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HPLC Principle,Instrumentation and Application

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HPLC Chromatography and its principle

Liquid chromatography

High Performance Liquid Chromatography ( HPLC )
The components of the high performance liquid chromatograph (HPLC).
The separation process.
The chromatogram

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HPLC Principle,Instrumentation and Application

  1. 1. High Performance LiquidHigh Performance Liquid ChromatographyChromatography ALAKESH PRADHANALAKESH PRADHAN COCHIN UNIVERSITY OFCOCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGYSCIENCE AND TECHNOLOGY School of IndustrialSchool of Industrial FisheriesFisheries M.Sc IInd Sem.M.Sc IInd Sem.
  2. 2. Overview:  Chromatography and its principleChromatography and its principle  Liquid chromatographyLiquid chromatography  High Performance Liquid Chromatography ( HPLC )High Performance Liquid Chromatography ( HPLC )  The components of the high performance liquid chromatograph (HPLC).The components of the high performance liquid chromatograph (HPLC).  The separation process.The separation process.  The chromatogram.The chromatogram.
  3. 3. Latest instrument
  4. 4. BackgroundBackground Chromatography and its Principle  Chromatography is a separation technique which is used toChromatography is a separation technique which is used to separate a mixture of compounds into its individual componentsseparate a mixture of compounds into its individual components based on certain physical and chemical properties.based on certain physical and chemical properties.
  5. 5. Some important terms:Some important terms:  Mobile phase: The solvent system which carries the mixture to beMobile phase: The solvent system which carries the mixture to be separated.separated.  Stationary phase: Immobile surface which is particulate in nature. This is theStationary phase: Immobile surface which is particulate in nature. This is the region over which the compound gets separated.region over which the compound gets separated.
  6. 6. Principle:  The process involves the interaction of the compounds in the analyte (whichThe process involves the interaction of the compounds in the analyte (which travels along with a mobile phase) across an immobile surface (stationarytravels along with a mobile phase) across an immobile surface (stationary phase).phase).  The compounds bind at specific regions of stationary phase based onThe compounds bind at specific regions of stationary phase based on certain physical and chemical properties. These bound molecules are thencertain physical and chemical properties. These bound molecules are then eluted with a suitable buffer and the same are collected with time.eluted with a suitable buffer and the same are collected with time. These are –These are –  PolarityPolarity  ChargeCharge  Molecular weightMolecular weight  Present of functional groupPresent of functional group
  7. 7. IntroductionIntroduction  HPLC is a form of liquid chromatography used toHPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution.separate compounds that are dissolved in solution.  HPLC instruments consist of a reservoir of mobileHPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and aphase, a pump, an injector, a separation column, and a detector.detector.  Compounds are separated by injecting a sample mixtureCompounds are separated by injecting a sample mixture onto the column.onto the column.  The different component in the mixture pass through theThe different component in the mixture pass through the column at differentiates due to differences in theircolumn at differentiates due to differences in their partition behavior between the mobile phase and thepartition behavior between the mobile phase and the stationary phase.stationary phase.  The mobile phase must be degassed to eliminate theThe mobile phase must be degassed to eliminate the formation of air bubbles.formation of air bubbles.
  8. 8. Continued… What is Liquid Chromatography?  Liquid chromatography is a separation technique that involves:Liquid chromatography is a separation technique that involves: •• the placement (injection) of a small volume of liquid samplethe placement (injection) of a small volume of liquid sample •• into a tube packed with porous particles (stationary phase)into a tube packed with porous particles (stationary phase) •• where individual components of the sample are transported along thewhere individual components of the sample are transported along the packed tube (column) by a liquid moved by gravity.packed tube (column) by a liquid moved by gravity.  The components of the sample are separated from one another by theThe components of the sample are separated from one another by the column packing that involves various chemical and/or physical interactionscolumn packing that involves various chemical and/or physical interactions between their molecules and the packing particles.between their molecules and the packing particles.  The separated components are collected at the exit of this column andThe separated components are collected at the exit of this column and identified by an external measurement technique , such as aidentified by an external measurement technique , such as a spectrophotometer that measures the intensity of the color , or by anotherspectrophotometer that measures the intensity of the color , or by another device that can measure their amount.device that can measure their amount.  Note:Note: The modern form of liquid chromatography is now referred to asThe modern form of liquid chromatography is now referred to as ““flash chromatographyflash chromatography
  9. 9. Principles of Liquid Chromatography
  10. 10. Terminologies for HPLC  HPLC : High Performance Liquid Chromatography : High Pressure LCHPLC : High Performance Liquid Chromatography : High Pressure LC  Now, before we go in depth of principle, lets have a basic look at fewNow, before we go in depth of principle, lets have a basic look at few terms as follows:terms as follows:  Resolving Power: The extent of separation of the compounds presentResolving Power: The extent of separation of the compounds present in the mixture across the column.in the mixture across the column.  Theoretical plates : An imaginary division of the column intoTheoretical plates : An imaginary division of the column into equilength plates.equilength plates.
