectable between 25 and 2000oc.
 The plate heater is protected from overheating.

15. 6.SAMPLE
APPLICATION
 Usual concentration of applied samples 0.1 to 1 μg / μl for qualitative Analysis and
quantity may vary in quantitation based on UV absorption 1 to 5 μl for spot and 10
μL for band application.
SAMPLEAPPLICATORS
1.MANNUAL 2.SEMI-AUTOMATIC 3.AUTOMA
TIC
Calibrated capillaries Applicators
 Applicators use spray on or touch and deliver technique for application.

16. a) 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.
b) Semi automatic sample applicator
 The instrument is suitable for routine use of medium samples
throughout.
 Linomat requires presence of an operator.
 With the linomat , samples are sprayed onto the chromatographic layer in
the form of narrow bands.
 During the spraying the solvent of the sample evaporates almost entirely
concentrating the sample into a narrow band of selectable length.

17. c) Automatic sampleapplicator
 Samples are either applied as spots through contact transfer
(0.1-5 micro lit) or as bands or rectangles (0.5->50microlit)using the spray
on techniques.
 Application in the form of rectangles allow precise applicationsof
large volume with out damaging the layer.
 ATSallows over spotting.
 Glass: Borosilicate
 Precision: <+/- 1% of volume
 Needle: Especially developed for the need of linomat III
and IV

18. 7. DEVELOPING
CHAMBER
1.Twin trough chamber
2.Automatic developing
chamber
Low solvent consumption
Reproducible pre –equilibrium
with Solvent vapour
Start of development
fully automatic and independent of
environmental effects.

19. 8. DERIVATIZATION DEVICE
a) Spraying
b) Dipping
c) Derivatization through gas phase
 Reasons for Derivatization:
•Changing non-absorbing substance into detectable derivatives.
•Improving the detectability.
•Detecting all sample components.
•Selectivity detecting certain substance.
•Inducing fluorescence.

20. a) Spraying:
a) Spraying headA
 For solutions of normalviscosity
Eg. Lower alcohol solution
b) Spraying head B
 For solutions of higherviscosity
Eg.Sulphuric acid reagent
b) Dipping:
 For proper execution of the dipping technique the chromatogram must be immersed andwithdrawn at a
controlled uniform speed. By maintaining a well defined vertical Speed and immersion time,
derivatization Conditions can be standardized and tide Marks , which can interferewith
densitometry evaluation , are avoided.

21. c)Derivatization through gas phase
 It offers rapid and uniform transfer of the reagent.
 It is unfortunate that only few reagents are suitable they include I, Br, Cl, as well as volatile
acids, bases and some other gases like H2S , NO.
 It offers rapid and uniform transfer of the reagent.
9.SCANNING DENSITOMETER
The scanner is connected to a computer.
The scanner features three light sources ,a deuterium lamp, a tungstenlamp
and a high pressure mercury lamp.
The scanning speed is selectable between 1 and 100 mm/s

22. STEPS INVOLVED IN
HPTLC
SAMPLE AND
STANDARD
PREPARA
TION
SELECTION OH CHROMATIGRAPHIC PLATES
LAYER PRE-WASHING
LAYER PRE-CONDITIONING
APPLICATION OF SAMPLE
CHROMATOGRAPHIC DEVELOPMENT
DETECTION OF SPOTS SCANNING AND DOCUMENTATION OF
CHROMATOPLATE USING PC CAT
SOFTWARE

23. SCHEMATICPROCEDUREFORHPTLC[4]

24. A. Selection of chromatographic plates-
• Hand made plates which are made up of cellulose and other materials which
are not used much now a day.
• Pre-coated plates- The plates with different support materials and sorbent
layers with different format and thickness are used for qualitative and
quantitative analysis.
• Support materials used in plates- Glass
Polyester/polyethylyne Aluminium
• Sorbents used in plates-Silica gel 60F, Aluminium oxide, Cellulose, silica gel
chemically modified –a)amino group(NH2), b) CN group
• Smaller particle size of silica helps in greater resolution and sensitivity.

25. B.Layer pre-washing-
Choloroform: Methanol: Ammonia (90:10:1 )
• It is purification step.
• 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.
• In case of silica 60F( most widely used sorbent) the major disadvantage of this sorbent is that it
contain iron as impurity.
• This iron is removed by using Methanol : water (9:1), this is the major advantage of the step of
pre-washing.
• Some commonmethods are- Asending
Dipping Continuous
• Solvents used for pre-washing-
Methanol Chloroform:Methanol ( 1:1)

26. C.Activation of pre coated plates-
• Freshly opened box of HPTLC plates doesn’t need activation.
• If plates exposed to high humidity or kept in hand for longer time then
activation is required and it’s activation results by removing moisture.
• The plates are activated by placing in an oven at 110- 1200 C for 30 min,
this step will removes water that has been physically absorbed on surface at
solvent layer.
• Activation at higher temp and for longer time is avoided which leads to
very active layer and there is risk of sample being decomposed.

