HIGH PERFORMANCE LIQUID CHROMATOGRAPHY INSTRUMENTATION. Mrs. G. Aruna Mpharm (PhD) Dept. of PA & QA KTPC

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1 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY INSTRUMENTATION Mrs. G. Aruna Mpharm (PhD) Dept. of PA & QA KTPC 1

2 HPLC INSTRUMENTATION CONSIST OF Solvent Reservoir( hplc solvent reservoir systems) Pumps Pre Guard Column Sample injection system Columns Detector Recorder and integrators 2

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5 HPLC SOLVENT RESERVOIR SYSTEMS 5

6 HPLC SOLVENT RESERVOIR SYSTEMS These are the glass bottles use to store the mobile phase. The mobile phase is pumped under pressure from one or several reservoirs and flows through the column at a constant rate. Desirable feature in the solvent delivery system is the capability for generating a solvent gradient. 6

7 HPLC SOLVENT RESERVOIR SYSTEMS Degasser is needed to remove dissolved air by subjecting the mobile phase under vacuum, distillation, spurging with fine spray of an inert gas at lower solubility such as Argon and Helium or by heating and ultrasonic stirring. Filtration is needed to eliminate suspended particles and organic impurities. 7

8 PUMPS Pass mobile phase through column at high pressure and at controlled flow rate. Performance of pump directly affects the Rt, reproducibility, detector sensitivity 8

9 PUMPS IDEAL CHARCETRISTIC OF A PUMP Non corrosive and compatible with solvent. Provide High pressure to push mobile phase Provide constant flow rate to mobile phase. Easy to change for one mobile phase to another. 9

10 PUMPS Should have reproducible flow rate and independent of column back pressure. Should not leak. High pressure generated by pump should not lead to an explosion. It should be easy to dismantle and repair. 10

11 TYPE OF PUMP USED IN HPLC Reciprocating pump Displacement pump Pneumatic pump 11

12 RECIPROCATING PUMP 12

13 RECIPROCATING PUMP WORKING Contains reciprocating piston that moves back and forth in hydraulic chamber. By the movement of piston solvent flow into the column under high pressure. When piston moves backward inlet valve open while exit valve closes. This result in mobile phase being 13 drawn into the main chamber (cylinder).

14 RECIPROCATING PUMP When the piston moves to the front the inlet valve closes and the exit valve opens. The reduction in volume in main chamber due to forward motion of piston result in mobile phase moving out of the exit valve under high pressure. DISADVENTAGE Pulsed flow which must be damped as they produce a base line noise on the chromatogram 14

15 ADVANTAGES RECIPROCATING PUMP Generate high output pressure (upto10000 poise). Ready adaptability to gradient elusion. Provide constant flow rate. Pressure generated is so high that any back pressure generated in the column due to higher viscosity of stationary phase can be easily overcome. 15

16 DISPLACEMENT PUMP / SYRINGE PUMP 16

17 DISPLACEMENT PUMP / SYRINGE PUMP WORKING Works on the principle of positive solvent pressure. Consist of screw or plunger which revolves continuously driven by motor. Rotatory motion provides continuous movement of the mobile phase which is propelled by the revolving screw at greater speed and pushes solvent through small needle like outlet. Consist of large syringe like chamber of capacity ml.

18 DISPLACEMENT PUMP / SYRINGE PUMP ADVANTAGES Flow is pulse free. Provide high pressure upto atm. Independent of column back pressure and viscosity of solvent. Simple operation. DISADVENTAGE Limited solvent capacity Gradient elution is not easy. 18

19 PNEUMATIC PUMP WORKING Pressure from a gas cylinder delivered through a large piston drivers the mobile phase. Pressure on the solvent is proportional to the ratio of piston usually 50: 1. 19

20 PNEUMATIC PUMP The driving air is applied, piston moves, inlet closes & outlet open pushing mobile phase to the column. A lower pressure gas source of 1-10 atm can be used to generate high liquid pressure.( atm ) About 70 ml of the mobile phase is pumped from every stroke. 20

