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Instrumentation: Instrumentation Modern HPLC essentially consist of following main components: Solvent delivery systems Pumping systems Sample Injector systems HPLC Column(s ) Detector Data System 9/22/2013 2 HPLC Instrumentation Overview: HPLC Instrumentation Overview Principle Pattern An Example Solvent Reservoirs Controller Solvent Cabinet Vacuum Degasser Binary Pump Auto sampler Column Compartment Detector 9/22/2013 3 Solvent delivery systems: Solvent delivery systems Continuously provide eluent (solvent). Provide accurate mobile phase compositions . Includes solvent reservoirs , inlet filter , and degassing facilities which works in conjugation. 9/22/2013 4 Solvent Reservoirs: Solvent Reservoirs 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). 9/22/2013 5 Inlet Filters: Inlet Filters Type of filter. Stainless Steel or glass with 10 micron porosity . Removes particulates from solvent . 9/22/2013 6 Degassing System: Degassing System 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. 9/22/2013 7 Pumping System: Pumping System C onstant, reproducible, and pulse free supply of eluent to the HPLC column. Flow rate in between 0.1-10 cm 3 min -1 . Operating pressures from 3000 psi to 6000 psi. 9/22/2013 8 Types of Pumping System: Types of Pumping System Mainly three types Constant flow reciprocating pump Syringe (or displacement) type pump. Pneumatic (or constant pressure) pump. 9/22/2013 9 Constant flow reciprocating pump: Constant flow reciprocating pump 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. 9/22/2013 10 Reciprocating pump: Reciprocating pump Working: 9/22/2013 11 Reciprocating Pump Continuous… : Reciprocating Pump Continuous… 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. 9/22/2013 12 Syringe (or displacement) type pump : Syringe (or displacement) type pump Consist of large syringe like chamber (capacity up to 500 Cm 2 ). Plunger activated by screw-driven and hydraulic amplifier machine. Suitable for small bore column. 9/22/2013 13 Displacement Pump Continuous…: Displacement Pump Continuous … Working: 9/22/2013 14 Displacement Pump Continuous… : Displacement Pump Continuous… 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 9/22/2013 15 Pneumatic Pump: Pneumatic Pump Gas is used to pressurize the mobile phase present in a collapsible solvent container. 9/22/2013 16 Pneumatic Pump Continuous…: Pneumatic Pump Continuous… Working: 9/22/2013 17 Pneumatic Pump Continuous…: Pneumatic Pump Continuous… 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. 9/22/2013 18 Sample Injection system: Sample Injection system I ntroduce 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: Manual injection( Rheodyne / Valco injectors ) Automatic injection 9/22/2013 19 Manual Injector: Manual Injector 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. 9/22/2013 20 Manual Injector : Manual Injector Working: 9/22/2013 21 Automatic Injector System : Automatic Injector System Also know as A utosampler . 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. F lushes 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. 9/22/2013 22 HPLC Columns: HPLC Columns 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< 2 µm ) retain packing material. 9/22/2013 23 HPLC Columns Continuous: HPLC Columns Continuous Efficiency or performance of a column may be measured by fallowing expression : N = 16(V R /W B ) 2 …(a) H = L/N …(b) V R = Retention volume of the solute W B = Volume occupied by a solute ( For efficient column W B < V R ) 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. 9/22/2013 24 HPLC Columns Continuous: HPLC Columns Continuous 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 9/22/2013 25 HPLC Columns Continuous…: HPLC Columns Continuous… 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. 9/22/2013 26 HPLC Columns Continuous…: HPLC Columns Continuous … 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. 9/22/2013 27 HPLC Columns Continuous…: HPLC Columns Continuous… On the basis of chromatographic objective HPLC column can be categorized as follows: 9/22/2013 28 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) Stationary Phase (column packing): Stationary Phase (column packing) 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. B onded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing . C hemically-modified silicas , unmodified silica or cross-linked co-polymers of styrene and divinyl benzene, commonly used as stationary phase. 