1. HPTLC (High performance thin layer chromatography)
VAISHNAVI U.S.D.N
M.Pharmacy(Pharmaceutical Chemistry)
UNDER THE GUIDENCE:
CEEMA MATHEW,M.pharmacy Asst.Professor

2. INDEX INTRODUCTION ADVANTAGES OF HPTLC OVER HPLC & TLC
FEATURES OF HPTLC
STEPS INVOLVED IN HPTLC
DIFFERENCES BETWEEN TLC AND HPTLC

3. INTRODUCTION

4. CHROMATOGRAPHY is a broad range of physical methods used to separate and or to analyze complex mixtures. The components to be separated are distributed between two phases: a stationary phase bed and a mobile phase which percolates through the stationary bed.  

5. High performance thin layer chromatography (HPTLC) is an enhanced form of thin layer chromatography (TLC).
A number of enhancements can be made to the basic method of thin layer chromatography to automate the different steps, to increase the resolution achieved and to allow more accurate quantitative measurements.

6. Principle of separation is ADSORPTION
Automation is useful to overcome the uncertainty in droplet size and position when the sample is applied to the TLC plate by hand.
Principle of separation is ADSORPTION

7. Advantages of HPTLC over HPLC
Sample and standard, both can be used at a time.
Evaluation time is less.
Use of internal standard is not necessary.
Simultaneously 2 or 3 persons can work but not in case of HPLC.
Solvent degasing and removal of impurities is not necessary.

8. Advantages of HPTLC over TLC
Visual chromatogram
simplicity
Multiple sample handling
Low running and maintenance costs and disposable layer etc
Detection by TLC scanner
Spotting by applicator
Highly precised

9. FEATURES OF HPTLC

10. Simultaneous processing of sample and standard - better analytical precision and accuracy less need for Internal Standard
Several analysts work simultaneously
Lower analysis time and less cost per analysis
Low maintenance cost
Simple sample preparation - handle samples of divergent nature
No prior treatment for solvents like filtration and degassing
Low mobile phase consumption per sample
No interference from previous analysis - fresh stationary and mobile phases for each analysis - no contamination
Visual detection possible - open system
Non UV absorbing compounds detected by post-chromatographic derivatization

11. STEPS INVOLVED IN HPTLC

13. · 80% of analysis - silica gel GF
Selection of chromatographic layer
· Precoated plates - different support materials - different Sorbents available
· 80% of analysis - silica gel GF
· Basic substances, alkaloids and steroids - Aluminum oxide
· 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

14. To avoid interference from impurities and watervapours
Sample and Standard Preparation
To avoid interference from impurities and watervapours
Solvents used are
Methanol, Chloroform: Methanol (1:1)
Ethyl acetate: Methanol (1:1)
Chloroform: Methanol: Ammonia (90:10:1)
Methylene chloride : Methanol (1:1)
1% Ammonia or 1% Acetic acid
Dry the plates and store in dust free atmosphere

15. Layer pre-washing & pre-conditioning
If any impurities are present, those are need to be removed hence pre washing is done.
Conc.HCl : Methanol(1:9)
Plate is dipped in the soln, allowed for the solvent to pass throughout the plate. Hence impurities are washed out

16. 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 By placing in an oven at ºc for 30’ prior to spotting (pre conditioning)
Aluminum sheets should be kept in between two glass plates and placing in oven at ºc for 15 minutes.

17. · Above this causes poor separation
Application of sample and standard
· 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

18. Selection of Mobile phase
Can be explained by STAHL’S TRIANGLE
Hydrophilic
Non-Polar E
Polar
Lipophilic M S
Active
Inactive
EG:If
E- hydrophilic then
M- polar &
S- inactive
E- ELUENT
S- SORBANT
M- MIXTURE

19. Trial and error - one’s own experience and Literature
Normal phase - Stationary phase is polar - Mobile phase is non polar - Non-polar compounds eluted first because of lower affinity with stationary phase - Polar compounds retained because of higher affinity with the stationary phase
Reversed phase - Stationary phase is non polar - Mobile phase is polar - Polar compounds eluted first because of lower affinity with stationary phase - Non-Polar compounds retained because of higher affinity with the stationary phase

20. 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

21. Chromatographic Develpment
1. TWIN TROUGH CHAMBER

23. Detection and visualization
· 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
· Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1% iodine solution
· When individual component does not respond to UV - derivatisation required for detection

27. APPLICATIONS Pharmaceutical research Bio medical analysis
Clinical analysis
Environment analysis
food industry
Quantification of herbal extracts
Therapeutic drug monitoring to determine its concentration and metabolite in blood urine
Analysis of environment pollution level
Characterization of hazards in industrial waste
Quantitative determination of prostaglandins & thromboxanes in plasma.

28. DIFFERENCES BETWEEN TLC AND HPTLC

29. PARAMETER
TLC
HPTLC
Type of chromatographic plates
Hand made/pre coated
Pre coated
Absorbant layer mm mm
Particle size
5-20 micrometer
4-8 micrometer
Application of sample
Manual/semi automatic
Semi automatic
Shape of sample
spot
band

30. Spot size
3-6mm
1-2mm
Sample volume
1-10 microlit
0.1-2microlit
No of sample per plate
15-20
40-45
Optimal development distance
10-15cm
5-7cm
Development time
Depend upon mobile phase
40% less than TLC
Quantization
Manual
Manual/ instrumentation
Reproducibility of result
difficult
Reproducible

31. REFERENCES
Ajaz Ahmad, M Mujeeb, Bibhu Prasad Panda (2010) An HPTLC Method for the Simultaneous Analysis of Compactin and Citrinin in Penicillium citrinum Fermentation Broth. Journal of Planar Chromatography-modern TLC 23 (4), 282–285
1994 Rebecca Carrier and Julie Bordonaro - Intro to Biochemical Engineering Term Project, merged with the 1997 project by Kevin Yip
Puri, A., Ahmad, A. and Panda, B. P. (2010), Development of an HPTLC-based diagnostic method for invasive aspergillosis. Biomed. Chromatogr., 24: 887–892. doi: /bmc.1382
Wikipedia
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