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Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
Instrumentation Of HPLC
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Instrumentation Of HPLC

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Gives basic idea about instrumentation of high performance liquid chromatography

Gives basic idea about instrumentation of high performance liquid chromatography

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  • 1. Prepared by M.D.Dwivedi B.Pharm ɪvth Yr.
  • 2. Modern HPLC essentially consist of following main components:  Solvent delivery systems  Pumping systems  Sample Injector systems  HPLC Column(s)  Detector  Data System 10/16/2013 2
  • 3. Principle Pattern An Example Solvent Reservoirs Controller Solvent Cabinet Vacuum Degasser Binary Pump Auto sampler Column Compartment Detector 10/16/2013 3
  • 4.    Continuously provide eluent (solvent). Provide accurate mobile phase compositions. Includes solvent reservoirs, inlet filter, and degassing facilities which works in conjugation. 10/16/2013 4
  • 5.   10/16/2013 A good HPLC unit should have 3-4 solvent reservoirs to release eluent into a mixing chamber at varying rate. Inert container for holding the solvent (mobile phase). 5
  • 6.    10/16/2013 Type of filter. Stainless Steel or glass with 10 micron porosity. Removes particulates from solvent. 6
  • 7.   10/16/2013 Removed dissolved gases (such as oxygen and nitrogen). May consist of vacuum pump system, a distillation system, a heating and stirring device, or a system for spearing. 7
  • 8.    10/16/2013 Constant, reproducible, and pulse free supply of eluent to the HPLC column. Flow rate in between 0.1-10 cm3 min-1 . Operating pressures from 3000 psi to 6000 psi. 8
  • 9. Mainly three types 1. Constant flow reciprocating pump 2. Syringe (or displacement) type pump. 3. Pneumatic (or constant pressure) pump. 10/16/2013 9
  • 10.        10/16/2013 The term "reciprocating" describes any continuously repeated backwards and forwards motion. Widely used (~90% in HPLC system) type of pump. It gives a pulsating delivery of the eluent. Pulse damper is used to make the flow pulse free. Deliver solvent(s) through reciprocating motion of a piston in a hydraulic chamber. Solvent is sucked during back stroke and gets deliver to the column in forward stroke. Flow rates of eluent can be set by adjusting piston displacement in each stroke. 10
  • 11. Working: 10/16/2013 11
  • 12. Advantages:  Small internal volume (35-400 µL) High output pressures up to 10,000 psi.  Smart adaptability to gradient elution.  Constant flow rates independent of column back pressure, solvent viscosity and temperature.  10/16/2013 12
  • 13.    10/16/2013 Consist of large syringe like chamber (capacity up to 500 Cm2). Plunger activated by screw-driven and hydraulic amplifier machine. Suitable for small bore column. 13
  • 14. Working: 10/16/2013 14
  • 15. Advantages:  Flow is independent of viscosity, back pressure.  Deliver pulseless flow.  Provide pressure up to 78,000 psi. Disadvantages:  Costly  Low flow rate (1 to 100 mL/min).  Limited solvent capacity.  Inconvenience in frequent refilling i.e. in changing solvent 10/16/2013 15
  • 16.  Gas is used to pressurize the mobile phase present in a collapsible solvent container.  10/16/2013 16
  • 17. Working: 10/16/2013 17
  • 18. Advantages:  Not very costly.  Provide pulse free flow. Disadvantages:  Produce pressure only up to 2000 psi.  Not suitable for gradient elution.  Flow rate depends upon column back pressure, and viscosity.  Small capacity for filing of solvent. 10/16/2013 18
  • 19. Introduce required sample volume accurately into the HPLC system.  Introduction of sample without depressurizing the system.  Volume of sample must be very small (2 µL to 500 µL). Types of injection system: (a) Manual injection(Rheodyne/Valco injectors) (b) Automatic injection  10/16/2013 19
