high performance thin layer chromatography

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 110 o -120 o c 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. Photo documentation. Densitometry measurements. 26

27. Detection UV CABINET 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

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 1/10/2014 29 Measures visible, UV absorbance or Fluorescence. Convert the spot/band into chromatogram consisting of peaks

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

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. 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 , R x publications pages no 14.10 to 14.12 www.pharmainfo.com www.camagusa.com www.infoexpo.com