21 PNEUMATIC PUMP ADVANTAGES Pulse free flow. Generates high pressure. DISADVANTAGES Limited volume capacity (70 ml ) Pressure output and flow rate depends on the viscosity and column back pressure. Gradient elusion is not possible. 21

22 Pulse damper Important damping methods include A triple headed pump: - two heads in different stages of filling as the third is pumping. A tube with an air space or a flexible bellows or tube: - where a gas (air space) or a flexible metal vessel takes up some of the solution energy. When pump refills, this energy is released and a smooth pressure pulsation result. A restrictor: - I this method, a 25 cm length of 4 mm bore.stainless steel tubing. Packed with 20µm glass 22 beds, is placed between the pump and the column.

23 Sample injection system Septum injectors Stop flow septumless injection. Rheodyne injector / loop valve type. 23

24 Sample injection system Septum injection port. Syringe is used to inject the sample through a self sealing inert septum directly into the mobile phase. Drawback: - leaching effect of the mobile phase with the septum resulting in the formation of ghost peaks. Stop flow septumless injection. Flow of mobile phase through the column is stopped for a while. Syringe is used to inject the sample. Drawback: formation of ghost peak. 24

25 Sample injection system Rheodyne injector / loop valve type. Sophisticated modern method with good precision. Sample is introduced in the column without causing interruption to mobile phase flow. Volume of sample ranges between 2 µl to over 100 µl. 25

26 Sample injection system Operation of sample loop. sampling mode Injection mode. Sample is loaded at atmospheric pressure into an external loop in the micro volume sampling valve, and subsequently injected into the mobile phase by suitable rotation of the valve. Micro volume sampling valve operation of a Sampling loop. 26

27 27

28 COLUMN Made up of stainless steel or heavy glass to withstand the pressure. The columns are usually long (10 30 cm) narrow tubes. Contains stationary phase at particle diameters of 25 µm or less. The interior of column should be smooth and uniform. Column end fitting are designed to have a zero void volume. 28

29 CLASSIFACTION OF CLOUMN BASED ON APPLICATION column Main column Guard column Analytical column Preparative column Standard column Narrow bore Short fast column Micro preparative Preparative column Macro preparative 29

30 CLASSIFACTION OF COLUMN ON THE BASES OF COMPONENTS BONDED PHASE COLUMN COLUMN WHERE LIQUID IS INPERMAGNETED ON SOLID INERT SUPPORT 30

31 STANDARD COLUMN Internal diameter 4 5 mm and length cm. Size of stationary phase is 3 5 µm in diameter. Used for the estimation of drugs, metabolites, pharmaceutical preparation and body fluids like plasma. NARROW BORE COLUMN Internal diameter is 2 4 mm. ( signal is increased 4 times ) Require high pressure to propel mobile phase. Used for the high resolution analytical work of compounds with very high Rt. 31

32 SHORT FAST COLUMN Length of column is 3 6 cm. Used for the substances which have good affinity towards the stationery phase. Analysis time is also less (1-4 min for gradient elusion & sec for isocratic elusion). PREPARATIVE COLUMN Used for analytical separation i.e. to isolate or purify sample in the range of mg form complex mixture. Length cm Internal diameter 6 mm or more. 32

33 Preparative column are of three type : Micro preparative or semi preparative column Modified version of analytical column Uses same packaging and meant for purifying sample less then 100 mg. Preparative column Inner diameter 25 mm. Stationary phase diameter µm 33

34 Macro Preparative Column Column length cm Inner diameter 600 mm 34

35 GUARD COLUMN They are placed anterior to the separating column. Serve as a protective factor that prolongs the life and usefulness of the column. They are dependable column designed to filter or remove Particles that clog the separation column. Compounds and ions that could ultimately cause baseline drift, decrease resolution, decrease sensitivity and create false peaks. 35