  11. 11. Principles of HPLC Principle:Principle:  The table shows relation between variousThe table shows relation between various parameters of HPLC.parameters of HPLC.  Trendline:Trendline:  Stationary phase have small particulate size andStationary phase have small particulate size and high surface areas.high surface areas.  Columns: 20 cm or lessColumns: 20 cm or less  Mobile phase pumped at high pressures ofMobile phase pumped at high pressures of 200Bar, 3000 psi.200Bar, 3000 psi.  Flow rates: 1-3 cmFlow rates: 1-3 cm33 per minper min Column length No. of theoretical plates per unit area Resolving power Column length Particle size Surface area
  12. 12. What is HPLC?  HPLC is a separation technique that involves:HPLC is a separation technique that involves: ••the injection of a small volume of liquid samplethe injection of a small volume of liquid sample ••into a tube packed with tiny particles (3 to 5 micron ( μm ) in diameterinto a tube packed with tiny particles (3 to 5 micron ( μm ) in diameter called thecalled the stationary phase)stationary phase) ••where individual components of the sample are moved down thewhere individual components of the sample are moved down the packed tube (packed tube (column) with a liquid (mobile phase) forced throughcolumn) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump.the column by high pressure delivered by a pump.  TheseThese components are separated from one another by the columncomponents are separated from one another by the column packing that involves various chemical and/or physical interactionspacking that involves various chemical and/or physical interactions between their molecules and the packing particles.between their molecules and the packing particles.  These separated components are detected at the exit of this tube (These separated components are detected at the exit of this tube (column)column) by a flow-through device (detector) that measures their amount. Anby a flow-through device (detector) that measures their amount. An output from this detector is called a “liquid chromatogram”.output from this detector is called a “liquid chromatogram”.   In principle, LC and HPLC work the same way except the speed ,In principle, LC and HPLC work the same way except the speed ,  efficiency, sensitivity and ease of operation of HPLC is vastlyefficiency, sensitivity and ease of operation of HPLC is vastly  superior.superior.
  13. 13. HPLC systemHPLC system Flow chart of HPLC mechanism
  14. 14. Varian 9010 Solvent Delivery System Rheodyne Injector %A %B %C Flow Rate Pressure {H2O} {MeOH} (mL/min) (atmos.) Ready Ternary Pump A C B from solvent reservoir Column to detector to column through pulse dampener to injector through pump load inject
  15. 15. Picture of HPLC instrument
  16. 16. COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEMCOMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEM SolventSolvent Solvent Delivery System (Pump)Solvent Delivery System (Pump) InjectorInjector SampleSample ColumnColumn DetectorsDetectors Waste CollectorWaste Collector Recorder (Data Collection)Recorder (Data Collection)
  17. 17. Instrumentation of HPLC ( Describing the 5 major components and their functions….) 1 2 3 4 5 Solvent reservoirs and degassing Not shown here 1 – Pump 2 – Injector 3 – Column 4 – Detector 5 – Computer
  18. 18. 1. Pump: •The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatograph at a specific flow rate, expressed in milliliters per min (mL /min). •Normal flow rates in HPLC are in the 1-to 2-mL/min range. •Typical pumps can reach pressures in the range of 6000- 9000 psi (400-to 600-bar). •During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient).
