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  • 1. High Performance Liquid Chromatography(HPLC)-an IntroductionBhupander Kumar (Scientist)National Reference Trace Organic LaboratoryCentral Pollution Control Board, Delhi
  • 2. Liquid Chromatography Origins• Michael Tswett (1906) separated the plantpigments by organic solvent as mobile phase &chalk as stationary phase• Martin and Synge (1941) won the Nobel prize inchemistry in 1952 for performing liquid-liquidpartition chromatography
  • 3. HPLC- Introduction• HPLC is a form of column chromatography used inchemistry to separate, identify and quantify compoundsbased on polarities (affinity) and interactions with column’sstationary phase• HPLC utilized column’s stationary phase, a pump thatmoves the mobile phase and analyte through column and adetector that provides a retention time for the analyte• A pump provides high pressure to propels the mobile phaseand analyte through packed column and allows for betterseparation, so it is also called high pressure liquidchromatography
  • 4. Liquid Chromatography - Types• Partition Chromatographyi.e. paper chromatography and Thin layerchromatography (TLC). Partitionchromatography uses a retained solvent onthe solid support and separates analytesbased on polar differences. The polar analytesdiffuses into a stationary water layerassociated with the polar stationary phase andare thus retained. This method uses polarstationary phase (silica or alumina) and a nonpolar water miscible mobile phase.
  • 5. •Adsorption or Normal chromatographyThis NP-HPLC uses a polar stationary phase and anon-polar, non aqueous mobile phase for separatinganalytes readily soluble in non-polar solvents. The useof more polar solvents in mobile phase decrease theretention time of a analyte. Very polar solvents inmixture deactivate the stationary phase by creating astationary bound water layer because its behavior ispurely an adsorptive mechanism
  • 6. •Ion exchange chromatographyThis chromatography is based on theinteraction between solute ions and chargedsited bound to the stationary phase. Ions ofsame charge are excluded. Types of ionexchangers includes:Polystyrene resinsCellulose and dextran ion exchangers (gels)Porous silicaThis form of chromatography is widely used inwater purifications
  • 7. •Size exclusion chromatographyThis is also known as gel permeationchromatography (GPC), separates particleson the basis of size. The large moleculessimply pass by the pores as they are too largeto enter the pores. There fore large moleculesflow quicker through the column than smaller,that is smaller the molecule, larger theretention time. This technique is widely usedfor the molecular weight determination ofpolysaccharides and proteins
  • 8. •Reverse phase chromatographyRP-HPLC has a non-polar stationary and anaqueous, moderately polar mobile phase. Onecommon stationary phase is a derivatized silicawith R, where R is a alkyl group such as C8 H17or C18H37. With these stationary phases,retention time is longer for molecules whichmore non-polar, while polar molecules elutesmore readily
  • 9. HPLC vs LCHPLC as compared with LC is characterized by:• High resolution• Small diameter (4.6 mm), stainless, glass column• Column packing with very small (3,5, and 10 µm)particles• relatively high inlet pressures and controlled flow ofthe mobile phase• Continuous flow detectors capable of handling smallflow rates and detecting very small amounts• Rapid analysis
  • 10. HPLC – Analytical ScopeIn addition, it is used for analyzing air and water pollutants,and for monitoring pesticide levels and other POPs in theenvironment.HPLC commonly used for analysis of– Biological compounds: Amino acids, proteins, lipids– Pharmaceuticals: Antibiotics, steroids, analgesics– Inorganic compounds: Antioxidants, Afflatoxin– Environmental pollutants: PAHs, PCBs, Herbicides, Pesticides,Carbamates & Phenol– Forensic: Drugs, Poisons, NarcoticsThis technique is used for chemistry and biochemistryresearch analyzing complex mixtures, purifying chemicalcompounds, isolating natural products, or predicting physicalproperties.
  • 11. HPLC ComponentsPumpRheodyne InjectorColumnfrom solventreservoirtodetectorTo injectorTo columnLiquidMobile Pump Injection Separation Detector RecorderPhase Valve Column
  • 12. HPLC Pumps Pressure to 6000 psi-In order to achieve flowrates of 0.5 to 5.0 ml/min for a 10 -30 cm column,pressure of 70 to 400atm (1000 to 6000 psi) arerequiredBinary, tertiary or quaternary pump for delivery ofmultiple solventsControl flow rate from 0.1 to 10 ml/minControlled pressure range1 to 5000psi
  • 13. Injection valveThe function of the injector is to place the sample into thehigh pressure flow in narrow volume so that the sampleenters the column as a homogenous Injection valve consists of a rotor and fixed sample tubecalled “loop” (1 – 100 µl) Reproducibility: 0.1 %
  • 14. HPLC ColumnsGuard Column: A 0.5 to 2.0 µm Guard Columns are placedanterior to the separating column. This serves as a protectivefactor that prolongs the life and usefulness of the separationcolumn.1) particles that clog the separation column;2) compounds and ions that could ultimately cause "baselinedrift", decreased resolution, decreased sensitivity, andcreate false peaks;3) compounds that may cause precipitation upon contact withthe stationary or mobile phase; and4) compounds that might co-elute and cause extraneouspeaks and interfere with detection and/or quantification.
