HPLC -High Performance Liquid Chromatography

HPLC-Brief Discussion on HPLC Technique ,an advanced analytical technique used to separate, identify, and quantify components in a mixture using a liquid mobile phase under high pressure.

1. HPLC By – Dr. Wrushali P a ncha le , Associate Professor, DRGIOP Amravati

2. What Is HPLC? HPLC stands for “High- performance liquid chromatography (sometimes referred to as High-pressure liquid chromatography ) Defined as: “ A chromatographic technique used to separate components of mixture for the purpose to identify, quantify or purify the individual components of the mixture ” . Widely used in the field of biochemistry and analytical chemistry. Dr.Wrushali Panchale 2

3. Salient features Accuracy of quantitative measurement; Speed of separation; Higher resolving power; Needs a small sample for analysis. Powerful, adaptable and automated process, results obtained quickly and easily. Affordable repetitive and reproducible analysis using the same column; Continuous monitoring of the column effluent; Automation of the analytical procedure and data handling. Provides data management, security features, and reporting and instrument validation. Productivity can be increased by managing all the areas of analysis from sample to instrument and from separation to reporting results. Dr.Wrushali Panchale 3

4. Principle of HPLC The principle of HPLC is to force the sample through the column by pumping the mobile phase along with sample at high pressure a according to relative affinities . Dr.Wrushali Panchale 4

5. Principle of HPLC Lower adsorption affinity Greater adsorption affinity travel faster. travel slower. Phase preference can be expressed by distribution co- efficient,K: Kx= C Stat /C Mob Here, C Stat =Concentration of compound X in the stationary phase . C Mob = Concentration of compound X in the mobile phase . Kx = Partition co- efficient. Dr.Wrushali Panchale 5

6. Instrumentation There are 5 major components Solvent delivery system (includes a pump, flow controller and a filter. Commonly glass bottles with caps are used. Vaccum for 5- 10 min is also used for degassing Pump: It forces the mobile phase to pass through colu mn . Flow rate is 1- 2 ml/ min . Dr.Wrushali Panchale 6

7. Injector Manually (syringe) or automated. Sample volume 5- 20µl. Column Heart of HPLC. Separate sample components on basis of physical and chemical parameters. Lenght 10-30cm. Diameter 4- 10nm. Packing material 5-10nm thick. Dr.Wrushali Panchale 7

8. 4 ) Detector; Detection of elutes from column. Quantitative analysis of sample components. Output transferred to recorder/ computer. 5) Computer for data processing. Data system that controls modules of HPLC . Signals from detector are determine elution time, quantitative interpreted to and qualitative analysis of sample. Dr.Wrushali Panchale 8

9. Dr.Wrushali Panchale 9

10. Dr.Wrushali Panchale 10

11. Characteristics of solvent cost Absorbance Solvents used- acetonitrile. Methanol, water Fig. 1. Absorption spectrum of acetonitrile Fig. 2. Absorption spectrum of methanol Dr.Wrushali Panchale 11

12. Solvents All solvents should be ‘HPLC’ grade. This is a type of reagent grade material. It has been filtered using a 0.2 μm filter. Filtered solvent helps extend pump life by preventing scoring. It also reduces the chances of a column plugging. Dr.Wrushali Panchale 12

13. Fig. 4. Elution of caffein Fig. 5. Elution of cholesterol. Mobile Phase- Organic solvent: water (95:5) 4. Pressure (ACN is lower) 3. Elution Strength (ACN is generally higher) Dr.Wrushali Panchale 13

14. 5. Selectivity of separation Fig. 6. Separation of phenol, benzoic acid and p- toluic acid. Dr.Wrushali Panchale 14

15. 6. Peak shape For compounds such as salicylic acid (phenol with carboxyl or methoxy group in the ortho position), acetonitrile can cause significant tailing , which could be suppressed by using methanol. This is probably caused by differences in the way the mobile phases relate to the mutual interaction (adsorption) between the silica surfaces and target components , due to the chemical properties of the organic solvent molecules. Also, polymer- based reversed- phase columns generally tend to result in broader peaks than silica- based columns. This is particularly common for aromatic compounds in polystyrene columns. Dr.Wrushali Panchale 15