  19. 19. Pump Module–typesPump Module–types::  Isocratic pump - Delivers constant mobile phase composition;Isocratic pump - Delivers constant mobile phase composition; ••solvent must be pre-mixed;solvent must be pre-mixed; ••lowest cost pumplowest cost pump  Gradient pump - Delivers variable mobile phase composition;Gradient pump - Delivers variable mobile phase composition; ••can be used to mix and deliver an isocratic mobile phase or acan be used to mix and deliver an isocratic mobile phase or a gradient mobile phasegradient mobile phase ––Binary gradient pumpBinary gradient pump –delivers two solvents–delivers two solvents ––Quaternary gradient pumpQuaternary gradient pump –four solvents–four solvents
  20. 20. 2. Injector: •The injector serves to introduce the liquid sample into the flow stream of the mobile phase. •Typical sample volumes are 5-to 20-microliters (μL). •The injector must also be able to withstand the high pressures of the liquid system. •An auto sampler is the automatic version for when the user has many samples to analyze or when manual injection is not practical .
  21. 21. Sample Injection ……how is a sample actually put into an LC system Manual InjectorManual Injector:: 1.User manually loads sample into the injector using a syringe1.User manually loads sample into the injector using a syringe 2.and then turns the handle to inject sample into the flowing mobile2.and then turns the handle to inject sample into the flowing mobile phase… which transports the sample into the beginning (head) of thephase… which transports the sample into the beginning (head) of the column, which is at high pressurecolumn, which is at high pressure Auto samplerAuto sampler:: 1.User loads vials filled with sample solution into the auto sampler tray1.User loads vials filled with sample solution into the auto sampler tray (100 samples)(100 samples) 2.and the auto sampler automatically2.and the auto sampler automatically a. measures the appropriate sample volume,a. measures the appropriate sample volume, b. injects the sample,b. injects the sample, c. then flushes the injector to be ready for the next sample,c. then flushes the injector to be ready for the next sample, etc., until all sample vials are processed ……etc., until all sample vials are processed …… ………….for unattended automatic operation.for unattended automatic operation
  22. 22. 2323 Manual InjectorsManual Injectors Front View Inject Rear View Load - Inject Sample Loop
  23. 23. 2424 Automatic InjectorsAutomatic Injectors Step 1 Step 2 Step 3
  24. 24. 3.3. Column:Column: •• Considered the “heart of the chromatograph” the column’s stationaryConsidered the “heart of the chromatograph” the column’s stationary phase separates the sample components of interest using various physicalphase separates the sample components of interest using various physical and chemical parameters.and chemical parameters. ••The small particles inside the column are what cause the highThe small particles inside the column are what cause the high back pressure at normal flow rates.back pressure at normal flow rates. ••The pump must push hard to move the mobile phase through theThe pump must push hard to move the mobile phase through the column and this resistance causes a high pressure within thecolumn and this resistance causes a high pressure within the chromatograph.chromatograph.
  25. 25. Several Column Types ( can be classified as)  Normal phaseNormal phase  Reverse phaseReverse phase  Size exclusionSize exclusion  Ion exchangeIon exchange
  26. 26. Normal phase  In this column type, the retention is governed byIn this column type, the retention is governed by the interaction of the polar parts of the stationarythe interaction of the polar parts of the stationary phase and solute.phase and solute.  For retention to occur in normal phase, theFor retention to occur in normal phase, the packing must be more polar than the mobilepacking must be more polar than the mobile phase with respect to the samplephase with respect to the sample
  27. 27. 28 HO Si O O STATIONARY PHASES (NORMAL POLARITY) Silica or alumina possess polar sites that interact with polar molecules. Most polar…….Least polar Components elute in increasing order of polarity. Components elute in increasing order of polarity. Polar Group silica
  28. 28. Reverse phase  In this column the packing material is relatively nonpolar and the solvent isIn this column the packing material is relatively nonpolar and the solvent is polar with respect to the sample. Retention is the result of the interaction ofpolar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase.the nonpolar components of the solutes and the nonpolar stationary phase.  Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bondedTypical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous- organic mixtures such as methanol-water or acetonitrile-water.organic mixtures such as methanol-water or acetonitrile-water. Common Reverse Phase SolventsCommon Reverse Phase Solvents –– Methanol Acetonitrile Tetrahydrofuran Water CH3OH CH3CN H2O
  29. 29. 30 STATIONARY PHASES (REVERSE POLARITY) If the polar sites on silica or alumina are capped with non-polar groups, they interact strongly with non-polar molecules. Most non-polar…….Least non-polar Components elute in decreasing order of polarity. Components elute in decreasing order of polarity. C18 phase silica Si Me Me O Si O O
  30. 30. Size exclusion  In size exclusion the HPLC column is consisted ofIn size exclusion the HPLC column is consisted of substances which have controlled pore sizes andsubstances which have controlled pore sizes and is able to be filtered in an ordinarily phaseis able to be filtered in an ordinarily phase according to its molecular size.according to its molecular size.  Small molecules penetrate into the pores withinSmall molecules penetrate into the pores within the packing while larger molecules only partiallythe packing while larger molecules only partially penetrate the pores. The large molecules elutepenetrate the pores. The large molecules elute before the smaller moleculesbefore the smaller molecules..
