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CHAPTER 5HIGH PERFORMANCE LIQUIDCHROMATOGRAPHY (HPLC)Expected OutcomesExplain the basic principles and instrumentation of HPLCAble to state the function of each components of HPLC instrumentationCompare characteristics of Normal phase and Reverse phase HPLCDescribe HPLC methodologies in quantitative and qualitative analysisExplain the optimization of HPLC methodState the applications of HPLC
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5.1 Principles of HPLC in Chemical AnalysisTypes of chromatography(according to the nature of MP and SP)Stationary phaseMobile phase TypesSolidGasGas chroma. (GSC)SolidLiquidLiquid chrom. (LC)Liquid coated on asolidGasGas-liquid chroma.(GLC)Liquid coated ona solidLiquid underpressureHigh Performanceliquid chrom. (HPLC)3
Principles of HPLC in Chemical AnalysisBasic separation principle Chromatography is a technique employed for theseparation of mixtures of compounds in a sample. LC is a chromatographic method, which uses theliquid as MP (eluent/solvent reservoir). Separation of components occurs between mobilephase (MP, solvent) and stationary phase (SP, columnpacking material) under high pressure. Separation is based on different mechanism.(ion-exchange, size-exclusion, adsorption, partition)4
Basic Separating Principles inHPLC : Modes of SeparationIon-exchange separation based on the charge properties of themolecules. SP: a resin matrix whose surface displays ionic functional groupsthat interact with analyte ions of opposite charge; MP: a buffered aqueous solution; Suitable for separation of ions and polar molecules, which arewater soluble.COO- K COO- K COO- K 5cation exchange resinN OHN OHN OH-anion exchange resin
Cation Exchange ChromatographyCOO- K aNH4 COO- K AnalyteNH4 COO- K COO- NH4AnalytebCOO- NH4 K COO- K 6K
Cation Exchange ChromatographyCOO- NH4 cCOO-NH4COO- K Buffer mobile phasecontaining Na .AnalyteNa Na Na Na Na Na K K Na NH4 COO- Na Na dCOOCOO-AnalyteNa Na Na NH4 K K Na 7Elution of theanalyte from SP.Na
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Size exclusion separation according to molecular size SP: material having specific pore size controller MP: aqueous solution suitable to large molecules/macromolecularcomplexes eg. polymers(a)(b)analytesSPZoom9
Adsorption separation based on sorption and desorptionprocesses. SP: solid having unmodified surface, which is verypolar, such as silica or SiO2) MP: solvents, hexane, EA, CHCl3 and MeOH; Not suitable for the separation of strong polarcompoundsOH OHOH OHOHOHSiO2OHSilica10OH
Partition separation based on the difference of the dissolutionof solutes between MP and SP; SP: liquid coated or bonded on the packing particles; MP: solvents such as hexane, EA, CHCl3, MeOH, ACNand ultrapure H2O; Suitable for separating various compounds in themixture, and has broad application.SPMP(b) MPelute outanalyte(a)packing Analyteparticles dissolvedin SP11MP(c) MPelute outanalyte
Partition Chromatography (PC) Bonded phase PC SP is a liquid chemical group that covalently bonded tosilica packing particles, so as to avoid losing SP andincrease the thermal stability of SP.SP1.8-10 µmpackingparticlesPacking materialZoom in1212
Partition Chromatography (PC) How to bond SP on Silica-gel?Formed by the reaction of silica particles with anorgano-silane of the general formula Si(CH3)2RCl.1313
What is R? If R is a polar functional group – stationary phase is polar. Example cyano (-C2H4CN) amino (-C3H6NH2) diol (-C3H6OCH2CHOHCH2OH)14
If R is a non-polar functional group – stationary phase is non-polar. Example N-octyl (-C8) N-octyldecyl (C18)15
Basic Separating Principles inHPLC : Predicting Elution Order by MP Separation in HPLC is basically governed bymanipulating the polarity of both SP & MP.Mobile Phase Selection Elution order of HPLC is governed by polarity. Retention times are controlled by polarity ofmobile phase.16
Basic Separating Principles inHPLC : Polarity of MPMobile phase/solventPolarity index (P’)Cyclohexane0.04n-hexane0.1carbon tetrachloride1.6i-propyl ether2.4toluene2.4diethyl ether2.8tetrahydrofuran4.0ethanol4.3ethyl er10.217
Principles of HPLC in ChemicalAnalysis (Summary)HPLC: HPLC is a kind of LC, which uses highpressure to drive liquid MP through a columnof SP. Separation is basically governed bymanipulating the polarity of both SP & MP. Elution order of HPLC is governed bypolarity of analyte.1818
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Principles of HPLC in ChemicalAnalysis (Summary) Bonded phase PC SP is a liquid chemical group that covalentlybonded to silica packing particles, so as to avoidlosing SP and increase the thermal stability of SP.cyano (-C2H4CN)Polaramino (-C3H6NH2)diol (-C3H6OCH2CHOHCH2OH)Non-polaroctyl (-C8H17)octyldecyl (-C18H37)2020
5.2 Characteristics of Normal Phaseand Reverse Phase HPLCNormal-phase HPLC (NP-HPLC) Polar stationary phase Non/less polar mobile phaseNote: mixture of non/less polar solvents could be usedas MP. E.g. are hexane, EA, CHCl3, ether,dichloromethane Least polar solutes are first to elute from the column21