16. Degassing of Mobile phase Methanol is mixed with water, it releases heat , which tends to facilitate releasing any extraneous dissolved air as bubbles (facilitates degassing). Acetonitrile cools the temperature by absorbing heat , so air bubbles are generated later , as it gradually returns to room temperature. Dr.Wrushali Panchale 16

17. Techniques of Degassing Commonly used degassing practices for HPLC mobile phase are: Helium purging Vacuum degassing Sonication Pumps An ideal pump should have the following desirable characteristics Solvent compatibility and resistance to corrosion; Constant flow delivery independent of back pressure; Convenience of replacement of worn out parts; Low dead volume for minimum problems on solvent changeover. Dr.Wrushali Panchale 17

18. Types of pumps based on the amount of pressure that can be produced. Constant- volume pump (displacement pump) Constant- pressure pump (pneumatic pump Constant- volume pump. (a) Reciprocating pump Column Motor Piston Solvent Pulse damper Ball check valve Seal Single- piston reciprocating pump Dr.Wrushali Panchale 18

19. (b) Syringe pump Pressure up to 78,000 psi It consists of a cylinder (also known as syringe) that holds the mobile phase which is expelled by a piston. The piston is advanced by a motor connected through gears, to provide worm smooth pulseless flow. To column Motor Control electronics Cylinder Piston Mobile phase Dr.Wrushali Panchale 19

20. 2. Constant- pressure pump (pneumatic pump ) involve force of compressed gases /air to generate mechanical effect . Mechanism. MP is driven through the column with the use of pressure produced from gas cylinder. Due to solvents viscosity back pressure may be increased. Dr.Wrushali Panchale 20

21. 3. Displacement pump Low pressure inlet High pressure outlet Generally columns are protected by a guard column. These guard columns also adsorb undesirable sample Dr.Wrushali Panchale 21

22. 2. Injector Multiport valves (loop valve system) Manual Automatic Sample Manager Dr.Wrushali Panchale 22

23. Traditional Capillary Sample Volume 20- 25 μl 0.01- 8 μl Pumps Dual Piston Dual piston Column length 3- 15 cm 3- 25 m Column i.d. 2.1- 4.6 mm 300- 500 μm pH range 1.0 - 8.5 Flow rate 1ml/min 1- 20 μl/min Detector Volume <10 μl 500 nl Column (i) Pre-columns Generally columns are protected by a guard column. These guard columns also adsorb undesirable sample components. Pre- columns may be (i) Upstream of the injector, and (ii) Downstream of the injector. (i) Analytical columns Dr.Wrushali Panchale 23

24. Stainless steel tube Stainless steel tube Nut End fitting Ferrule Particle size of column Plate Numbers (N) 3 μm 100,000 plates/m 5 μm 55,000 plates/m 10 μm 35,000 plates/m Stationary phases Stationary phase microparticles with diameters 3 μm, 5 μm and 10 μm are used for analytical and guard columns. Typical quoted column efficiencies are: Table 3. Column efficiencies with respect to particle size Dr.Wrushali Panchale 24

25. Packings Originally, these were irregular silica and alumina. A range of synthetic, regularly shaped packings are now available. Porous - channels through packing Superficially porous - rough surface Smooth - bead like. Dr.Wrushali Panchale 25

26. Packing size As packing size is decreased, efficiency and pressure requirements are increased. ! Common diameters for analytical work diameter ! plates "" 10 μm "" 5000 "" 5 μm "" 9000 "" 3 μm" " 15,000 All are for a 15 cm x 4.6 mm column Dr.Wrushali Panchale 26

27. Column body Typically consist of stainless steel with a high precision internal bore. Some manufacturers offer column inserts - " don’t need to repurchase the column " fittings. Others offer columns where the external body can be compressed to improve packing efficiency. Dr.Wrushali Panchale 27