  • 15. Analytical columns: Theses are generally made outof stainless steel tubes with diameter of 3-5 mm anda length ranging from 10 to 30 cm. Normally columnsare filled with silica gel (2 – 10 µm in diameter) asstationary phase. On the basis of stationary phasecolumn types can be classified as: Normal phase Reverse phase Size exclusion Ion exchange
  • 16. The stationary phase in HPLC refers to the solidsupport contained within the column over whichthe mobile phase continuously flows. Columnscontaining various types of stationary phases arecommercially available. The chemicalinteractions of the stationary phase and thesample with the mobile phase, determine thedegree of migration and separation of thecompounds contained in the sample.Stationary phase
  • 17. Liquid-Solid PhaseLiquid-solid phase operates on the basis of polarity.Compounds that possess functional groups capable of stronghydrogen bonding will adhere more tightly to the stationaryphase than less polar compounds. Thus less polarcompounds will elute from the column faster thancompounds that are highly polar.Liquid-Liquid PhaseThis phase operates on the same basis as liquid-solid.However, this technique is better suited for samples ofmedium polarity that are soluble in weakly polar to polarorganic solvents.
  • 18. Normal phase:OH OH OH OHThis phase operates on the basis of hydrophilicityand lipophilicity by using the polar parts of thestationary phase and less polar mobile phase. Thepacking of the column must be more polar than themobile phase with respect to the sample. Thushydrophobic compounds elute more quickly thanhydrophilic compounds.SiO2
  • 19. Reverse phaseThis phase also operates on the basis of hydrophilicityand lipophilicilty but the stationary phase consists ofsilica based packing with long chain hydrocarboncompounds to decrease the polarity of columns. Forexample, C8 signifies an octyl chain and C18 anoctadecyl ligand in matrix. Typical stationary phasesare nonpolar and the solvents are polar aqueous-organic mixtures such as Methanol-Water orAcetonitrile-Water. Thus hydrophilic compounds elutemore quickly that do hydrophobic compounds.
  • 20. Size exclusion PhaseIn size exclusion the HPLC column stationaryphase consists of porous beads of substancewhich have controlled pore size and is able to befiltered in an ordinary phase according to itsmolecular size. Small molecules penetrate intothe pores within the packing while largemolecules only partially penetrate the pores. Thelarge molecules elute before the smallermolecules.
  • 21. Ion Exchange PhaseThis phase contains charge bearing functionalgroups attached to a polymer matrix and sampleanalytes are separated based upon attractive ionicforces between molecules carrying charged groupsof opposite charge to those charges on thestationary phase. Separations are made between apolar mobile liquid , usually water containing salts orsmall amounts of alcohols, and stationary phasecontaining either acidic or basic fixed sites.
  • 22. •relatively polar surfaceO O O| | |OSiOSiOSiOH| | |O O O| | |OSiOSiOSiOH| | |O O O•bulk (SiO2)x •surface• relatively nonpolar surfaceSilica GelO O O| | |OSiOSiOSiOR| | |O O O| | |OSiOSiOSiOR| | |O O O•bulk (SiO2)x•surfaceDerivatized Silica Gel•Where R = C18H37hydrocarbon chain•(octadecylsilylderiv.silica or “C18”)•“normal phase” •“reversed phase”
  • 23. Normal vs. Reversed Phase ChromatographyNormalPhase ReversedPhaseStationaryphase Polar(silicagel) Non-polar(C18)MobilephaseNon-polar(organicsolvents)Polar(aqueous/organic)Samplemovement Non-polarfastest PolarfastestSeparationbasedonDifferentpolarities(functionality)Differenthydrocarboncontent
  • 24. HPLC solvents for Mobile phaseExperience plays a major role in the selection of anHPLC solvents Generally there should be significant differencebetween the polarities of the S.P. and M.P., the reasonis that separation is based on the solubility differencesbetween the M.P. and S.P. Almost all reversed phase separations (polar m.p. andnonpolar s.p.) can be carried out with combination ofacetonitrile , and /or methanol, and water as M.P. The polarity of common HPLC solvent are as :Water > Acetonitrile >Methanol > Ethanol > Acetone >Ethyl acetate > cyclohexane > Hexane
  • 25. Practical Note Separation of most organic compounds can bedone by C - 18 stationary phases Solvents must be miscible e.g. water/methanol.An immiscible solvent such as water/toluene wouldcreate a mess in the column
  • 26. Mobile phase elution Isocratic elutions –This is equivalent to isothermal separations in GCConstant solvent composition, mobile phase polaristay constant throughout elution process. In isocratelution, peak width increases with retention time, thleads to the disadvantage that late eluting peaks verflat and broad, Their shape and width may keep thembeing recognized as peaks. In this elution, the selectividoes not change if the column dimensions (length aninner diameter) change – that is , peaks elute in thsame order
  • 27. Gradient Elutions –This is equivalent to temperature programming inGC. A separation in which the mobile phasecomposition is change during the separationprocess is described as gradient elution. Oneexample is a gradient starting at 10% methanol andending at 90% methanol after 20 minutes. Ingradient elution, the elution order may change asthe dimensions of the column or flow rates of themobile phase change.