  • 20.     Also know as Rheodyne / Valco injectors. User manually loads sample into the injector using a syringe. Overloading of column causes band broadening hence volume used must be very small (2 µL to 500 µL). Sample should be introduce without depressing the system. 10/16/2013 20
  • 21. Working: 10/16/2013 21
  • 22.      Also know as Autosampler. Programmed based sample delivery system. User loads vials filled with sample solution into the autosampler tray (100 samples). Autosampler automatically 1. Measures the appropriate sample volume, 2. Injects the sample, 3. Flushes the injector to be ready for the next sample, etc., until all sample vials are processed. Also controls the sequence of samples for injection from vials. 10/16/2013 22
  • 23.       Material: Stainless steel (highly polished surface). External diameter: 6.35 mm Internal diameter: 4-5 mm (usual 4.6 mm) Length: 10-30 cm (usual 25 cm) Packing particles size (3 µm, 5µm, 10 µm) Stainless steel frits or mess discs (porosity< 2µm) retain packing material. 10/16/2013 23
  • 24.  Efficiency or performance of a column may be measured by fallowing expression : N = 16(VR/WB)2 H = L/N …(a) …(b) VR = Retention volume of the solute WB = Volume occupied by a solute ( For efficient column WB < VR ) N = Plate number of the column (dimensionless) H = Height of the column (mm × µm) L = Length of the column (cm)  For more efficient column ‘N’ should be larger and correspondingly ‘H’ gets smaller. 10/16/2013 24
  • 25. Factors affecting efficiency of column:  Particle size  Flow rate  Thickness of stationary phase  Mobile phase viscosity  Diffusion of solute in mobile and stationary phases  How well a column is packed 10/16/2013 25
  • 26. For prolonged life of HPLC columns  Guard column  Scavenger column  Column thermostats Guard column:  Also know as pre-column.  Placed in between injector and analytical column.  Having same material as in column but with larger size particles ~ 30-40 µm. 10/16/2013 26
  • 27. Scavenger column:  Place between the pump and injection valve.  Saturate the aqueous eluent (specially high or low pH buffers)with silica. Column thermostats:  HPLC is performed at ambient temperature in number of cases.  Controls temperature of the column for better resolutions (chromatograms).  HPLC is performed at ambient temperature in many cases. 10/16/2013 27
  • 28.  On the basis of chromatographic objective HPLC column can be categorized as follows: Scale Chromatographic Objectives Analytical Information ( compound identification and concentration) Semi-preparative Data and small amount of purified compound[<0.5 g] Preparative Large amount of purified compound [>0.5 g] Process (industrial) Manufacturing quantities ( g to kg) 10/16/2013 28
  • 29.     The stationary phase is the substance fixed in place for the chromatography procedure. The stationary phase can be a solid, a liquid, or a bonded phase. Bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing. Chemically-modified silicas, unmodified silica or cross-linked co-polymers of styrene and divinyl benzene, commonly used as stationary phase. 10/16/2013 29
  • 30.      Silica particles as the basis of the support. Sizes 3 µm, 5 µm, and 10 µm (spherical and regular in shape). Pore size normally are in the 60 – 100 Å range. Pore size of 300 Å or larger being used for larger biomolecules. Columns are packed using high-pressure to ensure that they are stable during use. 10/16/2013 30
  • 31.  1. 2. 3. 4. Several types of particles are used in HPLC column packing. Micro porous (or diffusive) particle/Porous microsphere Perfusion particles Nonporous (or micropellicular) Chiral (bounded) stationary phase 10/16/2013 31
  • 32. Diffusive Pore Liquid or ion exchange film 5 µm Solid core Micropellicular particles 10/16/2013 Microporus Particle Thorough pore Perfusion Particle 32