Characteristics of Normal Phaseand Reverse Phase HPLCSP in NP-HPLC (2): Organic moieties with cyano-silane or amino-silanefunctional groups have replaced reactive silanolgroups (Si-OH) on the silica surface.2222
Characteristics of Normal Phaseand Reverse Phase HPL CReversed-phase HPL C (RP-HPLC) Non/less polar stationary phase Polar mobile phaseE.g. are MeOH, ACN, Ultrapure water Most polar solutes are first to elute from the column23
Characteristics of Normal Phaseand Reverse Phase HPL CSP in RP-HPLCIt uses a polar mobile phase and a non-polarstationary phase.The silanol groups ( Si-OH ) present in silica is treatedwith an organochlorosilane:CH3SiOSi(CH2)17CH324CH3ODS bonded groups
You are given one samples contains an analytesalchoholB-alkaneC-esterPredict the elution order for both NP and RPHPLC. Your answer should suggest suitable1. SP2. MP3. Elution order25A-
Basic Separating Principles inHPLC : Predicting Elution OrderExercise 1:Predict the order of elution for the separation of CH3C(O)CH3 CH2 CH2CH3 and C3H7OHusing a C8 bonded phased stationary phase.Explain your answer.26
Exercise 2A student set up a HPLC separation of the following compounds is runthrough a column with C2H4-CN functional group attached to the siloxanebackbone and n-hexane as the mobile phase.What is the order of elutionfor these compounds? Explain your reasoning.OOOHpropionic acidOpropyl acetateo-xylene Discuss the advantages of Reverse Phase HPLC compared to NormalPhase HPLC analysis27
Solution to exercise 1: C8 is non-polar so non-polar molecule will then beretent longest. So the alkene will be eluted last,followed by ketone and alcohol. Propanol Propanone Propene28
Characteristics of Normal Phaseand Reverse Phase HPL CReversed-phase HPLCAdvantages The mode has a very broad scope that allows sampleswith wide ranges of polarity to be separated. The mode is generally experimentally easier, fasterand more reproducible than other LC modes. It can be applied to the separation of ionic or ionizablecompounds by the use of ion-pairing techniques.PIA /Topic 4/AY08-0929
Characteristics of Normal Phaseand Reverse Phase HPL CReversed-phase HPLCDisadvantages For silica bonded phases, stable columns can bemaintained at pH 2-10. Below pH 2 the bondedgroups will be hydrolyzed, and above pH 10, thesilica is appreciably soluble in the mobile phase.H O-Si(CH3)2ROHsilicaOH-30O-Si(CH3)2R
Characteristics of Normal Phaseand Reverse Phase HPL CReversed-phase HPLCDisadvantages The presence of unreacted silanol groups on the silicasurface can often cause poor peak shape and nonreproducible behavior between columns due tosolute adsorption.31
5.3 Quantitative and Qualitative Analysis Retention Time Matching Standard Curve Method Internal Standard Method32
Methodologies in Qualitativeand Quantitative AnalysisRetention Time Matching Tr of standard and sampleare matched when bothare run under the sameconditions.3333
Methodologies in Qualitativeand Quantitative AnalysisDisadvantages As std and sample are injected consecutively,simultaneous analysis is not possible. Whichchromatography allows simultaneous analysis?Matching3434
Methodologies in Quantitativeand Qualitative AnalysisStandard curve method prepare a set of standard solutions containing a pureanalyte obtain a series of chromatograms plot a calibration curve of peak area/height versusconcentration determine the concentration of unknown sample fromthe calibration curve35
Methodologies in Quantitativeand Qualitative AnalysisInternal standard method prepare a set of standard solutions containing a pureanalyte Spike a known amount of an internal standard intothe standard and sample solutions obtain a series of chromatograms plot a standard curve of peak area ratio versusconcentration determine the concentration of unknown samplefrom the standard curve36
5.4 Components of HPLC instrumentation1) MP supply system2) Pump3) Sample injectionsystem4) Column5) Detector6) Workstation3737
HPLC instrumentation/Solventthermostat3838
HPLC instrumentation1) MP supply systemknown as MP (solvent) reservoirsFunction: to provide MP/solvent (s)for the runA. MP/Solvent reservoir(s): One: Filled with single solvent or the mixtureof the solvents of different polarities Reservoir filter 1: Filled with several solvents of differentpolarities, respectively3939
HPLC instrumentation1) MP supply systemB. Solvent(s): The solvent(s) used must be high pure; The solvent(s) must be filtered before use; The solvent(s) must be degassed before use; HAc, FA, H3PO4, TFA or ammonium acetate, phosphate,and ion-pairing chemicals which are soluble in MP can beused to control pH value.4040
HPLC instrumentationFiltration: To remove small particulate matter such as dust andinsoluble salt, which will greatly damage the pump andcollect on the top of the column. Micropore filter membrane (0.45µm usually and 0.2µm forbuffer salt solution) is commonly used for filtration.MPsmall particulate matterColumnclogged column4141
