High Performance Liquid Chromatography (HPLC)

Content include basic introduction to chromatography. Brief view of Liquid Chromatography. HPLC introduction, other names, types of HPLC, detailed instrumentation with image of each part, and applications. Sources of content described in 'References' entitled slide. This presentation was prepared for the partial fulfillment of Master of Pharmacy.

1. High Performance Liquid Chromatography (HPLC) Presented To Ms. Saumya Shukla By Sachin kumar Vishwakarma - M.Pharm ( Pharm.Chemistry )

2. CHROMATOGRAPHY Greek word= Chroma ( color ) + Graphein (to write) 1906, Mikhal Tswett (Russian Botanist ), separate plant pigments by passing through a calcium carbonate packed glass column. Chromatography is separation technique in which the solutes partition between a mobile phase and stationary phase . Mobile phase , the extracting phase that moves through the system . Stationary phase , the extracting phase remains in a fixed position . Chromatogram a plot of the detector’s signal as a function of time or volume of eluted mobile phase. 2

3. DEFINITION According to USP, ‘ Chromtography can be defined as a procedure by which solutes are separated by a differential migration process in a system consisting of two or more phases, one of which moves continuously in a given direction and in which the individual substances exhibit different mobilities by reason of differences in absorption, partition, solubility, vapour pressure, molecular size, or ionic charge density.’ 3

4. TYPES OF CHROMATOGRAPHY Sr.No . Mobile phase Stationary phase Type 1 Gas Liquid Gas-Liquid chromatography 2 Gas Solid Gas-Solid chromatography 3 Liquid Liquid Liquid-Liquid chromatography 4 Liquid Solid Liquid-Solid chromatography 4

5. LIQUID CHROMATOGRAPHY 1941, Martin and Synge , developed the theory of chromatographic separations, they were awarded the 1952 Nobel prize in chemistry for this work . Liquid chromatography (LC) is a separation technique based on the difference in the distribution of components between two non-miscible phases in which liquid mobile phase elutes through a stationary phase in a column. The three forms of high performance liquid chromatography most often used are based on mechanism of partition , adsorption and ion exchange. 5

6. TYPES OF LIQUID CHROMATOGRAPHY Liquid-solid chromatography (LSC ). Liquid-liquid (partition) chromatography (LLC ). Bonded-phase chromatography (BPC ). Gel permeation (exclusion) chromatography (GPC). 6

7. HPLC Developed by Huber in 1969. Principles ‘ Separation of mixtures in microgram to gram quantities by passage of the sample through a column containing a stationary solid by means of a pressurized flow of a liquid mobile phase; components migrate through the column at different rates due to different relative affinities for the stationary and mobile phases base on adsorption, size or charge .’ 7

8. HPLC (Key features) 1. High resolving power; 2. S peed of separation; 3 . Continuous monitoring of the column effluent; 4. A ccurate quantitative measurement; 5. Repetitive and reproducible analysis using the same column; and 6. Automation of the analytical procedure and data handling. 8

9. HPLC (Other names) High Performance Liquid Chromatography (HPLC) High Pressure Liquid Chromatography (HPLC) High Price Liquid Chromatography (HPLC) High Speed Liquid Chromatography ( HSLC ) High Efficiency Liquid Chromatography ( HELC ) 9

10. HPLC A PPRATUS HPLC apparatus includes following components :- pumping system, an injector, a chromatographic column with or without a column temperature controller, a detector and a data acquisition system (a computer, an integrator or a chart recorder ) NOTE - For ion exchange chromatography a suppressor column is installed between main column and detector. 10

11. HPLC INSTRUMENTATION 11

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13. HPLC Pumps D eliver metered amounts of the mobile phase from the solvent reservoirs to the column through high pressure. TYPES :- Reciprocating Piston pump Syringe type pump Constant pressure pump Pulse dampers Constant flow pump 13

14. Reciprocating Piston Pump Output pressure upto 10,000 psi Solvent is pumped back and forth by a motor driven piston Ready adaptability to gradient and constant flow rate Small internal volume 35-400 microlitre 14

15. HPLC Mixing Unit Mix the solvents of different proportions . TYPES :- Low pressure ( use He for degassing of solvents ) High pressure (no use of He ) Mixing of solvents is done by two methods :- Static mixer (packed with beads ) Dynamic mixer (use a magnetic stirrer and operates at high pressure ) 15

16. Gradient Controller Device that allows one to create a gradient program so as to alter the nature or polarity of solvents . It must form a homogenous mixture before it reaches to column . TYPES :- Isocratic separation (mobile phase is of same polarity throughout the process) Gradient elution (the polarity of solvents is gradually increased and hence the compositions of solvent to be changed ) 16

