Hplc presentation for class


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  • It is common practice to make up these organic solvents as mixtures with water or, in a lot of cases, have each pure solvent mixed under instrument control and changed at a certain rate with time (gradient). Gradients can be simple or complex. Simple - as a linear gradient (ramp); Complex as steps (start and hold) and ramps together. You can have a solvent or several solvents being controlled at the same time with a changing modifier such as a pH buffer. Methanol - Most common solvent. Close to water in structure. Miscible in all proportions with H 2 O so that for less polar organics you can have the power of 90% Methanol with 10% H 2 O. Acetonitrile - Highly polar, very low UV absorbance. Also completely miscible with H 2 O but lacking in hydrogen bonding capability thus affording a different partitioning effect. Tetrahydrofuran - Molecule has high dipole moment. More soluble with non-polar compounds. Water - Also a very common solvent. Used to make up solvent modifiers to adjust pH (buffers) as well as ion-pairing reagents. Emphasize degassing (“bends”- bubble in detector) and for particle-free (dust could be up to 10X size of particles of solid support). 555- 25mM H 3 PO 4 :MeCN(90:10)->10:90 605 - 0.1M pH4.7 OAc: MeCN (1:1) isocratic. 610 - MeCN:H 2 O -> MeCN 8316 - 100% H 2 O 8330 - MeOH:H 2 O isocratic 8331 - MeOH:H 2 O(HOAc-C 10 SO 3 H) 8332- MeOH:H 2 O (3:2-CN), (1:1-C-18) 8325-A=MeCN:0.01M OAc (75:25), B= MeCN -> 60%MeCN total. λ= 190 for MeCN, λ= 205 for MeOH, λ= 190 for H 2 O, λ~ 290 for THF, λ~ 255 for 1% HOAc and λ~ 260 NH 4 OAc (1M)
  • Here you can see the structure of the different packing materials of Merck. Starting from the irregular particles (e.g. LiChrosorb) there came the invention of spherical particles, used for the LiChrospher, Superspher, Purospher and Purospher STAR sorbents) and especially the latest innovation by Merck, the monolithic column technology in terms of the Chromolith column. On the different pictures you can compare the macropores (through-pores) and the mesopores of the different variations of these packing materials.
  • Hplc presentation for class

    1. 1. HPLCProf. P.RavisankarVignan Pharmacy collegeValdlamudiGuntur Dist.Andhra PradeshIndia.banuman35@gmail.com00919059994000
    2. 2. HPighressureL iquidC hromatographySince high pressure is used when compared to classical chromatographySeparation of the components from a mixture is achieved by pumpingmobile phase at High pressure using appropriate displacement pumpsor gas pressure. (Due to the small particle size (3-5 um).
    3. 3. HPigherformanceL iquidChromatographyMore ever it is improved performance when compared to otherconventional column Chromatographic techniques.
    4. 4. HPighricedL iquidC hromatography
    5. 5. • Michael Tswett (1906) -separation of plant pigments• Martin and Synge (1941) liquid-liquid partition chromatography• 1952 Nobel Prize• Other chromatography Techniques• Thin-layer chromatography (TLC)• Paper chromatography• Preparative column chromatography• Medium pressure chromatography• Gas chromatography• Ion-exchange chromatography• Size-exclusion chromatography (Gel Permeation Chromatography)• HPLC• VLC• Flash Chromatography• Affinity Chromatography• Chiral Chromatography• Super Critical Fluid Chromatography5
    6. 6. What means chromatography ?AABCACABBCC CBCBACBABCCCC CCCBBBBBBAAAAAASample separated componentsChromatography is the chemical / physical separation of components for qualitative andquantitative analysisPrinciplePrinciple6
    7. 7. Different chromatography methods7Name Mechanism Stat. Phase Mobile phasePaper- partition liquid liquidThin layer- adsorption solid liquidGas- adsorption /partitionsolid or liquid gasColumn- adsorption /partitionSolid/liquid liquid
    8. 8. Liquid Chromatography (LC)• Two different phases are used to separatecomponents of the mixture: stationary andmobile phase• The solute separates on the column viainteractions based on physical mechanisms likeretention8
    9. 9. Partitioning• Separation is based on the analyte’srelative solubility between two liquidphases9Stationary PhaseMobile PhaseSolvent Bonded Phase
    10. 10. 10
