HPLC

7,883 views
7,523 views

Published on

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY, ppt

Published in: Education, Technology
2 Comments
15 Likes
Statistics
Notes
No Downloads
Views
Total views
7,883
On SlideShare
0
From Embeds
0
Number of Embeds
17
Actions
Shares
0
Downloads
666
Comments
2
Likes
15
Embeds 0
No embeds

No notes for slide

HPLC

  1. 1. HIGH PERFORMANCE(PRESSURE) LIQUID CHROMATOGRAPHY
  2. 2. HPLC <ul><li>Presented By - </li></ul><ul><li>Mr. Shaise Jacob </li></ul><ul><li>Faculty </li></ul><ul><li>Nirmala College of Pharmacy </li></ul><ul><li>Muvattupuzha, Kerala </li></ul><ul><li>India </li></ul><ul><li>Email – jacobshaise@gmail.com </li></ul>
  3. 3. <ul><li>Liquid chromatography is a separation technique that involves: </li></ul><ul><li>the placement (injection) of a small volume of liquid sample into a tube packed with porous particles (stationary phase) </li></ul><ul><li>where individual components of the sample are transported along the packed tube (column) by a liquid moved by gravity. </li></ul>
  4. 4. <ul><li>The components of the sample are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. </li></ul><ul><li>The separated components are collected at the exit of this column and identified by an external measurement technique, such as a spectrophotometer that measures the intensity of the color, or by another device that can measure their amount </li></ul><ul><li> Note:The modern form of liquid chromatography is now referred to as “flash chromatography” </li></ul>
  5. 5. Four types of high performance liquid chromatography (HPLC): <ul><li>partition </li></ul><ul><li>adsorption (liquid-solid) </li></ul><ul><li>ion exchange </li></ul><ul><li>size exclusion or gel </li></ul>
  6. 6. ◊ improved performance <ul><li>◊ high pressure </li></ul><ul><li>HPLC- Separation is accomplished by partitioning b/w a M.P & Stationary column material. </li></ul><ul><li>Packing material </li></ul><ul><li>small, uniform particle </li></ul><ul><li>gives high column efficiencies </li></ul><ul><li>High pressure </li></ul><ul><li>to achieved desired flow rates </li></ul>
  7. 7. Types of HPLC techniques <ul><li>Based on Modes of chromatography </li></ul><ul><li>1. Normal phase mode: </li></ul><ul><li>S.P is polar </li></ul><ul><li>M.P is non polar </li></ul><ul><li>2. Reverse phase mode: </li></ul><ul><li>S.P is non polar </li></ul><ul><li>M.P is polar </li></ul><ul><li>Different columns used: ODS,C18,C8,C4… </li></ul>
  8. 8. Based on principle of separation <ul><li>1. Adsorption chromatography </li></ul><ul><li>2. Ion exchange “ </li></ul><ul><li>3. Ion pair “ </li></ul><ul><li>4. Gel permeation / Size exclusion “ </li></ul><ul><li>5. Affinity “ </li></ul><ul><li>6. Chiral phase “ </li></ul><ul><li>Based on elution technique </li></ul><ul><li>Isocratic separation </li></ul><ul><li>Gradient separation </li></ul>
  9. 9. Based on scale of operation <ul><li>Analytical HPLC </li></ul><ul><li>Preparative HPLC </li></ul><ul><li>Based on the type of Analysis </li></ul><ul><li>Qualitative analysis </li></ul><ul><li>Quantitative analysis </li></ul><ul><li>HPLC offers numerous advantages </li></ul><ul><li>♠ Capable of handling “macromolecules” </li></ul><ul><li>♠ Suitable for pharmaceutical compounds </li></ul><ul><li>♠ Efficient analysis of liable natural products </li></ul><ul><li>♠ Reliable handling of inorganic & ionic species </li></ul>
  10. 10. ♠ Dependable analysis of biochemical's <ul><li>PRINCIPLE </li></ul><ul><li>Adsorption </li></ul><ul><li>Particle size of the S.P material plays a crucial role in HPLC </li></ul><ul><li>Micro particulate column packing : </li></ul><ul><li>Silica particles -> uniform, porous, with spherical or irregular shape </li></ul><ul><li>Diameter -> 3.5 to 10 µm </li></ul>
  11. 11. HPLC instrumentation comprises: <ul><li>M.P reservoirs </li></ul><ul><li>Eluent degas module </li></ul><ul><li>Solvent delivery pumps </li></ul><ul><li>Manual / Auto injector </li></ul><ul><li>Analytical column </li></ul><ul><li>Detector </li></ul><ul><li>Data processor </li></ul>
  12. 16. Mobile phase reservoir <ul><li>stores M.P (HPLC grade solvents) </li></ul><ul><li>♠ Resolution & Speed of analysis } </li></ul><ul><li>Flow rate, polarity & pH of M.P </li></ul><ul><li>Can't use metallic reservoir </li></ul><ul><li>Eluent degas module </li></ul><ul><li>Dissolved gases in M.P pose a number of problems </li></ul><ul><li>∆ flow ∆ excessive detector noise </li></ul><ul><li>∆ Rt fluctuation </li></ul><ul><li>♠ Bubbling the pump & detector, </li></ul>