9/22/2013 29 Stationary Phase Continuous…: Stationary Phase Continuous… S ilica 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. 9/22/2013 30 Stationary Phase Continuous…: Stationary Phase Continuous… 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 9/22/2013 31 Stationary Phase Continuous…: Stationary Phase Continuous… 9/22/2013 32 Thorough pore Diffusive Pore Perfusion Particle 5 µm Microporus Particle Micropellicular particles Solid core Liquid or ion exchange film Stationary Phase Continuous…: Stationary Phase Continuous… 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. 9/22/2013 33 Stationary Phase Continuous…: Stationary Phase Continuous… 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). 9/22/2013 34 Stationary Phase Continuous…: Stationary Phase Continuous… Nonporous particles: Made from either silica or resin. Smaller in size (1.5 - 2.5 µm) with thin porous layer. 9/22/2013 35 Mobile Phase: Mobile Phase Also know as eluent. Solvent used in HPLC must be of HPLC grade i.e. Filtered using 0.2 μ m 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 . 9/22/2013 36 PowerPoint Presentation: 9/22/2013 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 Water >1 10.2 170 Detectors: Detectors The detector refers to the instrument used for qualitative and quantitative detection of analytes after separation . Monitors the eluent as it emerges from column. E stablishing 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). 9/22/2013 38 Detectors Continuous: Detectors Continuous Sensitivity: E xpressed as the noise equivalent concentration , i.e . the solute concentration , C n , which produces a signal equal to the detector noise level. The lower the value of C n 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). 9/22/2013 39 Detectors continuous…: Detectors continuous… Types of Detectors: Bulk property detectors Solute property detectors Bulk property detectors: M easure 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 9/22/2013 40 Detectors continuous…: Detectors continuous… Solute property detectors: R espond 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 9/22/2013 41 Detectors continuous…: Detectors continuous… Detectors U sed in HPLC: Type Limit of detection ( µg/cm 3) Response Flow rate sensitivity Temperature 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 o C No Conductometric 10 -2 Selective Yes ± 1 o C No Amperometric 10 -5 Selective Yes ± 1 o C _ Mass spectroscopy 10 -5 Universal No None Yes 9/22/2013 42 Refractive - index detector: Refractive - index detector 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 = speed of light in vacuum v = speed of light in medium 9/22/2013 43 Refractive - index detector continuous…: Refractive - index detector continuous… The RI of a few commonly used mobile-phase is stated below : 9/22/2013 44 Mobile-Phase Refractive-Index Benzene 1.501 Decane 1.410 Hexane 1.375 Octane 1.397 Tetrayhydrofuran 1.405 Refractive - index detector continuous…: Refractive - index detector continuous… Working: 9/22/2013 45 Refractive - index detector continuous…: Refractive - index detector continuous… Advantages: Universal response Independent of flow rate. Disadvantages: Less sensitivity Temperature dependent, strict temperature control (±0.001 °C ). Not suitable for gradient elution. 9/22/2013 46 Conductivity detectors: Conductivity detectors Conductivity measurement of effluent. M ainly 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. 9/22/2013 47 Conductivity detectors continuous…: Conductivity detectors continuous… Working: 9/22/2013 48 Ultraviolet Detector: Ultraviolet Detector B ased 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 . H igh 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. 9/22/2013 49 Ultraviolet Detector Continuous…: Ultraviolet Detector Continuous… Ultraviolet detector are of fallowing types: F ixed-wavelength detector V ariable-wavelength detector 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. 9/22/2013 50 Ultraviolet Detector Continuous…: Ultraviolet Detector Continuous… 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. A llow monitoring at any wavelength within the working range of the detector. 