17. Degassing Devices Used to remove dissolved gases from solvents . TYPES :- Vacuum filtration :-which can remove the air bubbles. But is not always reliable & complete He purging :-by passing He through the solvent. Very effective but He is expensive Ultrasonication :-which converts ultra high frequency to mechanical vibrations – this causes the removal of air bubbles 17

18. HPLC Injectors Used to introduce the sample into the system . TYPES :- Septum injectors : injecting through a rubber septum. Not common – has to withstand high pressure. Stop flow (online): in which the flow of mobile phase is stopped for a while & the sample is injected through a valve device. Rheodyne injector ( loop valve type): A means for injecting samples in which the sample is loaded into a short section of tubing and injected onto the column by redirecting the mobile phase through the loop. 18

19. Rheodyne injector ( loop valve type ) This has a fixed volume loop like 20 µl or 50 µl or more .Injector has 2 modes load position: when sample is loaded in the loop 2 . Inject mode: when the sample is injected 19

20. HPLC Columns Columns works as stationary phase holder in HPLC (heart of HPLC ). TYPES :- Guard column :- An inexpensive column used to protect a more expensive analytical column. Analytical column :- The most imp part of the HPLC technology which decides the efficiency of separation. Column length: 5 cm to 30cm .Column diameter: 2mm to 50 mm Particle size: 1 µ to 20 µ 20

21. Column material : Stainless steel ( mostly used), glass, polyethylene and PEEK (poly ethylene ether ketone) (latest). Particle nature : spherical, uniform , porous materials are used Guard and standard column for HPLC 21

22. HPLC Detectors A detector consists of a flow through cell mounted at the end of the column and capable of detecting various types of components in the eluate . Type 1 or Bulk property detector Refractive-index detectors Conductivity detectors TYPES :- Type 2 or Solute property detector Photometric/UV-detectors Fluorescence Detectors Electrochemical detectors 22

23. Bulk property detector Measure the difference in some physical property of the solute in the mobile phase compared to the mobile phase alone. Refractive index detector 23

24. Solute property detector R espond to a particular physical or chemical property of the solute, being ideally independent of the mobile phase . Photometric detectors :- Operate in ultraviolet region of spectrum. 24

25. Photometric detector TYPES :- Single wavelength D :-low pressure mercury discharge lamp; 254nm Multi-wavelength D :-combination with interference filter; allow number of wavelength to selected, e.g. 206,226,280,313,340 or 365 nm Variable wavelength D :-deuterium light source; grating monochromator ; any wavelength in deuterium continuum (190-360nm) Programmable D :-allow the automatic change of wavelength Diode array D :-microprocessor controlled photodiode array; light passes through the floe cell into a polychromator 25

26. HPLC Recorders and Integrators Recorders: are used to record the responses obtained from detectors after amplification. They record the baseline & all the peaks obtained with respect to time ( Rt ). But the area of the individual peaks cannot be known. Integrators : improved version of recorders with data processing capabilities Rt , height and width of peaks, peak area, % area . Integrators provide more information on peaks than recorders. 26

27. HPLC WORKING 27

28. HPLC METHODS Sr.No . Method Column Mobile Phase Remarks 1 Reverse Phase (RP HPLC) C-8, C-18, phenyl , cyano Mixtures of aqueous and organic phase Suitable for neutral or non-ionized compounds soluble in water/methanol/acetonitrile mixture 2 Normal Phase (n HPLC ) Cyano , diol, amino, silica Mixture of organic solvents Lipophilic compounds , insoluble in water 3 Ion pair HPLC C-8, C-18, cyano Like reverse phase, except addition of buffers to control pH Suitable for ionic or ionisable compounds 28

29. HPLC APPLICATIONS Quantitative analysis ( corresponding peak area or the peak height) Qualitative analysis Drug formulation and stability Multicomponent analysis Purity determination Isolation of natural pharmacologically active compounds Assay of Drugs ( Cephalosporins , Steroids and Frusemide etc.) Hyphenated Techniques (HPLC-MS ) Residual pesticide identification 29

30. REFERENCES “Indian Pharmacopoeia”; Ministry of health and Family Welfare, Government of India; Vol I, 2010 . Sethi , P. D .; HPLC Jeffery, G.H. ; Bassett ,J. ; Mendhan ,J. ; “Vogel`s Textbook od Quantitative Chemical Analysis”; 5 th Edition; John Wiley and sons, New York. Harvey, D. ; “Modern Analytical Chemistry”; McGraw Hill; New York. Mullertz , A. ; Perrie , Y. ; “Analytical Techniques in the Pharmaceutical Sciences”; Springer Nature; New York . Rouessac , F.; Rouessac , A.; “Chemical Analysis”; 2 nd Edition; John Wiley and sons, New York . Naidu, M.P .( Ph.D Research scholar); HPLC . 30

31. THANK YOU. 31