Hplc

powerpoint presentation on high performance liquid chromatography which include its definition, classification, principles of seperation, instrumentation and application.

1. H igh P erformance L iquid C hromatography 1

2. HPLC Prepared By- Miss. Chitralekha Sonawane Guided By- Dr. Sonali Mahaparale 2

3. Contents Introduction Advantages and Disadvantages Classification of HPLC Instrumentation Factors influencing Applications 3

4. Introduction Chromatography  is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. HPLC is really the automation of traditional liquid chromatography under conditions which provide for enhanced separations during shorter periods of time, utilizing very small particles, small column diameters, and very high fluid pressures. 4

5. Advantages Disadvantages Needs a small sample with a high accuracy and precise. Two interacting phases Room temperature Analysis Accurate results. Quick method  for analysis of sample. Need a skill to run the instruments Solvents consuming 5

6. Classification of HPLC Based on modes of chromatography Based on principle of separation Based on elution technique 6

7. Types of Modes Normal phase mode Stationary phase is polar, Ex. Silica, alumina Mobile phase is non-polar , Ex. Hexane ; dichloromethane; isopropanol ; methanol Reveres phase mode Stationary phase is non-polar , Ex. Silica gel with C18/C8 Mobile phase is polar Ex. water ; methanol; acetonitrile ; tetrahydrofuran (THF) 7

8. Principle of seperation Adsorption chromatography Ion exchange chromatography Ion pair Chromatography Affinity chromatography Molecular Exclusion Chromatography Partition Chromatography 8

9. Adsorption Chromatography It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibrations between the mobile and stationary phase accounts for the separation of different solutes. 9

10. Ion Exchange Chromatography   Resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. 10

11. Ion Pair Chromatography In this chromatography a reverse phase column is temporally converted into ion exchange column by using ion pairing agents like pentane, hexane, sulphonic acid etc. 11

12. Affinity Chromatography   It utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. For example, the immobilized molecule may be an antibody to some specific protein. 12

13. Molecular Exclusion Chromatography The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size. 13

14. Partition Chromatography This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid. 14

15. Elution Technique Isocratic elution Employs a single solvent or solvent mixture of constant composition. Gradient elution Here two or more solvent systems that differ significantly in polarity are employed. After elution is begun; the ratio of the solvents is varied in a programmed way, sometimes continuously and sometimes in a series of steps. 15

16. Instrumentation Solvent Reservoir Pumps Sample Injection System Columns Detector Waste 16

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18. Solvent Reservoir Glass reservoirs contain 500ml or more solvent. Degassers Vaccum pumping system A distillation system A device for heating and stirring A system for sparging Solvent treatment system Isocratic Gradient 18

19. Pumps General Requirements; Deliver high volumes (flow rates) of solvent (1to 10 mL /min) Deliver precise and accurate flow (<0.5% variation) Deliver high pressure (to 6000 psi) Inert toward solvents, pulse free flow, be reliable Types; Reciprocating pucmp Displacement pump Pneumatic pump 19

20. Reciprocating pump 20

21. Displacement Pump 21

22. Pneumatic Pump 22

23. Sample Injection System 23 LOAD INJECT

24. Columns Analytical Columns Length from 10 to 30 cm. Inside diameter is 4 to 10 mm. Particle size of packings is 5 or 10  m. Columns of this type contain 40,000 to 60,000 plates/meter. Types of columns packaging Porouse- Silica Gel , alumina, Ion exchange resine. Particular- Glass or polymer( Polymethacrylate ,  Polyvinyl alcohol) Guard Columns The composition is similar to that of the analytical column. Particle size is usually larger. 24

25. Detectors Bulk Property Detectors Refractive Index Detector Light Scattering Detectors Solute Property Detectors UV-VIS absorption Detector Fluorescence Detector Mass Spectroscopic Detector Electrochemical Detector 25

26. Refractive Index Detector Electrochemical Detector 26

27. Light Scattering Detector Fluorescence Detector 27

28. UV-VIS Absorption Detector Mass Spectroscopic Detector 28

29. Chromatogram 29

30. Factors Influencing Internal diameter of column Pump pressure Sample size The polarity sample, solvent and column Temperature 30

31. Applications Pharmaceuticals industry- Stability, Quantity of drug and testing of biological fluids. Analysis of natural contamination – Phenol and mercury from sea water. Forensic test – Steroid present in blood urine and sweat. Clinical test- Monitoring of hepatic chirosis patient Food and essence manufacture- Sweetener analysis in the fruit juice. 31

32. References https://www.shodex.com/en/kouza/b.html http://hplc.chem.shu.edu/NEW/HPLC_Book/Detect ors/det_ri.html https://en.wikipedia.org/wiki/High-performance_liquid_chromatography https://www.rpi.edu/dept/chem-eng/Biotech-Environ/IONEX/be_types.htm http://lab-training.com/landing/free-hplc-training-programme-9/ Skoog , Holler, Crounch “INSTRUMENTAL ANALYSIS”, India edition, Cengage learning, page no. 893-931. P. D. Sethi , “HPLC QUANTITATIVE ANALYSIS AND PHARMACEUTICAL FORMULATION, CBS Publication, page No.- 1-50. 32

33. Thank You 33