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1. HPTLC

2. INTR O D U CTION 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. PRINCIP L E 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 Pr e p a r a t i on Selection of chromatography layer Pre-washing Pre-conditioning Application of sample Chromatography development Detection of spots Scanning & documentation 4

5. Sample and Standard Preparation 5 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 6 Precoat e d plates di f ferent s u p p o r t m at e rials - 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

7. Pre coated plates 7 The plates with different support materials and sorbent layers with different format and thickness are used. 250μm are used for qualitative Plates with s orbent t hickn e ss o f 10 - and quantitative analysis.

8. Supports: Glass polyester sheets Aluminium sheets

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

10. Some of the binders used 10 🞄 G y psum (G) 🞄 Starch (S) 🞄 Layer co n t a ining fluores c ent indicator (F)

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 🞄 2 X 2 c m 🞄 1 X2 c m 🞄 5 X 10 cm 🞄 5 X 7.5 cm 🞄 Good cut edges of sheets is important to obtain constant Rf values . 12

13. Pre washing of pre coated plates 13 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.

14. Solvents used for pre-washing 14 1.Methanol 2.Chloroform: methanol ( 1:1 ) 3.C h olo r of or m: Meth a n o l: Amm o nia (9 :10:1 )

15. Activation of plates 15 F r eshly o p ened b o x of HPT L C p l ates d o esn ’ 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. 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 .

17. 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

18. 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.

19. 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.

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. Twin Trough Chambers 21

22. Post Chromatography Steps 22 1) Det e ctio n . 2) Photo documentation. 3) Densitometry measurements.

23. 2. C AMAG AUTO REAGENT SPRAYER

24. Dete c t i on 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

25. 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.

26. Densitometry measurements 9 / 2 9 / 2 01 4 29 Measures visible, UV absorbance or Fluorescence. Convert the spot/band into chromatogram consisting of peaks

27. Instrumentation of HPTLC consists of following: 🞄 La m p s e l e c t or 🞄 E n tr a nce l e ns s l it 🞄 Mo n oc h r om a t or entry s l it 🞄 Grat i ng 🞄 Mir r or 🞄 Slit aper t ure disc 🞄 Mir r or 🞄 Beam s plit t er 🞄 Ref e r e nce p h oto m ult i pli e r 🞄 Mea s uring p h oto m ultipli e r 🞄 Photo diode for transmission measurements.

28. IN S TRU M EN T A TION

29. 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

30. Photo-documentation With Digital Camera 38

31. 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.

32. v i deo scan so f tware f o r q u anti t ative e val u ation of images capture with digistore Documentation

33. Differences between TLC and HPTLC

34. 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 Ind u s t r i al Ap p l i c a t i o n s: Proce s s developm e nt and optimization, In-process Q.C. check, validation etc. Forensic : Poisoning investigations.

35. A) QUANTITATIVE DETERMINATION : Biochemical research/Biotechnology- Seperation of gangliosides Clinical- Inorganic & organic mercury in water & human serum. Caffeine in urine. Cosmetics- Hydrocortisone & cinchocaine in lanolin ointment E n viro n m ental A n a l ysi s - Pesticides in drinking water. Selenium in water. Food analysis- Vitamin C in fruit juices. Aflatoxins in food stuff

36. 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) FIN G ER PRI N T A N A L Y SI S - HPTLC finger print of Valerian. Finger print of garlic, Ashwaganda. Finger prints for identification of liquorice, ginseng.

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

38. 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