2. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

3. Idea of Chromatgraphyyy The mixture of substances is dissolved in a liquid or a gas and flowed through a tube (the column) containing a material to which the components of the mixture will be attracted to different degrees The attraction may be hydrogen bonding or van der Waals forces In some cases the attraction may be a chemical reaction In biochemistry, the difference often is that some components of the mixture will fit into pores in the column material whereas others will not; this is size- exclusion chromatography.

4. Thus … Chromatography basically involves the separation of mixtures due to differences in the distribution coefficient (equilibrium distribution) of sample components between two different phases. One of these phases is a mobile phase and the other is a stationary phase.

5. Developments in the field of chromatography YearScientist(s)Comments 1834Runge,F.F.Used unglazed paper and/or pieces of cloth for spot testing dye mixtures and plant extracts 1850Runge,F.F.Separated salt solutions on paper 1868Goppelsroeder. FIntroduced paper strip (capillary analysis) analysis of dyes, hydrocarbons, milk, beer, colloids, drinking and mineral waters, plant and animal pigments 1878Schonbein, C.Developed paper strip analysis of liquid solutions 1897 -1903Day,D.T.Developed ascending flow of crude petroleum samples through column packed with finely pulverized fuller ’ s earth. 1906-1907Tswett, M.Separated chloroplast pigment on CaCO 3 1931Kuhn,R. et al.Introduced liquid-solid chromatography for separating egg yolk xanthophylls 1940Tiselius, A.Earned Nobel prize in 1948; developed adsorption analyses and electrophoresis 1940Wilson, J.N.Wrote first theoretical paper on chromatography: assumed complete equilibrium and linear sorption isotherms; qualitatively defined diffusion, rate of adsorption, and isotherm nonlinearity 5

6. 1941Tiselius, A.Developed liquid chromatography and pointed out frontal analysis, elution analysis, and displacement development 1941Martin, A.J.P. and Synge, R. L. M. Presented first model that could describe column efficiency; developed liquid-liquid chromatography; received nobel prize in 1952 1944Consden, R., Gordon, A.H., and Martin, A..J.P Developed paper chromatography 1946Claesson,S.Developed liquid-solid chromatography with frontal and displacement development analysis; coworker A. Tiselius 1949Martin A.J.P.Contributed to relationship between retention and thermodynamic equilibrium constant 1951Cremer, E.Introduced gas-solid chromatography 1952Phillips, C.S.G.Developed liquid-liquid chromatography by frontal technique 1955Glueckauf, E.Derived first comprehensive equation for the relationship between HETP and particle size, particle diffusion, and film diffusion ion exchange 1956Van Deemter, J.J.Developed rate theoryby simplifying work of Lapidus and Ammundson to Gaussian distribution function 1957Golay, M.Reported the development of open tubular columns 1965Giddings, J.C.Reviewed and extended early theories of chromatography 6

7. MOBILE PHASE STATIONARY PHASE PRINCIPLE OF SEPARATION CONTAINERNAME OF THE CHROMATOGRAPHIC TECHNIQUE LIQUIDSOLIDDIFFERENCES IN ADSORPTION COLUMNLIQUID-SOLID ADSORPTION COLUMN CHROMATOGRAPHY LIQUIDSOLIDDIFFERENCES IN ADSORPTION THIN LAYERLIQUID-SOLID ADSORPTION THIN LAYER CHROMATOGRAPHY LIQUID DIFFERENCES IN PARTITIONING COLUMNLIQUID-LIQUID PARTITION COLUMN CHROMATOGRAPHY LIQUID DIFFERENCES IN PARTITIONING THIN LAYERLIQUID-LIQUID PARTITION THIN LAYER CHROMATOGRAPHY GASLIQUIDDIFFERENCES IN PARTITIONING COLUMNGAS-LIQUID-PARTITION COLUMN CHROMATIGRAPHY GASSOLIDDIFFERENCES IN ADSORPTION COLUMNGAS-SOLID ADSORPTION COLUMN CHROMATOGRAPHY 7

8. 8 GAS LIQUID/ SOLUTION CLASSIFICATION OF DIFFERENT CHROMATOGRAPHIC TECHNIQUES Stationary phase is solid Stationary phase is a liquid coated on the surface of an inert solid support gas - solid - adsorption chromatography gas - liquid - partition chromatography Mobile phase is Gas and all the solutes being separated are in gaseous phase under experimental conditions Mobile phase is liquid and all the solutes being separated are in liquid/solution state