  • 28. HPLC Chromatograms•Rt = 3.0 min.•faster moving•less retained•Rt = 5.2 min.•slower moving•more retained• 0 1 2 3 4 5 6 7•Time (minutes)•responseApproximationof peak area bytriangulationArea = base x height2•base•height•Peak A •Peak B
  • 29. Detectors in HPLCSelection of detector depends on analysis of interest Detector should be ideal for analyte Should give fast response to flowing systemCommonly used detectors in HPLC are: Refractive Index (RI) Detector UV-Visible Absorbance Detector (UV) Diode Array Detector (DAD) Fluorescence Detector (FLD) Fourier Transform Infrared Detector (FTIR) Evaporative Light Scattering Detector Electrochemical Detector
  • 30. Refractive Index (RI) detector Nearly universal but poor detection limit Passes visible light through 2 compartments, sample &reference When the solvent composition are the same, the lightpassing through the compartments is recorded as zero When a solute is passing through the samplecompartment, refractive index changes and sensed by thedetector
  • 31. UV-Visible Absorbance Detector Based on electronic transitions withinmolecules Most common type of detector for LC Work with fixed wavelengths Selectable for specific wavelengths
  • 32. Diode Array Detector Allows for the recording of the entire spectrum ofsolute as it passes through the DADFluorescence Detector Based on fluorescent light emission of excitedstate moleculesLightsourceManywavelengthExcitationmonchromatorOnewavelengthSamplecellLuminescence atmany wavelengthEmissionmonchromatorOne wavelengthDetector
  • 33. IR Detector IR Detector allows for spectrum records of flowing systemsimilar to DAD Water & Alcohol are the major interferences to solutedetectionEvaporative Light Scattering Detector Responds to any analyte that is significantly less volatilethan the mobile phase Elute is mixed with N2 gas and forms a fine mist Solvent (M.P.) evaporates leaving fine particles of analyte.The particles are detected by light scattering Response is proportional to analyte mass
  • 34. Electrochemical Detector Based on amperometry, voltametry and conductivityresponse of analyte to electrode usually held at constantpotential If the analyte is electro active, can be highly sensitivesince response is based on surface phenomenon ratherthan a solution bulk property (e.g. UV-Vis absorbance)Comparison of commercial HPLC detectorsDetector Approx LOD (ng) Useful with gradientUltraviolet 0.1 – 1.0 YesRefractive Index 100 – 1000 NoEvaporative Light Scattering 0.1 – 1.0 YesElectrochemical 0.01 – 1.0 NoFluorescence 0.001 – 0.01 Yes
  • 35. Conclusion HPLC most suitable system for analysis of nonvolatile,semi-volatile or thermally unstable compounds.Commonly used for analysis of environmental pollutants likePAHs, PCBs, herbicides, pesticides, Carbamates, phenol Most common mobile phases are Water, Acetonitrile,methanol and mixture of these solventsC - 18 containing stationary phase column is most suitablefor separation of all organic compoundsDad Array Detector (DAD) and Fluorescence Detector (FLD)are the most common detectors used for environmental
  • 36. Preventive maintenance•General Recommendations•Avoid solvents corrosive to stainless steel•Use only HPLC water•Consider miscibility of solvents, solubility of salts andbuffers•Filter mobile phase before use (0.4 micron)•Filter sample before use•Degas mobile phases before use (or use vacuumdegassing)•After use flush the HPLC to remove buffers and additives