27. D.Sample preparation and application-
Sample preparation-
• It’s important to prepare proper sample for successful separation.
• Sample and reference substances should be dissolved in the same solvent to
ensure comparable distribution at starting zones.
• It needs a high concentrated solution,as very less amount of sample need to
be applied.
• After that dry the plates and store in dust free atmosphere.
• Solvents used are-
• Methanol,
• Chloroform: Methanol (1:1),
• Ethyl acetate: Methanol (1:1),
• Chloroform: Methanol: Ammonia (90:!0:1),

28. Sample application-
• Usual concentration range is 0.1-1µg / µl,above this causes poor separation and
volume recommended for HPTLC-0.5-5μl .
• The size of sample spot applied must not exceed 1mm in diameter.
• Problem from overloading can be overcome by applying the sample as band.
• Some applicators used for application of sample-
Selection of applicator to be used depends on-
-Sample volume
-No. of sample to be applied
a) Capillary tubes
b) Micro bulb pipettes.
c) Micro syringes.
d)Automatic sample applicator.

29. E.Selection of mobile phase-
• Chemical properties of analytes and sorbent layer factors should be considered
while selection of mobile phase.
• Various components of Mobile Phase should be measured
separately and then placed in mixing vessel.
• The less amount of mobile phase is required then TLC .
• This prevents contamination of solvents and also error arising from volumes
expansion or contraction on mixing.
• Multi component mobile phase once used not recommonded for further use due to
diffirent evaporation and adsorption by layer.

30. F.Pre-conditioning (chamber saturation)-
• Un- saturated chamber causes high Rf values.
• Saturated chamber by lining with filter paper for 30min prior to development-
uniform distribution of solvent vapours-less solvent for the sample to travel-
lower RF values
• For low polarity mobile phase there is no need of saturation.However
saturation is needed for highly polar mobile phase.
• Chamber saturation influence separation profile.

31. G .Detection and visulization-
• Detection under UV light is first choice.
• Non destructive and 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.
• Non UV absorbing compounds like ethambutol, dicylomine dipping the plates in
0.1% iodine solution
.

32. Detectors-
UV cabinet
Diode –array detectors
• .

33. Instrumentation-
22
• Detector consistsof following-
Lamp selector Entrance lens slit
Monochromator entrance slit
Grating Mirror
Slit aperture disc Mirror
Beam splitter
Reference photo multiplier Measuring photo
multiplier
Photo diode for transmission measurements.

34. H .Scanning and documentation-
Scanning-
• The scanner converts band into peak and peak hieght or area is related to the
concentration of substance on spot/band.
• The peak height and area under spot are measured by instrument and recorded .
• 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.
Documentation-

35. Applications of HPTLC
• Pharmaceutical industry- Quality control,identity purity test etc.
• Food Analysis- : Quality control , additives , pesticides ,stability testing etc.
• Clinical Applications- Metabolism studies , drug screening ,stability
testing etc
• Industrial Applications- Process development andoptimization etc.
• Forensic- Poisoning investigations
• Biomedical Analysis- Seperation of gangliosides
• Environment Analysis-Pesticides in drinking water etc.
• Cosmetics-Hydrocortisone & cinchocaine in lanolin ointment etc.
• Natural products ,plant ingredients- Glycosides inherbal drugs,Piperine in piper
longum etc.
• Finger print Analysis-Finger prints for identification of liquorice, ginseng etc.
• Analysis of drugs in blood-

36. CONCLUSION
1. CHROMA
TOGRAPHY ISA SCIENCE OFSEPERA
TION USED EITHERFORIDENTIFICATIONOR
QUANTITATIONOFCHEMICALSUBSTANCES.
2. VARIOUSMODESOFCHROMATOGRAPHICTECHNIQUESWEREDEVELOPED BASEDONINITIALDISCOVERYBY
MICHAELT
.SWETT
.
3. HIGH PERFORMANCETHIN LA
YER CHROMA
TOGRAPHYIS MOST ADVANCEDTECHNIQUENOWADA
YS.
4. ITISTHEMODERNSEPARATIONTECHNIQUEWHICHISACCEPTRDASAN EXTREAML
YFLESIBLE,RELIABLE&
COSTEFFICIENTMETHOD.
5.BYTHISTECHNIQUE,ACCURATESEPARATIONOFSAMPLET
AKESPLACE.
6.ITCAN BECONSIDEREDASATIMEMACHINETHATCAN SPEEDUPOUR WORK& SOMANYTHINGSA
TATIME
USUALL
YNOTPOSSIBLEWITH OTHERANALYTICALTECHNIQUE.
7.HPTLCCANSIMULTANEOUSLYHANDLESEVERALSAMPLESEVENOF DIFFERENTNATURE& COPOSITION
SUPPORTINGSEVERALANALYTEOFA GIVENTIME.