9. 9 Liquid / Solution Chromatography ADSORPTION : Separation of solutes on the basis of their degree of adsorption on the surface of an adsorbent. PARTITION : Separation of solutes on the basis of their differences in partition coefficient of solutes in two mutually immiscible solvents. ION EXCHANGE : Separation of similarly charged ions based on the differences in the degree of their affinity towards the ion exchange resin. SIZE EXCLUSION : Separation of solutes based on the differences in their molecular weights or sizes. ELECTROPHORETIC : Separation based on the movement of charged solutes to opposite poles under the applied electrical field. AFFINITY : Separation of solutes based on specific affinity of solutes on the specific sites of the stationary phase.

10. Types of Chromatography 1.Liquid Column Chromatography 2.Gas Liquid / Solid Chromatography 3.Thin-layer Chromatography

11. Michael Tswett discovered liquid chromatography in 1906 and till late 1950,liquid chromatography lacked the attributes of gas chromatography and was being done on columns,papers and thin layer plates. 11

12. Modern Liquid Chromatography got a place in the early1960’s by a combination of the experiences gained with Gas Chromatography and Ordinary Column Chromatography. 12

13. Researchers working with liquid chromatography vigorously started making attempts to achieve all the attributes of gas chromatography to liquid chromatography. 13

14. IDEAL REQUIREMENTS OF HPLC Fast analysis : in minutes Sensitive :- easily detecting solutes at ppm or ppb levels Efficient :- providing high resolution Non destructive: Highly accurate :- qualitative and quantitative analysis Requires small samples :- micro litres Reliable and relatively simple Versatile:-can separate gases,liquids and solids ( up to Molecular weight 450 )

15. 1.Short time for analysis 2.Efficient 3.Sensitive 4.Non-destructive. 5.Accurate. 6.Small sample size. 6.Reliable 7.Relatively simple. 8.Relatively inexpensive. 9.Versatile. 15

16. Sequence of efforts taken place during the development of modern liquid chromatography To reduce separation time to achieve fast analysis. ( From hours to minutes) HOW? The solutes must move faster through stationary phase MEANS By increasing the mobility of the liquid mobile phase.

17. The MIGRATION VELOCITY of the liquid mobile phase should be HIGH. We can achieve faster migration velocities of liquid mobile phase by- 1. Applying vacuum at the other end of the chromatographic column 2.Applying high pressure on the liquid mobile phase. Application of higher pressure on liquid mobile phase seems to be an easier and more practical way for achieving fast analysis.

18. CHROMATOGRAPHIC COLUMN Glass is normally the choice for chromatographic column material due to its chemical inertness towards stationary phase, mobile phase and solutes being separated. We could not increase the pressure of a glass column? Hence glass will not be able to withstand high pressures of liquid mobile phase because glass has relatively small mechanical strength.

19. Which IDEAL MATERIAL should be selected in place of glass? STAINLESS STEEL due to its chemical inertness & mechanical strength.

20. 20 Previous condition :- Liquid mobile phase moving across the stationary phase in a glass column under atmospheric pressure. Present condition :- Liquid mobile phase with high migration velocity moving across the stationary phase in a stainless steel column under high pressure.. Will the column separation efficiency of the latter be good? NO

21. With the increase in the liquid mobile phase velocity under high pressures, solute molecules will not get enough time to interact with the stationary phase and separation efficiency will be reduced and we will not be able to achieve good separation of solutes. 21

22. How can we achieve good column separation efficiency? 22 1.By reducing the Particle size of the stationary phase support. 2. By reducing the width of chromatographic column.

23. We use Narrow bore (3mm, id) SS columns filled with small particles of stationary phase support (5µ) will give us high separation efficiency with low analytical time when liquid mobile phase moves through the column under high pressures of the order of 500 –5000 psi. 23

24. Can the physically coated stationary phases used in GC be used in narrow bore SS columns through which liquid mobile phase moves under high pressures? 24 Obviously… NO

25. Because under high pressures of liquid mobile phase, physically coated liquid stationary phases will be removed physically or by dissolution in liquid mobile phase. We need physically&chemically stable stationary phase. 25

26. Chemically treated inert (not strictly) particles have free silinol group at their surface. 26 Silica particle CCC Si - OH Free silinol groups

27. Early attempts were then made to chemically bonded long chain aliphatic alcohols with the free silinol groups present on the surface of silica particles. 27 Si - OH+ HO - R - H 2 O Si - O - R