  • 33. Microporus (or diffusive) Particles:  Main surface area is within the pores to interact with the stationary phase.  Small particles reduces the diffusion path length and thereby band broadening.  Zorbax Rx (Sil) (Silica sol) is a porous microsphere silica particle with 50% porosity and a pore size of 100 Å. 10/16/2013 33
  • 34. Perfusion Particles:  The particle consist of both small (diffusive) and large (through) pores in them.  Diffusive pore provide sorption power.  Through pore permits the mobile phase to pass directly through the packing.  Slightly larger than microporous particles (~ 12 µm). 10/16/2013 34
  • 35. Nonporous particles:  Made from either silica or resin.  Smaller in size (1.5 - 2.5 µm) with thin porous layer. 10/16/2013 35
  • 36.     Also know as eluent. Solvent used in HPLC must be of HPLC grade i.e. Filtered using 0.2 μm filter. Eluting power of the mobile phase is determined by its overall polarity, stationary phase polarity and the nature of the sample components. For 'normal-phase‘ separations eluting power increases with increasing polarity of the solvent, while for 'reverse-phase' separations eluting power decreases with increasing solvent polarity. 10/16/2013 36
  • 37. Solvent Solvent strength e° parameter, (adsorption) Solvent strength parameter, p’ (partition) UV cut-off (nm) n-Hexane 0.01 0.1 195 Cyclohexane 0.04 -0.2 200 Tetrachloromethane 0.18 1.6 265 Methylbenzene 0.29 2.4 285 Trichloromethane 0.40 4.1 245 Dichloromethane 0.42 3.1 230 Tetrahydrofuran 0.56 4.0 212 Propanone 0.56 3.9 330 Acetonitrile 0.65 5.8 190 iso-Propanol 0.82 3.9 205 Ethanol 0.88 4.3 205 Methanol 0.95 5.1 205 Ethanoic acid >1 4.4 255 Water10/16/2013 >1 10.2 170 37
  • 38. The detector refers to the instrument used for qualitative and quantitative detection of analytes after separation.  Monitors the eluent as it emerges from column.  Establishing both the identity and concentration of eluting components in the mobile phase stream. Characteristics of detectors:  Adequate sensitivity (10-8 to 10-15 g solute sec-1).  Desired stability and reproducibility.  Sort response time  Minimal internal volume (minimize zone broadening).  10/16/2013 38
  • 39. Sensitivity:  Expressed as the noise equivalent concentration, i.e. the solute concentration, Cn, which produces a signal equal to the detector noise level.  The lower the value of Cn for a particular solute, the more sensitive is the detector for that solute. A linear response:  The linear range of a detector is the concentration range over which its response is directly proportional to the concentration of solute. Type of response:  Detector is either universal or selective.  Universal (sense all the constituents of the sample).  Selective (respond to certain components). 10/16/2013 39
  • 40. Types of Detectors: 1. Bulk property detectors 2. Solute property detectors Bulk property detectors:  Measure the difference in some physical property of the solute present in the mobile-phase in comparison to the individual mobile-phase.  Not suitable for gradient elution. (a) Refractive - index detector (b) Conductivity detector 10/16/2013 40
  • 41. Solute property detectors:  Respond to a particular physical or chemical characteristic of the solute which should be ideally and absolutely independent of the mobile-phase being used. (a) UV - detectors (b) Fluorescence Detectors (c) Electrochemical detectors 10/16/2013 41
  • 42. Detectors Used in HPLC: Type Response Flow rate Temperature sensitivity sensitivity Gradient elution UV/Visible absorption 10-4 Selective No Low Yes Fluorescence 10-5 Selective No Low Yes IR absorption 10-3 Selective No Low Yes Refractive index 10-2 Universal No ± 10-4 oC No Conductometric 10-2 Selective Yes ± 1 oC No Amperometric 10-5 Selective Yes ± 1 oC _ Mass spectroscopy 10-5 Universal No None Yes 10/16/2013 Limit of detection (µg/cm3) 42
  • 43. Also know as ‘RI-Detector’ and ‘Refract meter’.  Based on refractive index measurement.  Determine change of refractive index of the eluant from the column with respect to pure mobile phase. Types: (a) Deflection refractometer (b) Fresnel refractometer  Referactive index (n) = 𝑐 𝑣 c = speed of light in vacuum v = speed of light in medium 10/16/2013 43