    11. 11. What is HPLC?• The most widely used analytical separations technique• Utilizes a liquid mobile phase & packed column toseparate components of mixture• uses high pressure to push solvent through the column• Popularity:– sensitivity– ready adaptability to accurate quantitative determination– suitability for separating nonvolatile species or thermallyfragile ones11
    12. 12. • Popularity:– widespread applicability to substances that are of primeinterest to industry, to many fields of science, and to thepublic• Ideally suited for separation and identification of aminoacids, proteins, nucleic acids, hydrocarbons,carbohydrates, pharmaceuticals, pesticides, pigments,antibiotics, steroids, and a variety of other inorganicsubstances12
    13. 13. History• Early LC carried out in glass columns– diameters: 1-5 cm– lengths: 50-500 cm• Size of solid stationary phase– diameters: 150-200 µm• Flow rates still low ! Separation times long!• Decrease particle size of packing causes increase in columnefficiency!– diameters 3-10 µm• This technology required sophisticated instruments– new method called HPLC13
    14. 14. Advantages of HPLC• Higher resolution and speed of analysis• Greater reproducibility due to close control ofthe parameters affecting the efficiency ofseparation• Easy automation of instrument operation anddata analysis• Adaptability to large-scale, preparativeprocedures14
    15. 15. Advantages to HPLC• Advantages of HPLC are result of 2 major advances:– stationary supports with very small particle sizes and largesurface areas– appliance of high pressure to solvent flow15
    16. 16. Chromatographic terms16BaselineInjectionmonitoringEluent peak;dead volumePeak widePeak widthat half-heightBaselinenoisePeakfronting TailingpoorresolutionBaselinedrift
    17. 17. Parameters used in HPLC• Retention parameters• Column efficiency parameters• Retention : When a component in a sample interactswith the stationary phase in the column and a delayin elution occurs• Column efficiency : Goodness of a column17
    18. 18. Column dead time, retention time18timesignaltt tt0R 1 R 2R 30tR 1 tR 2 tR 3t´R = tR - t0t0 = column dead time = time an unretarded compound needs to pass thecolumntR = retention time
    19. 19. Capacity and Selectivity19
    20. 20. Capacity Factor• Capacity factor20k´ =tR - t0t0t´Rt0=•If the substance is not retained by the stationary phase,the capacity factor is k = 0.•Small k (k < 1) values show that the components areonly retained slightly by the separation column. Theirpeaks are located close to the non-retained peak (k = 0).•the optimum separation range to be k values between 1and 15. Values for k > 5 mean long retention times withassociated band broadening.
    21. 21. Resolution21If the peaks are separated almost down to the baseline, R » 1.5.Higher resolutions than R = 1.5 are not desirable because theysignificantly extend the analysis time but do not result in additionalinformation.Generally the values of R » 1.0 are sufficient to achieve qualitative orquantitative results.Even values of R » 0.5 are sufficient to determine thenumber of components present. For quantitative analysis, however, thepeak areas overlap too much
    22. 22. Condition for Good separation22
    23. 23. Plate NumberPlate Number23The theoretical plate number or N is a quantitative measurefor the column efficiencyIn formulas (1) or (2) the peak base width WB or the half widthWH are compared with the retention time tR
    24. 24. Plate Number and Plate Height Since an efficient separation column delivers sharp peaks with narrow basewidths, a better column has relatively high value of N The concept of the theoretical plates is a useful tool to describe the efficiency of aseparation column The number of theoretical plates is proportional to the column length L. Thelonger a column, the more theoretical plates it has; however, the column backpressure increases To be able to compare separation columns of various lengths, the theoretical plateheight, H is used24H = L/N
    25. 25. 25
    26. 26. Five modes in HPLCLC mode Packing materials Mobile phase InteractionNormal phase chromatography Silica gel n-Hexane/IPE AdsorptionReversed phase chromatography Silica-C18(ODS) MeOH/Water HydrophobicSize exclusion chromatography Porous polymer THF Gel permeationIon exchange chromatography Ion exchange gel Buffer sol. Ion exchangeAffinity chromatography Packings with ligand Buffer sol. Affinity26