9/22/2013 51 Ultraviolet Detector Continuous…: Ultraviolet Detector Continuous… 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. 9/22/2013 52 Ultraviolet Detector Continuous…: Ultraviolet Detector Continuous… Photodiode-array detectors: 9/22/2013 53 Fluorescence Detectors: Fluorescence Detectors B ased 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 a nd a grating monochromator to isolate emitted fluorescent radiation. 9/22/2013 54 Fluorescence Detector Continuous…: Fluorescence Detector Continuous… 9/22/2013 55 Fluorescence Detector Continuous…: Fluorescence Detector Continuous… 9/22/2013 56 Electrochemical Detector: Electrochemical Detector The term 'electrochemical detector' in HPLC normally refers to amperometric or coulometric detectors. Measure the current associated with the oxidation or reduction of solutes. C omplete removal of oxygen is almost difficult, therefore , electrochemical detection is normally based upon the oxidation of the solute. A mperometric detector is presently considered to be the best electrochemical detector. 9/22/2013 57 Electrochemical Detector Continuous…: Electrochemical Detector Continuous… W orking 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. A uxiliary electrode: C urrent-carrying electrode and usually made of stainless steel. 9/22/2013 58 Electrochemical Detector Continuous…: Electrochemical Detector Continuous… 9/22/2013 59 Potentiostat From Column To waste Auxiliary Electrode Reference Electrode Working Electrode Electro Chemical Detector Electrochemical Detector Continuous…: Electrochemical Detector Continuous… Advantages: V ery small internal cell-volume, H igh degree of sensitivity, M ore limited range of applications, and E xcellent for trace analyses as UV-detector lacks adequate sensitivity . 9/22/2013 60 Applications of HPLC: Applications of HPLC Used for both qualitative and quantitative analyses of environmental, pharmaceutical , industrial, forensic, clinical, and consumer product samples . A few typical examples: I solation of natural pharmaceutically active compounds C ontrol of microbiological processes A ssay of cephalosporins A ssay of frusemide A ssay of theophylline A ssay of corticosteroids A ssay of dichlorphenamide A ssay of human insulin 9/22/2013 61 Applications of HPLC Continuous…: Applications of HPLC Continuous… Isolation of natural pharmaceutically active compounds Chromatographic Conditions : Column : Size-25 cm × 4.6 mm ID Adsorbent : Lichrosorb RP-8 Mobile-phase : Water/Acetonitrile-Gradient Elution Detector : UV 254 nm Category of Natural Products Constituents Used as Alkaloids Morphine; Codeine Analgesic, Antitussive Glycoside Digitalis glycosides Sennosides Cardiovascular diseases, Laxatives 9/22/2013 62 Applications of HPLC Continuous…: Applications of HPLC Continuous… Control of microbiological processes: Determine kinetics of the microbiological process M onitoring of the on-going process I solation and purification of active ingredients P urity control of active constituents M onitoring derivatization reactions 9/22/2013 63 Applications of HPLC Continuous…: Applications of HPLC Continuous… C ontrolled analysis of a microbiological process during Penicillin Production Chromatographic conditions: Column : Size-25 cm × 4.6 mm ID Adsorbent : Lichrosorb-NH 2 ( 10 μ m) Mobile-phase : 0.005 M H 2 SO 4 buffer (pH4.4)/acetonitrile (50:50) Flow rate : 3 ml min -1 Detector : UV-220 nm 9/22/2013 64 Applications of HPLC Continuous…: Applications of HPLC Continuous… A ssay 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 9/22/2013 65 Applications of HPLC Continuous…: Applications of HPLC Continuous… A ssay of Theophylline: Theophylline contains other related substances as impurities, namely : theobromine , caffeine and β- hydroxypropyltheophylline Chromatographic conditions: Sample size : 10 μ L Column : size – 250 × 4.6 mm ID Adsorbent : Lichrosorb (R) RP-8, 10 μ m Mobile-phase : 0.02 M KH 2 PO 4 Buffer ( pH 3.5)/Acetonitrile (95 : 5) Detector : UV-254 nm 9/22/2013 66 Applications of HPLC Continuous…: Applications of HPLC Continuous… 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, Some other applications: 9/22/2013 67 References: References Kar Ashutosh “Pharmaceutical Drug Analysis” , Revised Second E dition, New Age I nternal (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. 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