HPLC Principles,Operations, Maintenance and troubleshoting.pdf
0 likes151 views
This document discusses the principles and operation of high performance liquid chromatography (HPLC). It covers basic HPLC instrumentation configuration and separation modes, including reversed phase and normal phase systems. Methods for converting between normal and reversed phase systems are provided. Various HPLC detectors are outlined, including UV-Vis, PDA, and ELSD detectors. The document also discusses HPLC maintenance strategies and preventative maintenance for individual modules. Good laboratory practices for HPLC are reviewed, such as solvent preparation, filtration, and buffer preparation. Finally, common problems and their solutions are addressed.
HPLC Principles,Operations, Maintenance and troubleshoting.pdf
- 1. HPLC ( Basic Principles, Operations and Maintenance and Troubleshooting )
- 2. HPLC in recent time
- 5. Basic Configuration of HPLC
- 7. Reversed Phase HPLC System
- 8. Reversed Phase HPLC System
- 10. Factors!!!
- 12. Exercise!!
- 13. Answer!!!
- 14. Normal Phase HPLC System
- 15. Normal Phase HPLC System
- 16. Converting Normal Phase to Reverse Phase and Vice versa To convert a normal phase system/column to a reversed phase system/column, flush with a solvent that is miscible with both the current normal phase solvents and ideally Normal Phase : Hexane/Ethyl Acetate Flush : IPA then Methanol Finally : 50:50 Methanol/Water Reversed Phase : Buffered Aqueous Methanol To convert a reversed phase system/column to a normal phase system/column, follow a similar path to the one listed previously, but in reverse, for example, Reversed Phase : Buffered Aqueous Methanol Flush : 50:50 Methanol/Water Methanol then IPA Normal Phase : Hexane/Ethyl Acetate
- 17. HILIC System
- 19. UV-Vis Detector
- 21. PDA Detector
- 22. PDA Detector
- 23. Evaporating Light Scattering Detector (ELSD)
- 24. ELSD Mechanism
- 26. Maintenance
- 27. HPLC System Maintenance Strategy
- 30. Individual HPLC Module Preventive Maintenance
- 34. Possible problems from the Pumps
- 40. Replacement and cleaning of suction filter
- 41. Auto sampler common problems
- 42. UV-VIS and PDA detector common Problem
- 43. When to Change the Lamp
- 44. Good Laboratory Practice for HLPC 1. Preparation of Solvents : Correct solvent preparation is very important. It can save vast amounts of time spent troubleshooting spurious peaks, base-line noise etc. 2. Quality : All reagents and solvents should be of the highest quality. HPLC grade reagents may cost slightly more than lower grade reagents, but the difference in purity is marked. HPLC grade reagents contain no impurities to produce spurious peaks in a chromatogram baseline whereas AR grade reagents do contain trace levels of impurity, which may produce spurious baseline peaks. Important: Do not store HPLC grade water in plastic containers. Additives in the plastic may leach into the water and contaminate it. Always store HPLC grade water in glass containers
- 45. 3. Buffers : All buffers should be prepared freshly on the day required. This practice ensures that the buffer pH is unaffected by prolonged storage and that there is no microbial growth present. Changes in pH and microbial growth will affect chromatography. If buffer solutions are stored, be aware that they have a finite lifetime. Refer to pharmacopoeia monographs or similar for further guidance on buffer shelf life. Good Laboratory Practice for HLPC
- 48. 4. Filtration : Ideally, all HPLC solvents should be filtered through a 0.45 ÎĽm filter before use. This removes any particulate matter that may cause blockages. After filtration, the solvents should be stored in a covered reservoir to prevent contamination with dust etc. Filtering HPLC solvents will benefit both your chromatography and the wear and tear of the HPLC system. Pump plungers, seals and check valves will perform better and lifetimes will be maximized. Good Laboratory Practice for HLPC
- 49. Good Laboratory Practice for HLPC Wash Pump Injector Flow Cell Column
- 57. Please DO!!!
- 58. References • Maintaining and trouble shooting HPLC- David J. Lunser, copyright 1981 by Wiley and sons Inc. • Modern HPLC for practicing Scientist- Micheal W. Dong. • E.L Jhonson and R. Stevenson, Basic liquid Chromatography, Varian Associates, Palo Alto, California -1978. • L.R Snyder and J. J. Kirkland, Introduction to modern Liquid Chromatography, 2nd edition. • J. K Walker M.T. Jackson Jr., and J. B. Maynard-Chromatography System- Maintenance and Trouble Shooting, 2nd edition. • Trouble Shooting in HPLC –Agilent Technologies. • Theory and Principle of HPLC –Tranining Module Part – I-IV, 2013.
- 59. ThankYou