HPTLC

1. HPTLC BY Dr. PrawezAlam Department of Pharmacognosy College of Pharmacy Salman bin Abdulaziz University HPTLC

2. igh H erformance T hin P L ayer C hromatography

3. igh H atience T hin P L ayer C hromatography

4. igh H riced T hin P L ayer C hromatography

5. WHAT IS PLANAR /THIN LAYER CHROMATOGRAPHY Introduction: Chromatography is a physical process of separation in which the components to be separated and distributed between two immiscible phases-a stationary phase which has a large surface area and mobile phase which is in constant motion through the stationary phase. Introduction of HPTLC 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.

6. THE PRINCIPLE HPTLC takes place in high-speed capillary flow range of the mobile phase. There are three main steps HPTLC procedure: SAMPLE APPLICATION Sample to analyzed to chromatogram layer, volume precision and exact position are achieved by use of suitable instrument. CHROMATOGRAM DEVELOPMENT Solvent (mobile phase) migrates the planned distance in layer (stationary phase) by capillary action. In this process sample separated into it’s components. CHROMATOGRAM EVALUATION Separation tracks are scanned in densitometer with light beams in visible or uv region

7. TLC Plate HPTLC Plate

9. ADVANTAGES OFFERED BY HPTLC Difference between TLC and HPTLC

10. STEPS OF THE HPTLC PROCEDURE Wincats

12. Selection of HPTLCplates • Previously hand made plate is used in TLC for both qualitative and quantitative work, certain draw back with that is non uniformly layer, formation of thick layer paved for advant precoated plates. Now a days pre coated plates are available in different formet and thickness by different manufactures. These plates are used for both qualitative and quantitative purpose in HPTLC. • glass plates . • Polyester /polyethylene. • Aluminium plates

13. Application of sample and standard Sample application is one of the important and critical step for obtaining the good resolution for quantification by HPTLC.

14. LINOMAT 1V

15. AUTOMATIC TLC SAMPLER

16. DEVELOPMENT CHAMBER Chromatogram developement: After application of sample in HPTLC plate, chromatogram is developed by dipping in suitable solvent system taken in developing chamber. The solvent system rises over the layer by capillary action and separation of sample in different components take place.

17. LIGHT WEIGHT TWIN TROUGH CHAMBER

18. Automatic Developing Chamber ADC

19. DENSITOMETRIC CHROMATOGRAM EVALUATION Detection or visulation of spot/ band: There is no difficult in detecting the colored substance, or color les substance absorbing the uv radiation or with fluoresce (Riboflavin).

20. Reprostar. Photo & Video –Documentation / Video Densitometry

21. Qualitative Analysis: Development of TLC Methods for Differentiation between commonly used Umbelliferous Spices

22. UV λmax = 366 UV λmax = 254 After spraying with different reagents Fig. 2: TLC of CH2Cl2 and MeOH extracts (Hexane/EtOAc 8:2)

23. Qualitative Analysis: HPTLC fingerprints of successive extracts of M. Longifolia:

24. HPTLC fingerprint profiles of different extracts of M. longifolia:

25. HPTLC fingerprint profiles of different extracts of M. longifolia:

26. Quantitative Analysis: • Biomarker compound/ Standard compound • Pure compound/Single compound • Large amount • Therapeutic activity

27. Quantification of pulegone in methanolic extractof M. longifolia HPTLC chromatogram of standard Pulegone

28. HPTLC chromatogram of standard Pulegone HPTLC chromatogram of methanolic extract of M. longofolia

29. U V Spectra of standard pulegone and different extract of M. longofolia

30. TLC plate of pulegone standard and different extract of M. longofolia at UV λmax = 254 TLC plate of pulegone standard and different extract of M. longofolia after sraying with anisaldehyde suphuric acid

31. Determination of the amounts of Caffeine in Coffee seed subjected to different treatments Experimental: Sample preparations: The samples were purchased from the local market at Al-Kharj city. The seeds were powdered and 5 gm from each sample were extracted separate by boiling with water for two minutes. The resulted decoctions were filtered and filtrates were transferred to 100 ml volumetric flask. Mixture of EtOH and H2O were used to complete the volume with final ration of 1:1 EtOH and H2O.

32. Standard Solution: Standard solution was prepared by dissolving 10 mg of caffeine in 100 ml of 1:1 EtOAc/H2O mixture. A volume of 1, 2, 3, 4, 5, 6, 7, 8 mL were applied on silica gel plates to obtain the calibration curve.

33. Chromatographic Conditions: The TLC system composed of EtOAc/MeOH 85:15 was used as mobile phase. It resulted in a symmetric nice resolved spots corresponding to caffeine at Rf value = 0.38. Chromatogram of standard Caffeine

34. Chromatogram of standard and samples of Caffeine extracted from different coffee samples.

35. Fig. UV absorption spectrum of caffeine.

36. TLC plates of standard and caffeine extracted from different coffee samples.

37. Applications of HPTLC: • Pharmaceutical research. • Biomedical Analysis. • Clinical Analysis.  • Environment Analysis. • Food industry. • Therapeutic drug monitoring to determine its concentration and metabolites in blood urine etc. • Analysis of environment pollution level. • Quantitative determination of prostaglandin s and thromboxanes in plasma. • Determination of mercury in water.