    27. 27. 27HPLC Basic InstrumentationMobilephasePumpSolvent DeliveryInjectorSample InjectionColumnSeparationDetectorData ProcessorWaste
    28. 28. 28
    29. 29. Composition of HPLC System• Solvent• Solvent Delivery System (Pump)• Injector• Sample• Column• Detectors• Waste Collector• Recorder (Data Collection)29
    30. 30. Mobile Phase Degassing• Dissolved gases in the mobile phase can come out ofsolution and form bubbles as the pressure changesfrom the column entrance to the exit– May block flow through the system• Sparging is used to remove any dissolved gas fromthe mobile phase– An inert and virtually insoluble gas, such as helium, isforced into the mobile phase solution and drives out anydissolved gas.• Degassing may also be achieved by filtering themobile phase under a vacuum30
    31. 31. Are used to store Mobile-Phase. The solvent reservoir must bemade of inert material such as glass and must be smooth so as toavoid growth of microorganisms on its walls. It can be transparentor can be amber colored. A graduated bottle gives a roughestimate of mobile-phase volume in the bottle. Solvent reservoirsare placed above HPLC system (at higher level) in a tray. Theyshould never be kept directly above the system as any spillage ofsolvent on the system may damage electronic parts of HPLC.31Solvent Reservoir
    32. 32. Eluotropic series32
    33. 33. Mobile Phase Mixing• Solvent proportioning valve(3.) can beprogrammed to mix specificamounts of solventfrom the variousreservoirs toproduce thedesired mobilephase composition333.
    34. 34. • Isocratic elution:Use of a constant-composition mobile phase in liquid chromatography• Gradient elution:– Vary the mobile phase composition with time– If there is a wide polarity range of components to be eluted.– Allows for faster runs.– Ex: mobile phase composition can be programmed to vary from75% water: 25% acetonitrile at time zero to 25% water: 75%acetonitrile at the end of the run.• More polar components will tend to elute first.• More non-polar components will elute later in the gradient34
    35. 35. Common Reverse Phase Solvents• Methanol35CH3OH• Acetonitrile CH3CN• Tetrahydrofuran• Water H2O
    36. 36. Pumping system36
    37. 37. In order to reduce separation time and allow the use of smaller particle sizepackings (10 microns and below), we must force the liquid mobile phasethrough the column under pressure. This is the function of the pump (alsocalled the "solvent delivery system") to maintain a constant flow of mobilephase through the HPLC regardless of the pressure (back pressure) caused bythe flow resistance of the packed column.There are several types of pumps are available,Reciprocating Piston PumpsSyringe Type Pumps Constant Pressure Pumps.37
    38. 38. 38Reciprocating Piston PumpsConsist of a small motor driven piston which moves rapidlyback and forth in a hydraulic chamber that may vary from 35-400 µL in volume.On the back stroke, the separation column valve is closed, andthe piston pulls in solvent from the mobile phase reservoir. Onthe forward stroke, the pump pushes solvent out to the columnfrom the reservoir.A wide range of flow rates can be attained by altering thepiston stroke volume during each cycle, or by altering the strokefrequency. Dual and triple head pumps consist of identicalpiston-chamber units which operate at 180 or 120 degrees outof phase
    39. 39. 39Reciprocating Piston Pumps
    40. 40. Syringe Type PumpsThe syringe pump is a large, electrically operated simulation of ahypodermic syringe.Are most suitable for small bore columns because this pump deliversonly a finite volume of mobile phase before it has to be refilled.These pumps have a volume between 250 to 500 mL. The pumpoperates by a motorized lead screw that delivers mobile phase to thecolumn at a constant rate. The rate of solvent delivery is controlledby changing the voltage on the motor.40
    41. 41. Constant Pressure PumpsThe mobile phase is driven through the columnwith the use of pressure from a gas cylinder A low-pressure gas source is needed togenerate high liquid pressures.The valving arrangement allows the rapid refillof the solvent chamber whose capacity is about70 mL. This provides continuous mobile phaseflow rates.41