  31. 31. 32 STATIONARY PHASES (SIZE EXCLUSION) Size exclusion gels separate on the basis of molecular size. Individual gel beads have pores of set size, that restrict entry to molecules of a minium size. Larger molecules…….Smaller molecules Large molecules elute fast (restricted path), while small molecules elute slowly (long path length) Large molecules elute fast (restricted path), while small molecules elute slowly (long path length)
  32. 32. Ion exchange  In this column type the sample componentsIn this column type the sample components are separated based upon attractive ionicare separated based upon attractive ionic forces between molecules carrying chargedforces between molecules carrying charged groups of opposite charge to those chargesgroups of opposite charge to those charges on the stationary phase.on the stationary phase.  Separations are made between a polar mobileSeparations are made between a polar mobile liquid, usually water containing salts or smallliquid, usually water containing salts or small amounts of alcohols, and a stationary phaseamounts of alcohols, and a stationary phase containing either acidic or basic fixed sites.containing either acidic or basic fixed sites.
  33. 33. 34 STATIONARY PHASES (CATION EXCHANGE) Silica is substituted with anionic residues that interact strongly with cationic species (+ve charged) Most +ve…….Least +ve +ve charged species adhere to the support and are later eluted with acid (H+ ) +ve charged species adhere to the support and are later eluted with acid (H+ ) Cations exchange Na+ silica S O O ONa
  34. 34. 35 STATIONARY PHASES (ANION EXCHANGE) Silica is substituted with cationic residues that interact strongly with anionic species (-ve charged) Most -ve…….Least -ve -ve charged species adhere to the support and are later eluted with acid (H+ ) -ve charged species adhere to the support and are later eluted with acid (H+ ) Anions exchange Cl- silica Me N Me Me CH2 Cl
  35. 35. HPLC Columns Within the Column is where separation occurs.Within the Column is where separation occurs. Key Point –Proper choice of column is critical for success in HPLCKey Point –Proper choice of column is critical for success in HPLC Materials of construction for the tubingMaterials of construction for the tubing  Stainless steel (the most popular; gives high pressure capabilities)Stainless steel (the most popular; gives high pressure capabilities)  Glass (mostly for biomolecules)Glass (mostly for biomolecules)  PEEK polymer (biocompatible and chemically inert to most solventsPEEK polymer (biocompatible and chemically inert to most solvents Packing material:Packing material: TheThe packing material is prepared from SILICA particle, ALUMINA particlepacking material is prepared from SILICA particle, ALUMINA particle and ion exchange RESIN.and ion exchange RESIN. Porous plug of stainless steel or Teflon are used in the end of the columnsPorous plug of stainless steel or Teflon are used in the end of the columns to retain the packing material.to retain the packing material. According to the mode of HPLC , they are available in different size ,According to the mode of HPLC , they are available in different size , diameters, pore size or they can have special materials attached ( such asdiameters, pore size or they can have special materials attached ( such as antigen or antibody ) for immuno affinity chromatography.antigen or antibody ) for immuno affinity chromatography.
