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High Performance Thin Layer Chromatography (HPTLC) instrumentation
- 1. -MADHURA NEWREKAR M. Pharm (QA) -INSTRUMENTATION 1
- 2. INTRODUCTION • It is the major advancement of TLC principle. • The technique is more modernized. • Advantages over TLC. • Short time duration. • Better resolution. • More sensitivity. • Ease of quantification. 2
- 3. INSTRUMENTATION • Stepwise procedure include: 1.Preparation of plates 2.Sample application 3.Chromatogram development 4.Derivatization 5.Chromatogram evaluation 6.Scanning and documentation 3
- 4. PLATES • Pre-coated plates: • Wider choice for stationary phase.eg : Silica , alumina , cellulose , C18 , C8. • Particle size is 2-7 μm. • Pore size is smaller and more uniform. • Thickness is 0.2 mm. • If the plates are stored for longer duration of time, they need to be activated before use. 4
- 5. MARKETED EXAMPLES OF PRECOATED PLATES 5
- 6. SAMPLE APPLICATION • Samples are carefully taken with special syringe (Hamilton syringe). • The volume ranges available are 0.5μl ,1μl , 2μl , 5μl. • The syringe is filled carefully to avoid the presence of air bubbles as it will affect the volume of sample. 6
- 7. • It is connected to a nitrogen gas chamber . • The pressure to be used is 60-90 psi (4-6 bar). 7
- 8. • Plate size will vary according to sample number. • Samples are dried in the dessicator. 8
- 9. CHROMATOGRAM DEVELOPMENT • Chamber saturation is required for effective separation. • Before the development of chromatogram the chamber is saturated with vapors of mobile phase. • If the chamber saturation is not done the mobile phase rising through plate will get evaporated and the separation will not be effective. 9
- 10. • Flat bottom chamber: • Twin trough chamber: 1.Low solvent consumption 2.Reproducible preconditioning of the layer 10
- 11. • Horizontal developing chamber: • The HPTLC plate is developed from both opposing sides towards the middle, provided the separation distance of 45 mm. 11
- 12. DERIVATIZATION AND CHROMATOGRAM EVALUATION • The developed plates are observed in the UV cabinet using the wavelengths 254 nm or 366 nm ,for identifying the compounds. 12
- 13. • If the separated compounds are fluorescent in nature , they can be detected using UV cabinet, If the separated compound are not fluorescent , then the fluorescent material is incorporated into stationary phase. • Post chromatogram derivatization e.g. concentrated sulfuric acid. Many organic compound get charred due to sulfuric acid and produce black or brown spots. • Other reagents used : Iodine,Ninhydrin reagent. 13
- 14. DOCUMENTATION SYSTEM • Digistore 2 ( CAMAG ): • It has a digital camera with high resolution which is connected to winCATS software. • It has an illumination unit (Reprostar 3) which will provide the short , long wavelengths and white light. 14
- 15. SCANNER • For quantitative estimation of different compounds at their λmax can be carried out ,since the scanner provides the measurement at wavelengths in the range of 200-400nm. 15
- 16. • In the scanner , each sample band is brought in the path of a required wavelength. • The peaks are recorded and compared with standard. • Thus scanner makes in situ and accurate quantification by HPTLC. • Advantage : There is no need of removal of the separated components unlike TLC which pose the chances for errors. 16
- 17. • A scanning densitometer has the following components: • Light source: hydrogen lamp ,mercury vapor lamp , xenon lamp . • Collimating lens: It makes the beam parallel before entering it into monochromator. • Monochromator: Either a single or double monochromators are used according to the type of scanning densitometer. 17
- 18. • A converging lens: A beam is converged to focus on the separated compounds, When it gets reflected back it falls on the detector. • Detector : An effective detector such as photomultiplier tube is employed for measurement of reflected beam. A signal produced by the detector indicates the concentration of compound present in that band. 18
- 19. • A stepping motor: In order to bring each spot in the focus of a converged beam of radiation it is used in densitometers. It moves a plate under the scanned light beam in order to scan the plate. 19
- 20. CONCLUSION • The automation at different steps provide more accuracy , better resolution . Lower limit of detection in HPTLC. • One recent approach to automation has been the use of piezoelectric devices and inkjet printers for applying sample. • Being more advanced , the usage of HPTLC is well appreciated and accepted all over the world. 20
- 21. REFERENCES • Dr Supriya S Mahajan;Instrumental methods of analysis;2010;Page no: 270-274. • CAMAG; Basic tools for thin layer chromatography ;Edition 2017;Page no:10-17. • Skoog , Holller , Nieman; Instrumental Analysis; 7th edition ;2009;Page no:217-225. • Dr P D Sethi; HPTLC;1st edition ;1996,Page no:25,30-48. 21
- 22. THANK YOU 22
Editor's Notes
- #12: In case a longer separationDistance is desired, the Horizontal Developing Chamber can be used For development from one side. In the Horizontal Developing Chamber, a plate can be developed in the sandwich configuration as well as in the tank configuration. This permits the number of samples to be doubled as compared with development in A tank,