    42. 42. Tubing• Very small innerdiameter• Consistent i.d.• Very strong• Easy to cut• Fittings available42
    43. 43. Auto samplersAre fully automatic injection systems enabling greater productivityand the highest level of precision.The HPLC Autosampler incorporates an elegant swivel headthat allows a manual injection and a special Rheodyne injectionvalve to give accurate full or partial loop fill injections.43
    44. 44. Injection PortThe sample introduction device such as injector to introduce the sample in aflow of mobile phase at high pressure. It is not possible to use direct syringe injection on column like GC as theinlet pressure in LC is too high.The valve injection through fixed or variable loop is a common way ofintroducing the sample.The Rheodyne valve is the mostly used devise. The loop can be partially orfully filled. There are both the types of injectors available.The advantage of partial filling is the possibility of using small amount ofsample, when there is scarcity of sample.The precision of the injection is 1% RSD44
    45. 45. Samples are injected into the HPLC via an injection port. The injection port of an HPLC commonlyconsists of an injection valve and the sample loop. The sample is typically dissolved in the mobilephase before injection into the sample loop. The sample is then drawn into a syringe and injected intothe loop via the injection valve. A rotation of the valve rotor closes the valve and opens the loop inorder to inject the sample into the stream of the mobile phase. Loop volumes can range between 10µl to over 500 µl. In modern HPLC systems, the sample injection is typically automated45
    46. 46. ColumnHPLC Column HardwareStationary Phase46
    47. 47. Columns• Solid Support - Backbone for bonded phases.– Usually 10µ, 5µ or 3µ silica or polymeric particles.• Bonded Phases - Functional groups firmly linked(chemically bound) to the solid support.– Extremely stable– Reproducible• Guard column - Protects the analytical column:– Prolongs the life of the analytical column47• Analytical column - Performs the separation.
    48. 48. HPLC ColumnsParticle size Column ID Sample LoadAnalytical 3 – 5 µ 0.3 − 4.6 mm ng – µgSemi-prep 10 µ 8 – 10 mm 1 – 100 mgPreparative 10 – 30 µ 5 – 200 mm gramscale48•An HPLC column consists of a stainless steel tube which is sealed with fittingson both ends. Steel frits in the end fittings keep the packing material in thecolumn.•Analytical columns have inner diameters of 1 - 10 mm and lengths of 25 - 250mm. They are operated at flow rates of 60 µl - 5.0 ml/min.•To protect the actual separation column from chemical contamination, a guardcolumn with the same packing material as the separation column is installed.
    49. 49. Column Packing– Usually spherical silica particles of uniformdiameter (2-10µm)• The smaller particles yield higher separationefficiencies.– The silica particles are very porous• Allows for greater surface area for interactionsbetween the stationary phase and the analytes.– Other packing materials may also be used:• Zirconia (ZrO2) 49http://hplc.chem.shu.edu/NEW/HPLC_Book/Adsorbents/ads_part.html
    50. 50. Particle Diameter• Has a greater effect on resolution thancolumn length• Short columns with small particles ideal• 5µm is standard size• 3µm better, but restricted range of packingsavailable• Downside is high back pressure and issueswith retention of small particles inside thecolumn, blockages50
    51. 51. Cap nut withcompressionGuardcolumnSplit colletsHousing withouter threadcartridge51
    52. 52. Refers to the solid support contained within the column over which themobile phase continuously flows. The sample solution is injected into the mobile phase through the injectorport. As the sample solution flows with the mobile phase through thestationary phase, the components of that solution will migrate according tothe non-covalent interactions of the compounds with the stationary phase.The chemical interactions of the stationary phase and the sample with themobile phase, determines the degree of migration and separation of thecomponents contained in the sample. For example, those samples which have stronger interactions with thestationary phase than with the mobile phase will elute from the column lessquickly, and thus have a longer retention time, while the reverse is also true.Columns containing various types of stationary phases are commerciallyavailable.Stationary Phases52