  • 44.  The RI of a few commonly used mobile-phase is stated below : Mobile-Phase Benzene 1.501 Decane 1.410 Hexane 1.375 Octane 1.397 Tetrayhydrofuran 10/16/2013 Refractive-Index 1.405 44
  • 45. Working: 10/16/2013 45
  • 46. Advantages:  Universal response  Independent of flow rate. Disadvantages:  Less sensitivity  Temperature dependent, strict temperature control (±0.001 °C).  Not suitable for gradient elution. 10/16/2013 46
  • 47.     Conductivity measurement of effluent. Mainly measure inorganic ions and small organic substances, including organic acids and amines. Conductivity detector measures electronic resistance and measured value is directly proportional to the concentration of ions present in the solution. Employed as a detector in an ion chromatography. 10/16/2013 47
  • 48. Working: 10/16/2013 48
  • 49.    Based on the principle of absorption of UV visible light as the effluent from the column is passed through a small flow cell placed in the radiation beam. High sensitivity (detection limit of about 1x10-9 g mL-1 for highly absorbing compounds). Detector cells are generally 1 mm diameter tubes with a 10 mm optical path length. 10/16/2013 49
  • 50. Ultraviolet detector are of fallowing types: 1. Fixed-wavelength detector 2. Variable-wavelength detector 3. Photodiode-array detectors Fixed-wavelength detector:  Simplest UV absorption detector.  Mercury lamp source, optical filters to select a limited number of wavelengths 220, 250, 254, 280, 313, 334, 365, 436, and 546 nm. 10/16/2013 50
  • 51. Variable-wavelength detector:  Deuterium lamp (for UV region) or Tungsten filament light source (for visible region) a diffraction grating monochromator for wavelength selection and a photomultiplier detector.  Allow monitoring at any wavelength within the working range of the detector. 10/16/2013 51
  • 52. Photodiode-array detectors:  A photodiode array (PDA) is a linear array of discrete photodiodes on an integrated circuit (IC) chip.  A photodiode is a type of photodetector capable of converting light into either current or voltage, depending upon the mode of operation. 10/16/2013 52
  • 53. Photodiode-array detectors: 10/16/2013 53
  • 54.      Based on filter-fluorimeters or spectrofluorimeters. Flow cell has a capacity 10-25µL with a narrow depth (1.07 mm) and large surface area for excitation-emission collection. The fluorescent radiation emitted by the sample is usually measured at 90° to the incident beam. Simplest detector: mercury excitation source, and filters (one/more). Advanced detector: xenon source and a grating monochromator to isolate emitted fluorescent radiation. 10/16/2013 54
  • 55. 10/16/2013 55
  • 56. Ta rr i p gk g e c E m io s s i n Ma om n t o o c r h r o s il g n a & sm po e d c e t r a L e n s (d X cs or nE e ) n o X e n o n lM i T t P S lM S i t E S lX i t E f L l a ap s, h m Pc M T r d e t e t o 1 5 W Ln E e dM ne ) s n ( cs or o E x io tn a t i Ma om n t o o c r h, r o s il g n a & sm po ed c e t r a M D i r r o ie r fr f u s RD ee f ce e i r o e d n ao t o 8w - g µ e rn l C ei Fltc n l l oi ou , 10/16/2013 56
  • 57.     The term 'electrochemical detector' in HPLC normally refers to amperometric or coulometric detectors. Measure the current associated with the oxidation or reduction of solutes. Complete removal of oxygen is almost difficult, therefore, electrochemical detection is normally based upon the oxidation of the solute. Amperometric detector is presently considered to be the best electrochemical detector. 10/16/2013 57
  • 58. Working electrode:  Commonly made of glassy carbon, is the electrode at which the electro active solute species is monitored. Reference electrode:  Usually a Ag-AgCl electrode, gives a stable, reproducible voltage to which the potential of the working electrode is referred. Auxiliary electrode:  Current-carrying electrode and usually made of stainless steel. 10/16/2013 58