28. However these chemically bonded phases with Si - O - C bonds are stable only in acidic media. 28

29. We cannot restrict our separation work always in acidic media 29 In basic media Si– O- C bond undergoes hydrolysis. Si - O - R basic medium Si - OH + R - OH

30. We therefore would like to use chemical bonding with ‘ Si ’ of free silinol groups which will be hydrolytically stable over a wide acidic to basic pH range. 30

31. Attempts were then made to carry out chemical reactions with substituted silane ( Si H 4 ) having suitable long chain of hydrocarbons attached to it. 31

32. 32 Si - OH 1218 + Cl – Si – C – CC - H CH 3 H H H H H H Substituted siliane 1 218 – Si – C – CC - H CH 3 H H H H H H Si - O -H Cl

33. This chemically bonded phase ( Si-O-Si bond ) is therefore mechanically strong and hydrolytically stable over a wide pH range (2-9). 33

34. At this stage we can say that the combination of pumping systems capable of giving liquid mobile phase flow under high pressures and chemically bonded, mechanically stable liquid stationary phases with desired polarity, pore size,and their hydrolytic stability over acidic to basic pH range,has allowed us to achieve faster analytical times and versatility of separating solutes with diverse nature. 34

35. 35 About Pumping: Do our pumping systems allow us to have a pulseless flow of liquid mobile phase through chromatographic columns?

36. Different types of Pumps available are 1.Reciprocating Pumps 2.Motor driven Syringe type Pumps 3.Pneumatic Pumps.

37. A reciprocating piston pump operation results in a pulsating flow of liquid mobile phase. But for chromatographic separations we need pulseless flow of liquid mobile phase.

38. 38 A reciprocating pump for HPLC The piston expels the liquid through a one way valve (Check valve)

39. The pumping rate can be adjusted by controlling the distance the piston retracts or by the cam rotating speed. This limits the amount of liquid pushed out by each stroke. How do we achieve a pulse less flow? If we introduce a second reciprocating piston pump and synchronize its function with the first one the pulsation is reduced considerably.

40. 40 A dual piston reciprocating pump for HPLC Using common eccentric cam, it allows one piston to pump and other to refill. Thus we do get pulseless flow of liquid mobile phase with two synchronized piston pumps

41. 41 When the liquid mobile phase is moving under high pressures through chromatographic columns, introduction of a sample on to the column will require some specially designed injection system. Introduction of sample?

42. 42 The most popular and commonly used injector system, is the syringe –loop injector of the Rheodyne type. It is a fixed volume universal injector which allows introduction of micro litres of samples on to the chromatographic column.

43. 43 With Rheodyne injector we have achieved “small sample” attribute of GC with liquid chromatography.

44. 44 Thus so far with we have achieved the following attributes with LC Fast Analysis Small Sample Size Versatility Efficiency Non-destructive

45. NOW WHAT IS LEFT?

46. 46 DETECTORS: As soon as the solutes are eluted they should be detected and quantitated with sensitive and specific or universal detectors.

47. 47 We have universal detectors like : Refractive index detector is relatively less sensitive but can detect and quantitate all types of solutes Mass spectrometry detector is highly sensitive detector and can also help in structure elucidation.

48. 48 With specific and universal detectors we have achieved the following attributes: High sensitivity High reliability and accuracy

49. 49 Thus in place of glass column liquid chromatography now we have 145 2 3 1- Pumping system 2- Universal injector 3- Column 4- Detector 5- Recorder

50. 50 1.Pumping system capable of giving pulseless flow of liquid mobile phase under high pressures of the order of 500 to 5000psi. *This makes fast analysis.

51. 51 2.Universal injector which allows to introduce samples in microlitres. *This reduces sample size

52. 52 3.Narrow bore SS columns with suitable chemically bonded phases with small particle size (3-5µ), desired polarity and pore size and hydrolytically stable over a wide acidic to basic pH range. *This gives high separation efficiency, high versatility in separating solutes with diverse nature.

53. 53 4.Sensitive and Specific or Universal detector * This gives high sensitivity, reliability and accuracy of detection and quantitation of solutes.

54. 54 5.Computing system allows to record and compute chromatographic results.

55. 55 All the above factors add “High Performance” to normal Liquid Chromatography. It is therefore known as “High Performance Liquid Chromatography” abbreviated as HPLC.

56. Thank You