    53. 53. Monofunctional surface modification of SiO2OHSiOSiOSiOSiOOHOHOHOHX SiCH3CH3CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2C6H13OHSiOSiOSiOSiOOOHOOHSiCH3C18H37CH3SiCH3C18H37CH3SiCH3CH3CH3OHSiOSiOSiOSiOOOOOSiCH3CH3SiCH3CH3SiCH3CH3CH3EndcappingmonofunctionalmodificationEndcappingStationary Phases53
    54. 54. Reversed Phase Chromatography• Bonded phases made by covalentlybonding a molecule onto a solidstationary phase like silica• Typical stationary phases arenonpolar hydrocarbons, waxy liquidsor bonded hydrocarbons (such asC18, C8, C4, etc.)• pH range 2.5 to 7.5• 60-90% of all analytical LCseparations are done on bondedphases in the reverse phase mode.54
    55. 55. sphericalirregularStructure of Packing Materialmonolithic55
    56. 56. Synthesis of RP Packings56
    57. 57. Common RP Packings57columnWhatmanZorbaxPartisilInertsilStyragelUltraspherekromasikl
    58. 58. Reversed Phase ChromatographyReversed Phase Chromatographymodification: RP-8, RP-select B, RP-18, -CN, -Diol, -NH2base material: LiChrosorb®, LiChrospher®, Superspher®, Purospher®,Purospher®STAR, Chromolith®, Aluspher®, Polyspher®Normal Phase ChromatographyNormal Phase Chromatographymodification: Si, -Diol, -CN, -NH2base material: LiChrosorb®, LiChrospher®, Superspher®, Aluspher®,Purospher®STAR ,Chromolith®Ion Exchange ChromatographyIon Exchange Chromatographymodification: -NR+3, -N+HR2, NH2 for cations-SO3-, -COO-for anionsBase material: Polyspher®, LiChrosil®Size Exclusion ChromatographySize Exclusion ChromatographySorbents: LiChrogel®PS for SEC in organic solventsFractogel®for aqueous SEC58
    59. 59. Detector59•Detect various compounds as they elute out from column. Thedetector gives response in terms of a milivolt signal that is thenprocessed by the computer (integrator) to give achromatogram.•Basically detector consists of a flow-cell through which themobile phase and resolved sample moves optics shine throughthe detector cell and variation in optical properties aredetected.•A Ultra violet or UV detector detects absorbance of UV light bychromophores in the analyte compound.•A refractive index detector will sense variation in refractiveindex of mobile phase stream passing through flow-cell• Similarly Fluorescence Detectors checks for Florescence.
    60. 60. Various Detetectors are Listed below• Ultraviolet (UV)Fixed wavelength detectorVariable wavelength detectorDiode Array• Fluorescence• Electrical Conductivity• Refractive Index• Electrochemical• Light scattering• IR Absorbance• Mass-Spectrometric60
    61. 61. Fraction collector61http://www.lplc.com/old/instruments/img/cf1big.jpg
    62. 62. Automated Waste Collection62
    63. 63. Recorder63http://www2.okbu.edu/academics/natsci/chemistry/people/mrjordan/webtutorials/hplc/images/recorder.JPG
    64. 64. Picture of a Typical HPLC System64
    65. 65. 65
    66. 66. HPLC Data66http://www.mac-mod.com/tr/plumbing-tr.html
    67. 67. 67
    68. 68. • 2004: Further advances in column technologyand chromatography instrumentation– Utilized even smaller packing particle sizes (1.7µm)– Higher pressures (15000psi)– Allowed for significant increases in LC speed,reproducibility, and sensitivity.• New research utilizing particle sizes as small as1µm and pressures up to 100,000psi!68What is Ultra PerformanceLiquid Chromatography?
    69. 69. Chromatograms of simvastatin69
    70. 70. Why UPLC more efficient• Peak capacity (P) is the number of peaks thatcan be resolved in a specific amount of time• P is proportional to the inverse of the squareroot of the Number of theoretical plates (N):N = L/H• Plate heights are correlated through the VanDeemter equation70
    71. 71. Contrasting HPLC and UPLC• UPLC gives faster results with betterresolution• UPLC uses less of valuable solvents likeacetonitrile which lowers cost• The reduction of solvent use is moreenvironmentally friendly• Increased productivity can increase yourevenue in an industrial setting71
    72. 72. 72http://caffeineawareness.org/index.php
    73. 73. Waters (USA)Agilent (USA)Thermo (USA)Varian (USA)Dionex (USA)Jasco (Germany)Knauer (Germany)Shimadzu (Japan)Manufacturers73Ningbo Yujie Optical Instruments Co., Ltd.