  36. 36. 3737 Modes of High Performance LiquidModes of High Performance Liquid ChromatographyChromatography Types of Compounds Mode Stationary Phase Mobile Phase Neutrals Weak Acids Weak Bases Reversed Phase C18, C8, C4 cyano, amino Water/Organic Modifiers Ionics, Bases, Acids Ion Pair C-18, C-8 Water/Organic Ion-Pair Reagent Compounds not soluble in water Normal Phase Silica, Amino, Cyano, Diol Organics Ionics Inorganic Ions Ion Exchange Anion or Cation Exchange Resin Aqueous/Buffer Counter Ion High Molecular Weight Compounds Polymers Size Exclusion Polystyrene Silica Gel Filtration- Aqueous Gel Permeation- Organic
  37. 37. Types of columns in HPLCTypes of columns in HPLC::  Guard ColumnGuard Column  Fast ColumnFast Column  Preparative(i.d. > 4.6 mm; lengths 50 –250 mm)Preparative(i.d. > 4.6 mm; lengths 50 –250 mm)  Capillary(i.d. 0.1 -1.0 mm; various lengths)Capillary(i.d. 0.1 -1.0 mm; various lengths)  Nano(i.d. < 0.1 mm, or sometimes stated as < 100 μm)Nano(i.d. < 0.1 mm, or sometimes stated as < 100 μm)  Analytical[internal diameter (i.d.) 1.0 -4.6-mm; lengths 15 –250 mm]Analytical[internal diameter (i.d.) 1.0 -4.6-mm; lengths 15 –250 mm]
  38. 38. Guard Column  These are placed anterior to the separating column. This serves asThese are placed anterior to the separating column. This serves as protective factor.protective factor.  They are dependable columns designed to filter or remove :They are dependable columns designed to filter or remove : Particles that clog the separation columnParticles that clog the separation column Compounds and ions that could ultimately cause “ Baseline drift ”,Compounds and ions that could ultimately cause “ Baseline drift ”, decreased resolution, decreased sensitivity and create false peaks.decreased resolution, decreased sensitivity and create false peaks. These columns must be changed on a regular basis in order to optimizeThese columns must be changed on a regular basis in order to optimize their protective function.their protective function.
  39. 39. Fast Column  One of the primary reasons for using these column is to obtain improvedOne of the primary reasons for using these column is to obtain improved sample output ( amount of compound per unit time).sample output ( amount of compound per unit time).  Fast column are designed to decrease the time of chromatographic analysisFast column are designed to decrease the time of chromatographic analysis  Here internal diameter is same but length is short and packed with smallerHere internal diameter is same but length is short and packed with smaller particles , that are 3particles , that are 3 μm diameter.μm diameter.  Advantages-Advantages- Increased sensitivityIncreased sensitivity Decreased analysis timeDecreased analysis time Decreased mobile phase usageDecreased mobile phase usage Increase reproducibilityIncrease reproducibility
  40. 40. Capillary Column  It is also known as micro columnsIt is also known as micro columns  It has a diameter much less than a millimeter and there 3 types:It has a diameter much less than a millimeter and there 3 types: Open tubularOpen tubular Partially packedPartially packed Tightly packedTightly packed They allow the user to work with nanoliter sample volume , decreasedThey allow the user to work with nanoliter sample volume , decreased flow rate and decreased solvent usage volume , led to cost effectivenessflow rate and decreased solvent usage volume , led to cost effectiveness
  41. 41. Preparatory Column  Used when objective is to prepare bulk ( milligrams) of sample forUsed when objective is to prepare bulk ( milligrams) of sample for laboratory preparatory application.laboratory preparatory application.  It has usually a large column diameter , which is designed to facilitateIt has usually a large column diameter , which is designed to facilitate large volume injections into the HPLC systemlarge volume injections into the HPLC system
  42. 42. 4.4. DetectorDetector:: •• The detector can see (detect) the individual molecules that comeThe detector can see (detect) the individual molecules that come out (elute) from the column.out (elute) from the column. ••A detector serves to measure the amount of those moleculesA detector serves to measure the amount of those molecules so that the chemist can quantitatively analyze the sampleso that the chemist can quantitatively analyze the sample components.components. ••The detector provides an output to a recorder or computerThe detector provides an output to a recorder or computer that results in the liquid chromatogram(i.e., the graph of thethat results in the liquid chromatogram(i.e., the graph of the detector response).detector response).