36 Bonded phase column Here the molecules, comprising the stationary phase i.e. the surface of the silica particles, are covalently bonded to a silica based support particles. The most popular bonded phase,siloxanes, are formed by heating the silica particles in dilute acid for the day so as to generate the reactive Silonol group. 36

37 OH OH OH ו ו ו - Si O Si - O - Si I I I 37

38 Silonal group is the treated with organochlorosilane. CH3 CH3 ו ו OH + Cl Si R - Si O Si R + HCl Si CH3 CH3 These bonded phases are stable between the ph range 2 9 and upto temperature of 80º C. Bonded phase is made with a linear C 18 hydrocarbon, also know as ODS (octadecyl silane) bonded phase. Used in pharmaceutical analysis or separation of less polar 38 components.

39 An alkyl nitrile column or cyano column which has 12 carbon atoms with the last atom appearing as a nitrile group (CN).moderately polar column. Amino alkyl bonded phase column which is normally C 8, last C atom bearing NH2 group. Polar column. Use full in separation of CHO, peptides, amino acids. Advantages Can withstand high pressure exerted by mobile phase. Life of column is more. No bleeding effect Disadvantages Very expensive Manually can not be fabricated 39

40 COLUMN WHERE LIQUID IS INPERMAGNETED ON SOLID INERT SUPPORT. These are not use widely now days. stationary phase dose not have the strength to stay in the column on account of the physical forces exerted by the mobile phase at very high pressure. Amount of loading on inner support is minimum stationary phase starts bleeding out of the column and can cause resistance to mass transfer. 40

41 METHOD OF PACKING Depends on the mechanical strength of stationary phase. Particle size of the stationary phase. Particles of greater then 20 µm dry packing Particles of lesser then 20 µm slurry packing / wet packing. DRY PACKING Particle size greater then 20 µm filled into vertical clamped column in small quantity. Deposition is done by tapping or vibrating the column. Column is unclamped and the tapped on the firm surface to obtain dense and reproducible packing. 41

42 WET / SLURRY PACKING Particle size with diameter less then 20 µm can only be placed wet as a suspension. Suspension should be stable, it should not sediment, and agglomentation should be avoided. 42

43 43

44 DETECTORS Based on the application, the detectors can be classified into Bulk property detectors Solute property detectors. Response time should be least (ten times less than the peak width of the solute in time units. 44

45 BULK PROPERTY DETECTORS Compare an over all change in physical property of mobile phase with or without an eluting solute. These types of detectors tend to be relatively insensitive and require temperature control. e.g. Refractive index detector. SOLUTE PROPERTY DETECTORS They respond to a physical property of the solute that is not exhibited by the pure mobile phase. These detectors are more sensitive, detect the sample in nanograms quantity. e.g. uv visible detector, electrochemical detector, fluorescence detector. 45

46 ULTRAVIOLET VISIBLE DETECTOR UV detectors are the most commonly used detector. They measure the ability of a sample to absorb light. This can be accomplished at one or several wavelengths. A variable wavelength UV detector, capable of monitoring from 190 to nm will be found suitable for the detection of the majority of samples. 46

47 Mobile phase from the column is passed through a flow cell held in the radiation beam of uv / visible spectrophotometer. Selective in nature, detect only those solutes that absorb uv/ visible radiation e.g. alkenes, aromatic compounds and compound having multiple bonds between C and O, N or S. BASICALLY TWO TYPES OF ABSORBANCE DETECTORS ARE AVAILABLE fixed wavelength detector variable wavelength detector 47

48 Fixed wavelength detector HPLC detectors which do not allow changing the wavelength of the radiation called fixedwavelength detectors. 48

49 Fixed wavelength detector Low-pressure mercury lamp emits very intense light at nm. By filtering out all other emitted wavelengths, utilize only 254 nm line to provide stable, highly sensitive detectors capable of measuring subnanogram quantities of any components which contains aromatic ring. The 254 nm was chosen since the most intense line of mercury lamp is 254 nm, and most of UV absorbing compounds have some absorbance at 254 nm. 49