    75. 75. DETECTORS UV-Vis/PDA Refractive index Electrochemical Conductivity Fluorescence Radioactivity Evaporative light scattering Corona CAD PDA-MS PDA-NMR FTIR75
    76. 76. OVERVIEWDetector Analyte /attributes sensitivity NatureUV-VisWorks with all molecules containingchromophores absorbing UV-VisngSpecificPDA Works for all wavelengths in UV-Vis ngFluorescenceCompound with native fluorescence orfluorescence tagfg-pgConductivityAnions, cations, organic acids,surfactantsngRadio activity Radioactive labeled compounds ng-pg76
    77. 77. Detector Analyte /attributes sensitivity NatureRefractive IndexTemperature sensitive; polymers,sugars, triglycerides, incompatiblewith gradients0.1-10 µgUniversalEvaporative lightScattering(ELSD)Uniform response; nonvolatile to semivolatile compounds; compatible togradientsngElectrochemical Redox reactions pgCorona CADCan detect non UV absorbingchromophoresngMass Spectrometer Definite analyte identification fg-pgBoth universal andspecificIR Works with all molecules mg77
    78. 78. Uv- visible detectorSenstiveWide linear rangeUnaffected by changes in temp.And mobile phase compositionPrinciple :• Lambert-Beer’s law:A= ε b cAt constant cell thickness and constant wavelengtha linear connection between the Absorbance A andthe Concentration C is achieved.78
    79. 79. Types of uv-visible detectorsA. Fixed wavelength detectorsB. Variable wavelength detectorsC. Photodiode array detectors79
    80. 80. Fixed wavelength detectors Operates only at 1 wavelength (254 nm) Best overall precision is noted for peakarea measurements as compared tovariable wavelength detectors. Uses a discrete source:– Low pressure mercury lamp (253.6 nm)– Silicone photodiode light detector It can also be used at other wavelengths byfiltering the emission source to give other lines oreven using phosphor screens to give lines notavailable from mercury.80
    81. 81. Variable wavelength detectors Uses continuous source of light• Deuterium or xenon lamp or tugston lamp• Desired wavelength is isolated by monochromator Improved sensitivity from operating at λmax of solute ofinterest Drawback: limited lamp lifetime as compared tomercury lamp (Deuterum lamp: 1000-2000 hrs) Dual wavelength detection feature:– Useful for simultaneous monitoring of twosubstances– Baseline noise is higher because this feature isachieved by toggling the monochromator between 2wavelengths 81
    82. 82. 82
    83. 83. Photodiode array detectorsUses charge coupled DA with 512 to 1024 diodes capableof spectral resolution of 1 nmEarlier, they suffered from sensitivity problems, which hasbeen solved by advanced flow cell design using fiberoptics technology to extend path length withoutincreasing noise or chromatographic band dispersionPerforms a simultaneous measurement of absorption as afunction of analysis time and over a chosen wavelengthrange, so we get UV spectrum for each eluted peakUsed for method development where λmax of impurities indrug are unknownEvery compound can be quantitated at its λmax , so usefulfor trace analysisCompounds not resolved chromatographically cansometimes be resolved spectrallyMolecular absorption spectrum can be used for peakidentification and peak tracking83
    84. 84. Measureslightintensity ateach λMeasureslightintensity ateach λ84
    85. 85. Indirect photometric detectionUsed when solute of interest does not possesschromophoreHere, mobile phase possess a chromophore andabsorbs lightWhen analyte without a significantchromophore passes through the detector cell,the absorption of mobile phase is decreased andis recorded as negative peak85
    86. 86. 86
    87. 87. 87
    88. 88. Features of modern uv-visdetectorsDual or multiple wavelength detection and stopflow scanning featuresFront panel access to self alligned sources and flowcells for easy maintananceSelf validation features such as:– Power up diagnostics– Leak sensors– Time logs for lamps– Built in holmium oxide filters for wavelength calibration– Filter wheels for linearity verification88