28. HPLC column examples Dr.Wrushali Panchale 28

29. Columns (25- 50 cm long; i.d. 4.0- 10 mm) packed with smaller particles (5- 10 µm) flow rates of 1- 5 ml/min pressures reaching up to 8000 psi. Most of the work in analytical HPLC is, however, done using pressures between about 400 and 1500 psi . Column stationary phases Today, most packing fall into four classes. Silica or alumina Bound phases on either alumina or silica. Gels Controlled- pore glass or silica. Dr.Wrushali Panchale 29

30. Absorption phases - alumina ! common mobile phases ! hexane, chloroform, 2- propanol. ! example application - amines. silica ! common mobile phases ! hexane, chloroform, 2-propanol. ! example applications - ethers, esters, ! porphyrins , fat- soluble vitamins. Partition phases Can be broken down into Normal phase - polar materials bound to the support. Reverse phase - non- polar materials bound to the support. Mixed phase - ! may have some of each Dr.Wrushali Panchale 30

31. Partition phases Normal ! Amino (- NH2) ! Cyano (- CN) ! Diol (glycidoxy- ethylmethoxysilane) Reverse ! C- 2 or RP- 2 (-Si-CH2CH3) ! C- 8 or RP- 8 (-Si-(CH2)7CH3) ! C- 18 or RP- 18 (-Si-(CH2)17CH3) Increasing the C number results in a thicker, more retentive phase Ion exchange phases Strong cation ! - sulfonic acid group Strong anionic !- quarternary amine Weak anion ! - primary amine Weak cation ! - COOH Dr.Wrushali Panchale 31

32. Capillary and Microbore columns. Several companies have begun offering columns with smaller ID. Microbore column - 1 mm ID, packed column. Capillary column - < 1 mm ID, internal bound phase. These columns require smaller solvent flows, reduced sample size and improved detector design . Capillary and Microbore columns. Capillary and Microbore columns. Aromatic Compounds mobile phase!2% ethylacetate in hexane flow rate " 4 μ l/min column "" Fusica II, 300 μ m I.D. x ! ! ! 25 cm silica sample ! 1. toluene ! 2. nitrobenzene ! 3. acetophenone ! 4. 2,6-dinitrobenzene injection! ! 60 nl detection! ! UV 254 nm Dr.Wrushali Panchale 32

33. Capillary and Microbore columns. Capillary and Microbore columns. Aromatic Compounds mobile phase!2% ethylacetate in hexane flow rate " 4 μl/min column ""Fusica II, 300μm I.D. x ! ! ! 25 cm silica sample ! 1. toluene ! 2. nitrobenzene ! 3. acetophenone ! 4. 2,6-dinitrobenzene injection! ! 60 nl detection! ! UV 254 nm Silica based columns Silica is the ideal support for HPLC columns. It offers a large mechanical stability, excellent wide range of p hysicochemical surface properties, a bonding chemistries and is compatible with a broad range of organic solvents. Dr.Wrushali Panchale 33

34. pH stability In general, HPLC columns are stable within a pH range of 2 to 8. If you are measuring a pH value, the measurement must be done in the aqueous media before mixing the eluent with organic solvents. Modern HPLC columns can be used outside that pH range. The new bonding chemistries allow use down to pH 1 for some stationary phases. However, please check vendor’s product information before using silica based column outside the pH range of 2 to 8. However, best lifetimes are obtained between pH 2.0 and pH 6.8. Dr.Wrushali Panchale 34

35. Mechanical stability Stationary phases based on silica are mechanically very stable. The packed columns show no pressure limit and can be used at more than 40 MPa (6000 psi) without any problem. However, please avoid pressure shocks on the column. Pressure shocks lead to channeling in the column, which results in peak splitting in the corresponding chromatogram. Mobile phases (Eluents) Silica based stationary phases are compatible with all organic solvents in the above mentioned pH range. . Filter all prepared buffer through a 0.5 μm filter before using them in your HPLC system .