  43. 43. HPLC Detectors
  44. 44. 4545 Common HPLC Detectors •UV-VIS •Diode Array •Multiple Wavelength •Variable Wavelength •Mass Spectrometers •Refractive Index •Fluorescence •Light Scattering •Electrochemical •Radioactivity •Conductivity
  45. 45. 46 UV-Vis Detectors b c Detector Flow Cell I0 I Log I0 = A = abc I Principles: The fraction of light transmitted through the detector cell is related to the solute concentration according to Beer’s Law. Characteristics: Specific, Concentration Sensitive, good stability, gradient capability. Special: UV-Vis Spectral capability (Diode Array Technology ).
  46. 46. 47 Fluorescence Detection Emission Monochromator signal & spectra mode PMT detector Reference Diode 8 µl Flow Cell, auto-recognition Trigger pack Exitation Monochromator, signal & spectra mode Mirror Lens (condensor EX) Lens (condensor EM) Slit EM Slit PMT Slit EX Diffuser Xenon flash Lamp, 15 W
  47. 47. 48 Electrochemical Detectors • Gold for carbohydrates. • Platinum for chlorite, sulfate, hydrazine, etc. • Carbon for phenols, amines. • Silver for chloride, bromide, cyanide.
  48. 48. Variable UV/Vis DetectorVariable UV/Vis Detector ABS AUFS λ RunTime EndTime 0.001 2.000 238 0.00 min 10.0 min Ready
  49. 49. 5.5. ComputerComputer:: •• Frequently called the data system,Frequently called the data system, The computer not only controls all the modules of theThe computer not only controls all the modules of the HPLC instrument but it takes the signal from theHPLC instrument but it takes the signal from the detector and uses it to:detector and uses it to: 1. determine the time of elution (retention time) of the1. determine the time of elution (retention time) of the sample components (qualitative analysis) andsample components (qualitative analysis) and 2. the amount of sample ( quantitative analysis) .2. the amount of sample ( quantitative analysis) .
  50. 50. Varian 9060Varian 9060 PolychromPolychrom DetectorDetector UV Spectrum Chromatogram Reset Ready UV Spectrum {shows full UV abs Chromatogram {shows peaks, Rt} ABS. Time ABS. Wavelength UVmax UVmax Rt Rt
  51. 51. HPLC is optimum for the separation of chemical and biologicalHPLC is optimum for the separation of chemical and biological compounds that are non-volatile .compounds that are non-volatile . NOTE: If a compound is volatile (i.e. a gas, fragrance, hydrocarbon inNOTE: If a compound is volatile (i.e. a gas, fragrance, hydrocarbon in gasoline, etc.), gas chromatography is a better separation technique .gasoline, etc.), gas chromatography is a better separation technique . Typical non-volatile compounds are:Typical non-volatile compounds are:  Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol)Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol)  Salts like sodium chloride and potassium phosphateSalts like sodium chloride and potassium phosphate  Proteins like egg white or blood proteinProteins like egg white or blood protein  Organic chemicals like polymers (e.g. polystyrene, polyethylene)Organic chemicals like polymers (e.g. polystyrene, polyethylene)  Heavy hydrocarbons like asphalt or motor oilHeavy hydrocarbons like asphalt or motor oil  Many natural products such as ginseng, herbal medicines, plant extractsMany natural products such as ginseng, herbal medicines, plant extracts  Thermally unstable compounds such as trinitrotoluene (TNT), enzymesThermally unstable compounds such as trinitrotoluene (TNT), enzymes What is HPLC used for ? Separation and analysis of non-volatile or thermally unstable compounds
  52. 52. Separation Technique
  53. 53. 54 How can We Analyze the Sample? For example: Carbohydrates 1. fructose 2. Glucose 3. Saccharose 4. Palatinose 5. Trehalulose 6. isomaltose 1 2 3 4 5 mAU time 6
  54. 54. 55 Separations Separation in based upon differential migration between the stationary and mobile phases. Stationary Phase - the phase which remains fixed in the column, e.g. C18, Silica Mobile Phase - carries the sample through the stationary phase as it moves through the column. Injector Detector Column Solvents Mixer Pumps High Performance Liquid Chromatograph Waste
  55. 55. 56 SeparationsInjector Detector Column Solvents Mixer Pumps Chromatogram Start Injection mAU time High Performance Liquid Chromatograph
  56. 56. 57 SeparationsInjector Detector Column Solvents Mixer Pumps Chromatogram Start Injection mAU time
  57. 57. 58 SeparationsInjector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time
  58. 58. 59 SeparationsInjector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time
  59. 59. 60 SeparationsInjector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time
  60. 60. 61 SeparationsInjector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time
  61. 61. 62 SeparationsInjector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time
  62. 62. 63 SeparationsInjector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time
  63. 63. 64 SeparationsInjector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time
  64. 64. 65 SeparationsInjector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time
  65. 65. 66 SeparationsInjector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time
  66. 66. 67 SeparationsInjector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time
  67. 67. 68 SeparationsInjector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time
  68. 68. 69 SeparationsInjector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time
  69. 69. 70 SeparationsInjector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time
  70. 70. 71 SeparationsInjector Detector Column Solvents Pumps Mixer Chromatogram Start Injection mAU time
  71. 71. 72 The Chromatogram Injection to tR mAU time tR to - elution time of unretained peak tR- retention time - determines sample identity Area or height is proportional to the quantity of analyte.