    89. 89. Refractive index detectorsMeasurement response is a function of the refractionindex difference between pure mobile phase and themobile phase with the dissolved separatedcomponentsDue to the strong temperature dependence of therefraction index, good temperature control of themeasurement cell must be ensuredThe detection limit is in the range of 10-6- 10-8g/mlTo achieve a high sensitivity, in practice solvents are selected that havea very high or very low refraction indexThis ensures that the difference between the refractive indices of89
    90. 90. 90
    91. 91.  One half of the measurement cell is purged with the flowing mobile phase, the secondhalf of the measurement cell (reference cell) is filled with mobile phase. The refractionindex n is at first identical in both cells. If a sample component is added to the eluent, the refraction index n in themeasurement cells changes. The light beam experiences a deflection on the pathOperating principle91
    92. 92. 92
    93. 93. Fluorescence detectorsDuring fluorescence detection,the sample is irradiated withUV light of suitable wavelengthand excited for emission of longwave light.The Xenon lamp continuously sends light of 325 -410 nm.Provides LOD values 100 times lower thanabsorption detectorsHighly selective93
    94. 94. 94
    95. 95. 95
    96. 96. 96
    97. 97. 97
    98. 98. Electrochemical detectorsElectron transfer processes offer highly selective andsensitive methodEasily adaptable for use with microcolumnsAs background noise is dependent on mobile phaseconditions, it is difficult to utilize these detectorswith gradient elution separations2 types:1. Amperometric detection: fixed potential is applied tothe electrode (glassy carbon) and a solute which willoxidize at that potential yields an output current2. Coulometric detection: 100% of the solute species isconverted, which offers advantages of no mobile phaseflow dependence on signal and absolute quantitationthrough Faraday’s law98
    99. 99. `99
    100. 100. Conductivity detectors Measurement of electrical conductance is a subset ofECD, although it is generally considered separatelysince it is non-Faradaic electrochemistry; i.e. noelectron transfer reaction takes place The electrical conductivity of the mobile phase is usedas characteristic for detection. Universal technique for ionic solutes and are usedmainly for the separation of ions in water or polareluents Since the conductivity can change approx. 2 % per °C,conductivity detector are often equipped withautomatic temperature compensation100
    101. 101. With the removal of electrolytes contained in the eluent systemwhen using ion exchangers, a significant increase in the sensitivitycan be achieved (otherwise it creates difficulty in measuring lowconcentration of an ionic solute in the presence of highlyconducting mobile phaseThe resulting extremely low basic conductivity makes it possible, togenerate good measurement signals from the smallest samplevolumes.During conductivity detection the specific conductivity is measuredcontinuously.The individual eluted components of the material sample are eachdisplayed through a change in conductivity.101Conductivity detectors
    102. 102. Principle of 4 electrode methodThe two outer electrodes are used to transmit anelectronically stable alternating current through the cellThe electro-motor force (E), which is measureddeenergized on the two inner electrodes, is a measure forthe conductivity of the cell content according to theequation.χ = (K/E)*Iχ = electric conductivityK = cell constant between the 2nd and 3rd electrodeE = electro-motor forceI = current strengthThe deenergized measurement of the electro-motor forceat the two inner electrodes caused no passivity, hence theinstrument operates stably for a long period of time.102
    103. 103. 103
    104. 104. 104
    105. 105.  ELSD can outperform traditional detectors when analysing non-chromophoric samples by HPLC Traditional HPLC detectors such as UV and RI have limited capabilities UV and RI are not compatible with a wide range of solvents RI detection is not gradient compatible Different analytes produce different UV responses depending on theirextinction co-efficient ELSDs can detect anything that is less volatile than the mobile phase ELSD is universal and compatible with a wide range of solventsEvaporative Light Scattering Detector105
    106. 106. Unique Method of DetectionThree steps:• Nebulization• Mobile Phase Evaporation• Detection106
