High performance liquid chromatography (HPLC)

1. Highperformance liquid chromatography(HPLC) Dr. J. Rajesh Asso. Prof Dept of Chemistry MSEC, Kilakarai

2. A form of column identify, chromatography to and quantify the separate, • compounds. • Developed in1970s. • The most widely separationtechnique. used analytical

3. Chromatography • Chromatography is a technique which separates components in a mixture dueto the differing component time taken for each to travel through a stationary phase by a mobilephase. when carried throughit

4. Basically, all chromatographic systems consists of twophases. • Mobile phase - liquid or gaseous and flows over or through the stationaryphase • Stationary phase - solid, liquid or a solid/liquid mixture which is immobilized

5. Some chromatographyterms • Analyte • Substance that is to be separated during chromatography • Immobilizedphase • Stationary phase which is immobilized on the support particles or on the inner wall of the column tubing

6. Some chromatographyterms • Mobile phase • Phase which moves in a definite direction. (liquid/gas/fluid). • Consists of the sample being separated/ analyzed and the solvent that moves the sample through thecolumn. • Effluent • Mobile phase leaving the column.

7. Different types of chromatographymethods • Paperchromatography • Liquidchromatography • Gaschromatography • High performance liquidchromatography

8. High performance liquidchromatography • HPLC is an extension of conventional liquidchromatography. • Powerful tool in analyticaltechniques • Columns are tightly packed, and the eluent is forced through the column under high pressure(up to 5,000 psi) by apump.

9. Allows touseaverysmallerparticlesize • for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing throughit. • Allows a much better separation of the components of themixture.

10. HPLCTechnique • Utilizes liquid mobile phase to separate the mixture • Analytes are first dissolved in a solvent then through the column under high pressure of up to 400atm • Mixture is resolved into its components in the column

11. Thetotalseparationtimeisoften5or10 • minutes rather than hours or even days required for some separations by gravity flow with the largersystems.

12. Components ofHPLC • Pump • Injector • Column • Detector • Recorder or datasystem

13. A Flow Scheme forHPLC

15. Pump • A pump forces the mobile phase through the column at a much greater velocity than gravity-flowcolumns. • The pump can be pneumatic, syringe- type, reciprocating, or hydraulicamplifier.

16. Pump(cont.) • Pneumatic pumps preoperativepurposes. are used for • The most widely used pump today is the multihead pump with two or more reciprocatingpistons.

17. Pumps are designed in order to maintain a stable flow rate, avoiding pulsations even when the composition of the mobile phase varies • flow range – 0.01-10ml/min

18. HPLCPump

19. Injectors • Inject the liquid sample within range of 0.1- 100 ml of volume under highpressure • Produce minimum bandbroadening • Produce possible flowdisturbances • Volume mustbesmall (0.1-500 uL)

20. A modelinjector

21. Injector

22. Columns • Smooth-bore stainless walled glasstubing. steel or heavy- • Hundreds of packed columns differing in size and packing are available from manufacture.

23. Columns • E.g. Column packing vary in size (3 to 20 um) with the smaller particles used mostly for analytical separations and the larger ones for preparativeseparation. • The most common material used for column packing is silicagel.

24. HPLCcolumns

26. Detector • HPLC detectors monitor the elute as it leaves the column • Produce an electronic signal proportional to the concentration of each separated component

27. Detector • Crucial in traceanalysis • High sensitivity • Fastresponse • Simplifiesquantitation • Insensitive to changes in type of solvent, flow rate andtemp.

28. The most widely used detectionmethods • Spectrophotometers • Fluorometers • Electrochemicaldetectors • Massspectrometer • Refractive indexdetector

29. Detectors used inHPLC

30. Detectors used inHPLC

31. Depending on the relative polarity of the solvent and stationary phase, there are two variants in use in HPLC • 1.Normal phaseHPLC • Utilize polar adsorbent surface and non- polareluent • Polar substance in the mixture sticks to polar adsorbent thannon-polar • Non-polar ones will pass more quickly through thecolumn

32. 2. Reversed phaseHPLC • Utilize non-polar adsorbent polareluent surface and • Attraction between non-polar compound in the mixture and non-polaradsorbent

33. 2. Reversed phase HPLC(cont.) • Polar molecules will travel through the column more quickly because there is strong attraction between polar solvent and polar molecules when pass through the column • Reversed phase HPLC is the most commonly used form ofHPLC

34. Solvents used in mobilephase • hexane, heptane, cyclohexane,carbon tetrachloride, benzene, toluene, diethyl ether, chloroformetc. • Adsorbents used in stationary phase • silica gel, alumina, celite, cellulose powder, ion-exchange, cellulose,starch

35. Retentiontime • The time taken for a particular compound to travel through the column to the detector • From the time at which the sample is injected to the point at which the display shows a maximum peak height for that compound.

37. Types of chromaticseparation • AdsorptionChromatography • Ion- exchangeChromatography • Size ExclusionChromatography

38. AdsorptionChromatography • Competition for adsorption sites occurs between the molecules of the mixture to be separated and the molecules of the mobilephase • Mobile phase can be either a single solvent or two or more solvents depend on the analytes to bedesorbed • Speed of migration of the component along the column depend on adsorptive affinity

39. Ion- exchangeChromatography • Molecules can be separated by their ionic charges in a process known as Ion- exchangeChromatography. • Ion-exchange resins are used asthe column packingmaterials. • This method is used for separation of ionic species, such as aminoacids.

41. Size ExclusionChromatography • Known as gel permeation chromatography or gel filtrationchromatography. • Packing material with very small pore is used. • Precisely controlled pore size materials in the column • Large molecules, such as polymers are physically prevented from passing through the column

42. Applications HPLC is usedfor • Chemistry and biochemistry research • analyzing complexmixtures, • Purifying chemicalcompounds • Quality control to ensure the purity of raw materials • Analyzing air and water pollutants,

43. Applications(cont.) • Monitoring materials that may jeopardize occupational safety orhealth • Monitoring pesticide levels in the environment. • To survey food and drugproducts, • To identify confiscatednarcotics • To determine the amount of such chemical compounds found in new drugs in pharmaceutics

44. HPLC as compared with the classical technique • Small diameter, reusable stainless steel columns • Column packing with very small particles • Control flow of mobilephase • Precise sample introduction

45. HPLC as compared technique(cont.) • Good pumpingsystem • Special continuous flow detectors- can handle small flow rates and detect very smallamounts • Rapidanalysis • High resolution with the classical

46. Disadvantages ofHPLC • Cost • Complexity • Low sensitivity for somecompounds • Irreversibly adsorbed compounds not detected • Co-elution difficult to detect

47. Summary • The modern day technique is greatly • enhanced in terms of selectivity, resolution, through miniaturization and the use of very elaborate stationaryphases. • Therefore HPLC is widely used for separation of molecules in biological, pharmaceutical, food, environmental and industrialprocess

48. References: • Harold Varley practical clinicalchemistry • http://scimedia.com • http://www.forumsci.co.il/HPLC

49. THANKYOU!