36. ELUTION PATTERN IN NORMALPHASE AND REVERSE PHASE HPLC The us e of non pure solvents in HPLC causes irreversible adsorption of impurities on the column head. These impurities block adsorption sites, change the selectivity of the column and lead to peak splitting in the chromatogram. In gradient elution, impurities cause so called “ Ghost Peaks ”. Ghost peaks are peaks that always appear in the same position on the chromatogram. Their origin is not the sample, but the impurities from the solvents or solvent additives. Therefore, it is highly recommended to run a gradientwithout injection in the beginning of each method to determine the ghost peaks Dr.Wrushali Panchale 36

37. To avoid irreversible adsorption at the column head, you should always use a pre- column. The use of a pre- column increases the life time of a column dramatically. In addition, a pre- column can filter solid parts stemming from pump seals or injection rotors. An alternative to a pre- column is an in- line filter. These filters are attached directly to the column. These filters get rid of solid parts in the eluent but will not avoid irreversible adsorption of organic impurities Dr.Wrushali Panchale 37

38. Detector Systems Virtually every chemical and physical property that can be measured in solution has been look at. Detectors fall roughly into two classes Bulk property - measures an overall change in the mobile phas Solute property - measures a solute specific property. Properties of a good detector A detector must provide - high sensitivity, low detection limits, linearity, Reproducibility. This is true for any detector. Each detector will have specific advantages and will vary as to peak shape and spread, noise and flow/temperature dependence they have. Dr.Wrushali Panchale 38

39. Columns Solid Support - Backbone for bonded phases. Usually 10µ, 5µ or 3µ silica or polymeric particles. Bonded Phases - Functional groups firmly linked (chemically bound) to the solid support. Extremely stable Reproducible Guard - Protects the analytical column: Particles Interferences Prolongs the life of the analytical column Analytical - Performs the separation. Dr.Wrushali Panchale 39

40. Bonded Phases C- 2 C- 8 Ethyl Silyl - Si- CH 2 - CH 3 CN Cyanopropyl Silyl - Si-(CH 2 ) 3 - CN C- 18 Octadecyl Silyl -Si-(CH 2 ) 17 - CH 3 Octyl Silyl - Si-(CH 2 ) 7 - CH 3 Dr.Wrushali Panchale 40

41. Instrumentation Pump Injector Column Detector Mobile Phases Gradient Controller • Dr.Wrushali Panchale 41

42. Applications Pharmaceutical: Tablet dissolution. Shelf life determinations. Quality control. Environmental: Phenols in Drinking Water. Identification of diphenhydramine. Assessment of TNT toxicity. Dr.Wrushali Panchale 42

43. Clinical Quantification of DEET in Human Urine. Analysis of antibiotics. Food and Flavor Ensuring soft drink consistency and quality. Sugar analysis in fruit juices. Dr.Wrushali Panchale 43

44. Advantages It can be applied to the separation and analysis of very complex mixtures. Accurate quantitative measurements. Continuous monitoring of the column effluent. High resolution. Quick analysis. Separation of volatile and non- volatile components. Thermally unstable compounds isolated. Dr.Wrushali Panchale 44

45. Disadvantages Tedious to detect co- elution. High cost. Complex to operate. Dr.Wrushali Panchale 45

46. Columns C2 Silica Gel Rarely Used. Used For Plasma, Urine, And Aqueous Samples C4 Silica Gel Hydrophobic Substances. C8 Silica Gel Highly Polar Compounds (Steroids, Nucleosides, Cyclodextrins, Pharmacological Plant Constituents) C18 Silica Gel Nonpolar To Moderately Polar Compounds (Fatty Acids, Glycerides, Esters, Fat- Soluble Vitamins, Steroids Amino (NH2) for sugars with acetonitrile / water mobile phase. Cyano Cyclosporins from serum/blood, Antidepressants from serum, Benzalkonium Chloride from aqueous solution Diol for aqueous GPC applications Phenyl fatoxins from food, Caffeine from beverages, flavors, Phenols from water, trace elements from water Dr.Wrushali Panchale 46

47. Partitioning Separation is based on the analyte’s relative solubility between two liquid phases Stationary Phase Mobile Phase Solvent Bonded Phase Dr.Wrushali Panchale 47