    107. 107. Step 1: Nebulization Column effluent passes through nebulizer needle Mixes with nitrogen gas Forms dispersion of droplet107
    108. 108. Step 2: Mobile Phase Evaporation Droplets pass through a heated zone Mobile phase evaporates from the sample particle Dried sample particles remain108
    109. 109. Step 3: Detection Sample particles pass through an optical cell Sample particles interrupt laser beam andscatter light Photodiode detects the scattered light109
    110. 110. ELSD – A Powerful Detector for HPLCUniversalSensitiveGradient CompatibleAN ELSD IS AN EFFECTIVE REPLACEMENT OR A PERFECT COMPLEMENT TO EXISTINGLC DETECTORS RI UV MS Fluorescence110
    111. 111. Four Reasons to Replace an RI with anELSD:1. Better sensitivity2. Gradient compatible3. Stable baselines4. No solvent front peaks111
    112. 112. The ELSD Improves Baseline Stability and Detection Sensitivity Comparedto RI112
    113. 113.  Reasons to operate an ELS detector in series with aUV detector:1. Obtain a more accurate representation ofsample mass than UV2. See what may be missing from your UVchromatogram113
    114. 114. The ELSD responds to all components in this antihistamineformulation1. Diphenhydramine2. PEG, Gelatin3. Sorbitan 4. Glycerol5. Sorbitol114
    115. 115. Detect Difficult Samples like Triglycerides without Derivatizing1. LLO2. LLP3. OOL4. POL5. PPL6. OOO7. OOP8. PPO9. OOS 115
    116. 116. Although the Mass Spectrometer is a UniversalDetector, ELSDs offer many Benefits over MS:1. Lower investment and operating costs2. Less complicated operation3. Less maintenanceSince chromatographic requirements for ELSDand MS are similar, methods developed forELSD are usually transferable to MS withoutUse ELSD in Parallel with MS to Obtain Maximum Structural and ConcentrationInformationUse ELSD in Parallel with MS to Obtain Maximum Structural and ConcentrationInformation116
    117. 117. Replace Fluorescence Detectors with ELSD to Simply Methods byEliminating Pre- or Post-column Derivatization1.  Glycine (Gly)2.  Serine / Asparagine (Ser/Asn)3.  Aspartic Acid (Asp)4.  Glutamine (Gln)5.  Alanine / Threonine (Ala/Thr)6.  Glutamic Acid (Glu)7.  Cysteine / Lysine (Cys/Lys)8.  Histidine (His)9.  Proline (Pro)10.  Arginine (Arg)11.  Valine (Val)12.  Methionine (Met)13.  Tyrosine (Tyr)14.  Isoleucine (Ile)15.  Leucine (Leu)16.  Phenylalanine (Phe)17.  Tryptophan (Trp)117
    118. 118. Summary:Benefits of ELSD as a Replacement orComplement to Existing Detectors• See what you might be missing• More sensitive and stable than RI• Get a more accurate representation of samplemass than UV• Simplify methods by eliminating derivatization118
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    127. 127. 127APPLICATIONS OF HPLC : Assay of cephalosporins Assay of theophylline Separation of barbiturates, phenothiazines, benzodiazepinederivatives, rauwolfia alkaloids etc. by C-18 reversed phasesQuantitative analysis of several analgesics like aspirin, caffeine,paracetamol, phenacetin, etc.Analysis of urine and serum samplesSeparation of antipyrine and benzocaine in ear drops
    128. 128. 128Separation and purification of nucleic acids. (eg: purines and pyrimidine compounds by reversed phase technique.)Determination of preservatives and antioxidants (eg: sorbic acid)Analysis of vitaminsSeparation of coal and oil productsAnalysis of pesticidesAnalysis of carcinogens. (eg: aflatoxins, metabolites of aspergillus flavus.)
    129. 129. 129Separation of steroids like cortisone and cortisol which are used in the treatment of Rheumatoid arthritis.Assay of hydro cartisone, fluocinolone acetonide, triamcinolone acetonide in tablets and other formulations.Analysis of oestrogen in female urine.Analysis of lipids.Separation of tropane akaloids, ergot alkaloids, indole alkaloids, cinchona alkaloids, etc.Analysis and separation of amino acids and proteins.Analysis of carbohydrates.
    130. 130. 1. REFERENCES:2. Instrumental methods of chemical analysis-B.KSharma.3. Pharmaceutical analysis by Ashutosh Kar4. Fundamentalsof AnalytticalchemistrySkoog,West,Holler5. Instrumental analysis of chemical analysis-Gurudeep R Chatal6. www.ctsholc.com/tools7. 6.www.wikipedia.org/hplc8. www.chemguide co.uk/analysis9. www.hplcindia.com 130
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