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1 : HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY (HPTLC) Presented By – Mr. Shaise Jacob Faculty, Nirmala College of Pharmacy Muvattupuzha, Kerala, INDIA Email –
2 : INTRODUCTION HPTLC is a sophisticated & automated form of TLC Efficient separation in short time
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4 : PRINCIPLE Adsorption Advantages of HPTLC Over Other Chromatographic Methods In HPTLC, simultaneous processing of sample and standard – better analytical accuracy & precision Lower analysis time & less cost per analysis HPTLC is very simple In HPTLC, the sample preparation is simple
5 : 5. Solvent used in HPTLC needs no prior treatment like filtration & degassing 6. In HPTLC, the M.P consumption for sample is extremely low 7. HPTLC allows the use of corrosive & UV absorbing M.P
6 : STEPS INVOLVED IN HPTLC 1.Sample preparation 2.Selection of chromatographic layer 3.Plates 4.Pre-washing 5.Conditioning 6.Sample application 7.Pre-conditioning 8.M.P
7 : 9.Chromatographic development 10.Detection of spots 11.Scanning & documentation
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9 : Sample preparation For normal phase chromatography using silica gel / alumina pre-coated plates, solvents – non polar RP chromatography , usually polar solvents Selection of Chromatographic layer » Depends on the nature of material to be separated Commonly used materials are Silica gel 60F, Alumina, Cellulose etc
10 : Plates Generally, plates of 20 x 20cm or 5 x 7.5cm size having 100-250mm adsorbent thickness. Silica gel 60F254 pore size 6mm with fluorescent indicator is a coat material Basic difference in TLC & HPTLC plates is particle size of coated materials which is 5-20µm – TLC, 4-8 µm - HPTLC
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13 : Pre-washing » to remove water vapors » volatile impurities Which might get trapped in the plates To avoid this, plates are cleaned by using methanol as solvent by ascending or descending etc.
14 : Conditioning Plates activated by placing them in an oven at 120°C for 15 to 20 minutes. Sample Application Application of 1.0 - 5µl for HPTLC Application carried out by Linomat applicator on the plates which give uniform, safe & std. results
15 : PRE-CONDITIONING (Chamber saturation) For effective separation of sample For RP chromatography – saturate the chamber with methanol or polar solvent MOBILE PHASE Selection of appropriate M.P is based on the trial and error.
16 : Chromatographic development Ascending, descending, horizontal, continuous, gradient, multidimensional… HPTLC – migration distance of 5-6mm is sufficient, after development, plates removed & dried. Common problems encountered during chro. Development are as follows… 1. Tailing: due to the presence of traces of impurities, this can be reduced by buffering the M.P
17 : 2.DIFFUSION: This is seen as zones on chromatographic plates. This may arise due to non-uniformity of M.P DETECTION OF SPOTS Detection can be done by iodine vapor in iodine chamber. Visual inspection at 254nm of UV region in UV cabinet
18 : Scanning & Documentation HPTLC plates are scanned at selected UV regions WL by the instrument & the detected spots are seen on computer in the form of peaks. The scanner converts band into peaks & peak height or area is related to the concentration of the substance on the spot.
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22 : APPLICATIONS HPTLC rapidly gaining importance in several fields of science like Pharmaceuticals analysis Biochemistry Pharmacokinetics studies
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