high performance thin layer chromatography

1. HPTLC

2. INTRODUCTION
 Sophisticated form of thin layer chromatography. It
involves the same theoretical principle of thin layer
chromatography.
 It is also known as planar chromatography or Flat-bed
chromatography.
 Traditional Thin Layer Chromatography & its modern
instrumental quantitative analysis version HPTLC are
very popular for many reasons such as
 visual chromatogram,
 simplicity,
 multiple sample handling,
 low running and maintenance costs, disposable layer
etc.

3. PRINCIPLE
HPTLC have similar approach and employ the same
physical principles of TLC (adsorption
chromatography) i.e. the principle of separation is
adsorption.
 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.

4. Steps Involving in HPTLC
Sample
Preparation
Selection of
chromatography layer
Pre-washing
Pre-conditioning
Application of sample
Chromatography development
Detection of spots
Scanning & documentation
4

5. 5
Sample and Standard Preparation
Solvents used are
•Methanol,
•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 .

6. Selection of chromatographic layer
• Precoated plates - different support materials -
different Sorbents available
•80% of analysis takes place by silica gel GF ·
• Amino acids, dipeptides, sugars and alkaloids - cellulose
•Non-polar substances, fatty acids, carotenoids, cholesterol
- RP2, RP8 and RP18
•Preservatives, barbiturates, analgesic and phenothiazines-
Hybrid plates-RPWF254s
6

7. Pre coated 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.
7

8. Supports:
• Glass
• polyester sheets
• Aluminium sheets

9. 9
Some of the sorbents used in HPTLC:
• Silica gel 60F (Unmodified )
• Alluminium oxide
• Cellulose (microcrystalline )
• Silica gel chemically modified

10. Some of the binders used
 Gypsum (G)
 Starch (S)
 Layer containing fluorescent indicator (F)
10

11. Selection of HPTLC plates
Hand plates were available which are made up
of cellulose and other materials which are not
used much now-a –days.
11

12. Plate size
 20X20cm
 10X20cm
 5X10 cm
 5X7.5 cm
 Good cut edges of sheets is important to obtain
constant Rf values.
12

13. 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.
Silica gel 60F is 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 in the ratio of 9:1.This is the major
advantage of the step of pre-washing.
13

14. : Some common methods involved in
pre-washing
 Ascending method
 Dipping method
 Continuous method
14

15. Solvents used for pre-washing
 1.Methanol
 2.Chloroform: methanol ( 1:1 )
 3.Choloroform: Methanol: Ammonia (90:10:1 )
15

16. Activation of plates
 Freshly opened box of HPTLC plates doesn’t need
activation.
 Plates exposed to high humidity or kept in hand for
long time require activation.
 Plates are placed in oven at 110o-120oc for 30 min
prior to the sample application.
• Aluminium sheets should be kept in between two
glass plates and placing in oven at 110-120ºc for 15
minutes.
16

17. Pre-conditioning
• Also called Chamber Saturation
•Un- saturated chamber causes high Rf values
Sample application
Usual concentration range is 0.1-1μg / μl
Above this causes poor separation
Linomat IV (automatic applicator) - nitrogen
gas sprays sample and standard from syringe
on TLC plates as bands
Band wise application - better separation.

18. Selection of mobile phase
•Trial and error
•one’s own experience and Literature
•Normal phase
Stationary phase is polar
Mobile phase is non polar
•3 - 4 component mobile phase should be
avoided
•Multi component mobile phase once used not
recommended for further use.
•Twin trough chambers are used only because
10 -15 ml of mobile phase is required

19. 19
Some applicators used for application of sample
a) Capillary tubes.
Samples applied in the form of spots.
Volume of 0.1-0.2μl
b) Micro bulb pipettes.

20. c) Micro syringes.
• Sample can apply either as
spot or band
• Volume- 1μl.
d)Automatic sample applicator.
• Sample can apply either as
spot or band.

21. Pre- conditioning (Chamber saturation)
•Un- saturated chamber causes high Rf values
•Saturated chamber by lining with filter paper for 30
minutes prior to development - uniform distribution of
solvent vapours - less solvent for the sample to travel -
lower Rf values.
Chromatographic development and drying
•After development, remove the plate and mobile phase is
removed from the plate - to avoid contamination of lab
atmosphere
•Dry in vacuum desiccator

22. H P T L C DEVELOPMENT
 Vertical Development.
 Vario method development.
 Horizontal development.
 Automatic Multiple
Development (AMD)/( Gradient ).
22

23. Twin Trough Chambers
23

24. Vario Chamber Development
VARIO CHROMATOGRAM
24

25. Horizontal Development
25
•HPTLC plate is developed from
both opposing sides towards the
middle.
•Plate sizes 10x10cm and 20x10cm

26. Post Chromatography Steps
1) Detection.
2) Photo documentation.
3) Densitometry measurements.
26

27. Detection
Detection under UV light is first choice - non
destructive
- 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
UV CABINET

28. Detection and visualization
•Detection under UV light is first choice - non
destructive.
•Non UV absorbing compounds like ethambutol,
dicylomine etc - dipping the plates in 0.1% iodine
solution.
Quantification
•Sample and standard should be chromatographed
on same plate after development chromatogram is
scanned.
•Concentration of analyte in the sample is
calculated by considering the sample initially
taken and dilution factors.