50 VARIABLE-WAVELENGTH DETECTORS Detectors which allow the selection of the operating wavelength called variable wavelength detectors. 50

51 VARIABLE-WAVELENGTH DETECTORS Sensitivity for any absorptive component by selecting an appropriate wavelength individual sample components have high absorptivity at different wavelengths and thus, operation at a single wavelength would reduce the system's sensitivity Depending on the sophistication of the detector, wavelength change is done manually or programmed on a time basis into the memory of the system. 51

52 FLOURIMETRIC DETECTORS Very sensitive, but very selective. It is possible to detect even a presence of a single analyte molecule in the flow-cell. Fluorescence occurs when compounds having specific functional groups are excited by shorter wavelength energy and emit higher wavelength radiation. 52

53 FLOURIMETRIC DETECTORS Fluorescence is often collected at right angle to excitation beam. Only one sixth of fluorescence is collected. If concave mirror is placed around the sample cell abut 75 % of the emission is collected. With all sample cells, scattered radiation from the excitation source is selectively removed with cur off or band pass filters placed before photomultiplier tube. 53

54 Refractive Index Detector/ Differential refractometer The detection principle involves measuring of the change in refractive index of the column effluent passing through the flow-cell. It responds to any solute whose refractive index is significantly different from that of the mobile phase. Principle: it is based on two principles. Deflection ( deflection type refractometer) Reflection (reflection type refractometer) 54

55 Deflection type refractometer. Measure the deflection of a beam of a monochromatic light by double prism. Eluent passes through one half of prism & pure mobile phase to other half known as reference compartment. Reference and sample compartment are separated by diagonal glass divider. Auto zero is used to set, out put signal to zero when mobile phase is in both the compartments. 55

56 Deflection type refractometer. Tungsten lamp provides beam of light collimated through lens and passes through Eluent and reference compartment. Reflected by the mirror through the same compartment again. The beam of light is focused on a beam splitter before passing into the photo detector. 56

57 Deflection type refractometer. Refractive index of the mobile phase is changed due to the presence of solute, the beam from the sample compartment is deflected which produces the change signal that is proportional to the concentration of solute. Advantages Wide range of linearity. Covers entire refractive index range. 57

58 Reflection type refractometer Measure change in % of reflected light at glass liquid interface as the reflective index of liquid changes. Based on the Fresnels law of reflection which states The amount of liquid reflected at a glass- liquid interface varies with the angle of incidence and the refractive index of the liquid 58

59 working: Reflection type refractometer Two collimated beams from the projector (light source & lens) illuminate the reference and sample cell. Cells are formed of Teflon gasket, which is clamped between the cell prism and a stainless steel reflecting back plate. 59

60 As the light of beam is transmitted through the cell interfaces, it passes through the liquid film and impinges on the surface of the reflecting back plate. Diffused, reflected light appears as two spots and passes through the lens and detected by photo detector. The ratio of the reflected light to transmitted light is function of refractive index of the two liquid, the illumination of the cell back plate is direct measure of the refractive index of the liquid in each chamber 60

61 Electrochemical Detector/ Amperometric detector It is based on the measurements of the current resulting from an oxidation/reduction reaction of the analyte at a suitable electrode. The level of the current is directly proportional to the analyte concentration 61

62 Recorder and integrators Recorders are used to record the response obtained from the detector after amplification. They record the baseline and all the peaks obtained, with respect to time. Retention time for all the peaks can be calculated. Integrators are improved versions of recorder with data processing capabilities. They can record the individual peaks with retention time height and width of peak, peak area, etc. 62

63 References Instrumental method of analysis (seventh edition ) Willard merritt and dean settle page no Instrumental method of chemical analysis B.K.Sharma page no-, 58,59. Pharmaceutical drug analysis -2nd edition Ashutosh kar page no ,459,466 Pharmaceutical analysis vol-2 Dr.A.V.kasture page no 52,53. 63

64 Thank you 64

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