48. Common Reverse Phase Solvents Methanol CH 3 OH Acetonitrile CH 3 CN Tetrahydrofuran (THF) Water H 2 O Dr.Wrushali Panchale 48

49. Structure of silica Gel Single Silanol group Two silanol groups (Geminal groups) Silicon atoms joined together with oxygen atoms by siloxane bonds (silicon- oxygen- silicon bonds). On the surface of each primary particle some residual, uncondensed hydroxyl groups from the original polymeric silicic acid remain. These residual hydroxyl groups confer upon silica gel its polar properties. These hydroxyl groups react with the silane reagents to form bonded phases. Dr.Wrushali Panchale 49

50. Silica bonded (porous) Type B Solid silica 0.5µm 1.7µm 2.7µm Dr.Wrushali Panchale 50

51. - (CH 2 ) 17 CH 3 δ- NH 2 CH 2 O - CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 3 - N - CH 2 CH 2 CH 2 CH 3 CH 2 CH 2 CH 2 CH 3 - + - OH δ+ CH 2 CH 2 CH 3 CH 3 CH 2 CH 2 CH 2 NH 2 Hydrophobic interaction Hydrogen bond Ionic bond Interactions at the ODS packing surface Dr.Wrushali Panchale 51

52. Based on size Based on chemical composition Pellicular particles Porous particles Normal phase chromatography Reverse phase chromatography Consists of Consists of silica alumina Consists of silica Consists of silica non porous, or ion exchange resin. alumina or an ion chemically linkage to a spherical Particles are often wetted exchange resin. These low polar functional glass or with thin organic film particles are often group through siloxane. polymer which is wetted with thin These phases are beads. physically/chemically organic films which are prepared by treating the Particle size: bounded to surface. physically or surface silanol groups of 30- 40 μm. Particle size: 3- 10μm chemically bounded to silica with the surface. organochlorosilane Particle size: 3- 10 μm reagent. Stationary phase is less polar than the mobile phase. Table 9. Column Packing Materials in HPLC Dr.Wrushali Panchale 52

53. Advantages of Reverse Phase Liquid Chromatography A broad spectrum of sample types with widely ranging polarities and molecular masses can be separated. Can be used for the separation of electrolytes (weak and strong) by either generating suppression) or ion-pairs. 3. Structural isomers can be separated. unionised species (ion Easy to use. Sample loading capacity is higher than for silicas (x10). Dr.Wrushali Panchale 53

54. Limitations Restricted pH range from 2 to 7.5 for some silica- based supports. Separation may not only be due to partition processes since polar mechanisms may contribute if unreacted silanol groups are present or the bonded moieties have also polar characteristics. Dr.Wrushali Panchale 54

55. Dr.Wrushali Panchale 55

56. Dr.Wrushali Panchale 56

57. (i) Conventional (polymethacrylate) monolith (ii) Epoxy- based polymer monolith (iii) Nanoparticle embedded polymer monolith (iv) Nanoparticle coated polymer monolith (v) Monolithic cryopolymer formed using unidirectional freezing (vi)Polymerised high internal phase emulsion (polyhipe) Dr.Wrushali Panchale 57

58. Pump Injector Column Detector Mobile Phases Gradient Controller • Dr.Wrushali Panchale 58

59. UV DETECTOR Dr.Wrushali Panchale 59

60. DAD DETECTOR Dr.Wrushali Panchale 60

61. Dr.Wrushali Panchale 61

62. REFRACTIVE INDEX DETECTOR Dr.Wrushali Panchale 62

63. E lectrochemical detectors Dr.Wrushali Panchale 63

64. based on the measurements of the current resulting from oxidation/reduction reaction of the analyte at a suitable electrode. Since the level of the current is directly proportional to the analyte concentration, this detector could be used for quantification.. Electrochemical detectors A number of properties have been evaluated Detector types Dielectic constant, Amperometric Conductometric , Polarographic , Potentiometric The eluent should contain electrolyte and be electrically conductive . Dr.Wrushali Panchale 64

65. Dr.Wrushali Panchale 65