29. Densitometry measurements
 Measures visible, UV absorbance or
Fluorescence.
 Convert the spot/band into chromatogram
consisting of peaks
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30. Instrumentation of HPTLC consists of following:
 Lamp selector
 Entrance lens slit
 Monochromator entry slit
 Grating
 Mirror
 Slit aperture disc
 Mirror
 Beam splitter
 Reference photo multiplier
 Measuring photo multiplier
 Photo diode for transmission measurements.

31. INSTRUMENTATION

32. • Theory: According to the theory, the transmission of
light in a translucent material can be described by:
I0 = Ia + It + Ir + Ix
Where,
I0 = Intensity of incident light.
Ia = Intensity of absorbed light.
It = Intensity of transmitted light.
Ir = Intensity of reflected light.
Ix = Intensity of light lost due to
scattering.

33.  The Densitometer work by 2 modes:
1. Transmission mode
2. Reflectance mode

34.  In transmission mode the ratio of It/Io is
measured and converted in to absorbance values
 In reflectance mode the ratio Ir/Io is measured
and converted in to absorbance values
 According to Goodman & Goodall transmission
measurements are more sensitive than
reflectance measurements

35. Fluorescence measurement in densitometry:
1) Measurement of direct fluorescence
2) Measurement of fluorescent quenching.
1. Direct fluorescent measurement:
• This method is followed if the spot exhibit
fluorescence when exposed to UV light.
• In this two monochromators are used for selection
of excitation & emission wavelength. The
fluorescence is measured in reflectance mode.

36. 2) Fluorescence quenching measurement:
• As the name indicates, it utilizes the ability of analyte to
absorb & quench fluorescence light.
• In this technique fluorescent background is incorporated
into the layer. When excited by short wavelength
radiation the plate fluorescence's uniformly.
• If UV absorbing substance is present in the plate, a
portion of the fluoresced light is absorbed &
consequently quenched.
• This fluorescence diminution is measured as a function
of amount of analyte in the spot.
.

37. Advantages of densitometer scanner:
 The purpose of scanner is to convert the
spot/band on the layer into densitogram
consisting of peaks similar in appearance
to HPLC.
 The position of the scanned peaks on the
recorder chart is related to Rf values.
 Quantitation is faster, reliable accurate &
reproducible

38. Photo-documentation With
Digital Camera
38

39. 7)DOCUMENTATION:
 1. Documentation is important because labeling every single
chromatogram can avoid mistake in respect of order of
application.
 2. Type of plate, chamber system, composition of mobile
phase, running time and detection method should be
recorded.
 3. TO assist the analysts and researchers E .merck has
introduced HPTLC pre-coated plates with an imprinted
identification codes.

40. Documentation
 video scan software for quantitative evaluation
of images capture with digistore

41. Differences between TLC and HPTLC

42. Applications of HPTLC
 Pharmaceutical industry: Quality control, content
uniformity, uniformity test, identity/purity check.
 Food Analysis: Quality control , additives ,
pesticides ,stability testing ,analysis of sub-micron
levels of aflotoxins etc.
 Clinical Applications: Metabolism studies , drug
screening ,stability testing etc
 Industrial Applications: Process development and
optimization, In-process Q.C. check, validation etc.
 Forensic : Poisoning investigations.

43. A) QUANTITATIVE DETERMINATION:
1) Biochemical research/Biotechnology-
Seperation of gangliosides
2) Clinical-
Inorganic & organic mercury in water & human
serum. Caffeine in urine.
3) Cosmetics-
Hydrocortisone & cinchocaine in lanolin ointment
4) Environmental Analysis-
Pesticides in drinking water.
Selenium in water.
5) Food analysis-
Vitamin C in fruit juices.
Aflatoxins in food stuff

44. 6) Pharmaceutical & chemical substance-
 Content uniformity test of diclofenac sodium.
 Vitamin B1 pharmaceutical products.
7) Natural products ,plant ingredients-
 Glycosides in herbal drugs.
 Glycyrrhizic acid in liquorice.
8) Doping analysis-
 Atenalol in urine.
B) FINGER PRINT ANALYSIS-
 HPTLC finger print of Valerian.
 Finger print of garlic, Ashwaganda.
 Finger prints for identification of liquorice, ginseng.

45. 9) Identification and separation of phenyl
thiohydantoin -amino acid.
10) analysis of drugs in blood
EX: 1)separation of phenothiazine drugs like
chlorpromazine, acetophenazine, perphenazine,
trifluperazine and thoridazine.
11) identification of mycotoxins in admixture :
EX: detection of sterigmatocystin, zearalenone,
citrinin, ochrotoxinA, patulin, penicillic acid.
12) determination of polycyclic aromatic
hydrocarbons in particulate sample.
EX; determination of chryesene, pyrene,
fluoronthene etc.

46. REFERENCES
 HPTLC - Quantitative Analysis of Pharmaceutical
Formulations by Dr.P.D.Sethi, Page No.3 – 72.
 Pharmaceutical Analysis vol-II by Dr. A. V. Kasture,
Dr. K. R. Mahadik Nirali Publishers page no 28-30.
 Textbook of pharmaceutical analysis, third edition
by S. Ravi shankar, Rx publications pages no 14.10
to 14.12
 www.pharmainfo.com
 www.camagusa.com
 www.infoexpo.com