  72. 72. HPLC used for Qualitative Analysis
  73. 73. HPLC used for Quantitative Analysis
  74. 74. HPLC uses This technique is used for -This technique is used for - chemistry and biochemistry research analyzingchemistry and biochemistry research analyzing complex mixturescomplex mixtures purifying chemical compoundspurifying chemical compounds developing processes for synthesizing chemicaldeveloping processes for synthesizing chemical compoundscompounds isolating natural products, or predicting physicalisolating natural products, or predicting physical properties.properties. It is also used in quality control to ensure the purity ofIt is also used in quality control to ensure the purity of raw materials, to control and improve process yields, toraw materials, to control and improve process yields, to quantify assays of final products, or to evaluate productquantify assays of final products, or to evaluate product stability and monitor degradation.stability and monitor degradation.
  75. 75. In addition, it is used for analyzing air and water pollutants, for monitoring materials that may jeopardize occupational safety or health, and for monitoring pesticide levels in the environment.
  76. 76. 77 HPLC Applications Chemical Environmental Pharmaceuticals Consumer Products Clinical polystyrenes dyes phthalates tetracyclines corticosteroids antidepressants barbiturates amino acids vitamins homocysteine Bioscience proteins peptides nucleotides lipids antioxidants sugars polyaromatic hydrocarbons Inorganic ions herbicides
  77. 77. References  HPLC instrumentation – Agilent TechnologiesHPLC instrumentation – Agilent Technologies  Introduction to HPLC – Agilent TechnologiesIntroduction to HPLC – Agilent Technologies  Principles and Technique of Biochemistry and Moleculer Biology –Principles and Technique of Biochemistry and Moleculer Biology – Wilson.Keith and Walker. JohnWilson.Keith and Walker. John  HPLC THEORY INTRODUCTION AND INSTRUMENTATION HARDWAREHPLC THEORY INTRODUCTION AND INSTRUMENTATION HARDWARE 6th October 2008. L1 - Dr Cristina Legido-Quigley, Lecturer in Pharmaceutical6th October 2008. L1 - Dr Cristina Legido-Quigley, Lecturer in Pharmaceutical Chemistry (Separation Science) at KCLChemistry (Separation Science) at KCL WEB REFERENCESWEB REFERENCES  http://192.215.107.101/ebn/942/tech/techfocus/1071main.htmlhttp://192.215.107.101/ebn/942/tech/techfocus/1071main.html  Skoog, Holler, and Neiman.Skoog, Holler, and Neiman. Principles of Instrumental AnalysisPrinciples of Instrumental Analysis. 5th ed.. 5th ed. Orlando: Harcourt Brace & Co., 1998.Orlando: Harcourt Brace & Co., 1998.  http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htmhttp://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm  http://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.htmlhttp://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.html  http://weather.nmsu.edu/Teaching_Material/SOIL698/Student_Material/HPLChttp://weather.nmsu.edu/Teaching_Material/SOIL698/Student_Material/HPLC HP1090/HPLCINJ.HTMHP1090/HPLCINJ.HTM  http://test-http://test- equipment.globalspec.com/LearnMore/Labware_Scientific_Instruments/Analytiequipment.globalspec.com/LearnMore/Labware_Scientific_Instruments/Analyti cal_Instruments/Chromatographs/HPLC_Columnscal_Instruments/Chromatographs/HPLC_Columns
  78. 78. • THANK YOU…

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