  13. 17. <ul><li>Degas module with reservoir of inert gases </li></ul><ul><li>He or N 2 </li></ul><ul><li>Vacuum filtration </li></ul><ul><li>Helium purging </li></ul><ul><li>Ultrasonication (converts ultra high frequency to mechanical vibrations.) </li></ul><ul><li>SOLVENT DELIVERY PUMPS </li></ul><ul><li>Reciprocating pumps: </li></ul><ul><li>» widely used </li></ul><ul><li>» maintain accurate flow rate </li></ul>
  14. 20. Cross-sectional diagram of a simple single – piston reciprocating pump
  15. 21. Solvent delivery systems <ul><li>two types: 1. Isocratic system </li></ul><ul><li>2. Low pressure gradient </li></ul><ul><li>3. High pressure gradient </li></ul><ul><li>Injection system </li></ul><ul><li>a. Syringe system:- results best </li></ul><ul><li>b. Injection valve :- [Rheodyne injector] </li></ul><ul><li>» Loading through the sample loop (20-50µl) </li></ul>
  16. 22. u <ul><li>c. Automated injection device :- </li></ul><ul><li>commercially available, automatically inject 100samples </li></ul>
  17. 23. Guard column <ul><li>Pre-filter :- useful for industry </li></ul>
  18. 24. Analytical column <ul><li>Heart of any chromatographic system </li></ul><ul><li>» Actual separation of components takes place </li></ul><ul><li>Several S.P available </li></ul><ul><li>depending upon tech. or mode of separation </li></ul><ul><li>Column material </li></ul><ul><li>S.S, glass, polyethylene, PEEK </li></ul><ul><li>Column length Column diameter Particle size </li></ul><ul><li>5-30 cm 2mm-50mm 1µ-20µ </li></ul>
  19. 25. Particle nature: <ul><li>Spherical, uniform sized porous material </li></ul><ul><li>Surface area </li></ul><ul><li>1g S.P provides surface area 100-860 sq.m </li></ul><ul><li>Functional group </li></ul><ul><li>Depends on the type of chromatographic separations. </li></ul><ul><li>Normal phase mode: hydroxy group </li></ul><ul><li>Reverse phase mode: C 18 (octa decyl silane) </li></ul><ul><li>Bondapak ( waters) </li></ul><ul><li>C 8 octyl column, C 4 butyl column, CN Nitrile column </li></ul><ul><li>NH 2 Amino column </li></ul>
  20. 27. b
  21. 28. Column packing <ul><li>three forms </li></ul><ul><li>1.Microporous support </li></ul><ul><li>5-10 µm in d.m </li></ul><ul><li>2. Pellicular </li></ul><ul><li>Porous & 40 µm in d.m </li></ul><ul><li>3.Bonded phase </li></ul><ul><li>S.P bonded onto an inert support </li></ul>
  22. 29. DETECTORS <ul><li>1. UV DETECTOR : Based on UV light ab. </li></ul><ul><li>> fixed WL detector (254nm) </li></ul><ul><li>> variable WL detector (190-600nm) </li></ul><ul><li>2. REFRACTIVE INDEX DETECTOR : </li></ul><ul><li>Non specific / Universal detector </li></ul><ul><li>↓ sensitivity & specificity </li></ul><ul><li>3. PHOTODIODE ARRAY DETECTOR (PDA) </li></ul><ul><li>similar to UV detector, non destructive </li></ul><ul><li>190-600 nm for quantization & identification </li></ul><ul><li>Spectra is 3D, Response vs time vs WL </li></ul>
  23. 30. Photodiode Array Detector
  24. 31. Flourimetric detector <ul><li>Excitation & emission WL can be selected </li></ul><ul><li>↑ sensitive than UV </li></ul><ul><li>Disadvantage: Some comp. are not fluorescent </li></ul><ul><li>Conductivity detector </li></ul><ul><li>based on electrical conductivity </li></ul><ul><li>Amperometric detector </li></ul><ul><li>Reduction / oxidation </li></ul>
  25. 32. RECORDERS & INTEGRATORS <ul><li>Recorders – to record the responses </li></ul><ul><li>Integrators - data processing capabilities </li></ul><ul><li>◊ record individual peaks with Rt, height, width </li></ul><ul><li>of peaks, peak area, % of area.. </li></ul><ul><li>Selection of solvent systems </li></ul><ul><li>Solvent compatibility with the detector </li></ul><ul><li>e.g.. Hexane, chloroform, ACN , Methanol… </li></ul><ul><li>Most widely used solvent in HPLC is water </li></ul><ul><li>Millipore Milli-Q apparatus produce water </li></ul>
  26. 33. Selection of Column <ul><li>Non polar & moderately polar comp. -> </li></ul><ul><li>ADSORPTION CHRO. </li></ul><ul><li>Highly polar molecules by -> R.P Chro. </li></ul><ul><li>Acids & Bases by -> Ion exchange Chro. </li></ul>
  27. 34. APPICATIONS OF HPLC <ul><li>♥ Pharmaceutical field </li></ul><ul><li>♥ Chemical & Petrochemical industry </li></ul><ul><li>♥ Forensic </li></ul><ul><li>♥ Biochemical separations </li></ul><ul><li>♥ Food analysis </li></ul><ul><li>Qualitative analysis </li></ul><ul><li>Checking the purity of a compound </li></ul>
  28. 35. Quantitative Analysis <ul><li>Direct comparison method </li></ul><ul><li>injecting the sample & std. separately & comparing their peak areas. </li></ul><ul><li>Area of the peak = peak height x width of peak at half height </li></ul><ul><li>Calibration curve method </li></ul><ul><li>Multi component analysis </li></ul><ul><li>Isolation & identification of drugs </li></ul><ul><li>Stability studies </li></ul>

×