  • 59. Auxiliary Electrode Potentiostat Reference Electrode From Column Working Electrode To waste Electro Chemical Detector 10/16/2013 59
  • 60. Advantages:  Very small internal cell-volume,  High degree of sensitivity,  More limited range of applications, and  Excellent for trace analyses as UV-detector lacks adequate sensitivity. 10/16/2013 60
  • 61. Used for both qualitative and quantitative analyses of environmental, pharmaceutical, industrial, forensic, clinical, and consumer product samples. A few typical examples:  Isolation of natural pharmaceutically active compounds  Control of microbiological processes  Assay of cephalosporins  Assay of frusemide  Assay of theophylline  Assay of corticosteroids  Assay of dichlorphenamide  Assay of human insulin  10/16/2013 61
  • 62. Isolation of natural pharmaceutically active compounds Category of Natural Products Constituents Used as Alkaloids Morphine; Codeine Analgesic, Antitussive Glycoside Digitalis glycosides Sennosides Cardiovascular diseases, Laxatives Chromatographic Conditions : Column : Size-25 cm × 4.6 mm ID Adsorbent : Lichrosorb RP-8 Mobile-phase : Water/Acetonitrile-Gradient Elution Detector : UV 254 nm 10/16/2013 62
  • 63. Control of microbiological processes:  Determine kinetics of the microbiological process  Monitoring of the on-going process  Isolation and purification of active ingredients  Purity control of active constituents  Monitoring derivatization reactions 10/16/2013 63
  • 64. Controlled analysis of a microbiological process during Penicillin Production Chromatographic conditions: Column : Size-25 cm × 4.6 mm ID Adsorbent : Lichrosorb-NH2 (10 μm) Mobile-phase : 0.005 M H2SO4 buffer (pH4.4)/acetonitrile (50:50) Flow rate : 3 ml min-1 Detector : UV-220 nm  10/16/2013 64
  • 65. Assay of Cephalosporins:  Several commercially available cephalosporin antibiotics have been adequately separated by HPLC methods under the following experimental parameters Column : ODS-SIL-X-II Mobile-phase : 0.95 M Ammonium Carbonate/Methanol (95 : 5) Detector : UV-220 nm 10/16/2013 65
  • 66. Assay of Theophylline:  Theophylline contains other related substances as impurities, namely : theobromine, caffeine and βhydroxypropyltheophylline Chromatographic conditions: Sample size : 10 μL Column : size – 250 × 4.6 mm ID Adsorbent : Lichrosorb (R) RP-8, 10 μm Mobile-phase : 0.02 M KH2PO4 Buffer (pH 3.5)/Acetonitrile (95 : 5) Detector : UV-254 nm 10/16/2013 66
  • 67. Some other applications: Field Typical Mixture Pharmaceuticals Antibiotics, Sedatives, Steroids Biochemicals Amino acids, Proteins, Carbohydrates, Lipids Food Products Additives, Artificial Sweeteners, Anti - oxidents Polluants Pesticides, Herbicides, PCBs Forensic Chemistry Drugs, Poisons, 10/16/2013 67
  • 68. 1. 2. 3. 4. 5. 6. 7. 8. Kar Ashutosh “Pharmaceutical Drug Analysis”, Revised Second Edition, New Age Internal (P) Limited Publishers, page no. 452-474. Jeffery G.H., Bassett J., Mendham J. , Denney R. C., “Vogel's Textbook Of Quantitative Chemical Analysis”, Fifth edition 1989, Longman Scientific & Technical, Page no. 220-229. Patnaik Pradyot, ”Dean’s Analytical Chemistry Hand Book”, Second Edition, McGRAW-HILL, Page no. 5.60-5.91. Kealey D. , Haines P. J. , “Instant Notes Analytical Chemistry” , Frist edition 2002, BIOS Scientific Publishers Limited, Page no. 155-173. Harvey David, “Modern Analytical Chemistry”, McGraw-Hill Higher Education, Page no. 578-589. Lee David C. and Webb Michael L. , “Pharmaceutical Analysis” , Frist Published 2003, Blackwell Publishing Ltd, Page no. 44-49. Kamboj P.C. , “Pharmaceutical Analysis volume ɪɪ Instrumental Methods” First Edition 2010, Vallabh Publication , Delhi, Page no. 239-280. WWW.Google.co.in 10/16/2013 68
  • 69. “Optimism is the faith that leads to achievement, nothing can be done without hope and confidence” —- Helen Keller 10/16/2013 69

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