HPLC instrumentation1) MP supply systemC. Degasser:To remove dissolved gases in MP such as N2 and O2 which maylead to the formation of gas bubbles when MP enters the detectorresulting in distortion of signals. Gas bubbles within the columncan also lead to very high pressure and unstable pressure.PIA /Topic 4/AY08-094242
HPLC instrumentation1) MP supply systemD. Mixing chamberPlace where the solvents aremixed in correctcompositions.Mixing chamber4343
HPLC instrumentation2) PumpFunction: to provide the high pressure (driving force) requiredfor the run, which is the core component of HPLC.4444
HPLC instrumentation3) Sample Injection System Six-port valve and sample loopSix-port valveSample loop4545
HPLC instrumentationColumn(MP out)Column(MP out)LoopLoop1Pump(MP in)2WasteWasteSample inSample inLoad PositionPump(MP in)Inject Position4646
HPLC instrumentation3) Sample Injection System Syringe & Needle4747
HPLC instrumentation4) Column: Analytical column Function: to separate the analyte in the run External packing is usually constructed fromstainless steel tubing. Why?PIA /Topic 4/AY08-094848
HPLC instrumentation4) Column: Analytical column Common dimensions of HPLC column– packings ( ): 1.8 10 m4949
HPLC instrumentation Analytical columns with internal diameter ofaround 5 mm packed with 5- m particles offera good compromise between sample capacity column efficiency applied pressure volume of MP used5050
HPLC instrumentation4) Column: Guard column To avoid the formation of clogged columninduced by small insoluble particles fromsample/MPGuardcolumnPIA /Topic 4/AY08-09Analytical Column5151
HPLC instrumentation5) Detector: Types of detector1. Ultraviolet-visible Detector (UV-VIS)Diode array detector (DAD)/Photodiode arraydetector (PDA)2. Refractive index detector (RI)3. Mass spectrometer detector (MSD)52
5.5 Optimization of HPLC AnalysisFactors that affect a HPLC analysis Flow rate of mobile phase Type of column Type of detector53
Factors that affect HPLC analysisFlow Rate of Mobile Phase As flow rate increases, retention time decreases. If the flow rate is too high, some of the compoundsmay elute at the same time. This leads to poorseparation efficiency. The flow rate must be adjusted and optimized toeffect good separation of the solutes in a sample.54
Factors that affect HPLC analysisType of Column Polarity of SP matced with an analyte This would effect greater interaction between theanalyte and the column leading to good separation. The selection of a specific column (stationary phase)depends on whether or not the planned separationis possible or logical with a given mechanism.55
Factors that affect HPLC analysisType of Column1. Particle size: prefer smaller particle Higher plate number Narrower peaks Higher pressure required to move theeluent through the column Manufactured columns have about 50,000 plates/m if packed with 5 mparticles, and about 25,000 plates/m if packed with 10 m particles.56
Factors that affect HPLC analysisType of Column2. Particle shapeColumns packed with spherical particles requiredless pressure for a given eluent velocity.57
Factors that affect HPLC analysisParameter IncreaseRetention TimeColumn LengthIncreaseColumn Internal DiameterIncreaseColumn Particle sizeDecrease58
Different Modes of LiquidChromatographyThere are two mobile - phase elution methods: Isocratic elution Gradient elutionOther than the polarity of SP and MP, we further make use of differentmethods to achieve good separation.59
Different Modes of LiquidChromatographyIsocratic elutionA single mobile phase composition is in use for the entire separation.Diagram extracted from: Skoog,West, Holler and Crouch, 2004,8th ed. Fundamentals of AnalyticalChemistry, pg.976)Q: How to resolvepeaks 1 to 3?7060
4.3.2 Different Modes of LiquidChromatographyGradient elution The composition of the mobile phase changes with time during theseparation, usually by mixing two solvents with different eluting powers incontinually changing proportions Gradient elution allows early eluting peaks to be adequately separatedwithout the later eluting peaks becoming too dispersed.61
Diagram extracted from: Skoog,West, Holler and Crouch, 2004,8th ed. Fundamentals of AnalyticalChemistry, pg.976)PIA /Topic 4/AY08-0962
Common Problems in HPLCAnalysisColumn Troubleshooting1. Noisy Baseline Possible causes : Dirty flow cellDetector lamp failingair bubbles passing through detectortemperature effects on detector63
Common Problems in HPLCAnalysis2.NormalDrifting baseline Possible causes : Gradient elutionTemperature unstable (RI detector)Contamination in mobile phaseContamination in system64Drifting
Common Problems in HPLCAnalysis3.Ghost peaks - peakswhich appear even when nosample is injected Possible causes : dirty mobile phase4.Unusually high pressure Possible causes : air pockets trapped in the column pump malfunction clogged column65
Common Problems in HPLCAnalysis5.Fronting peaks Possible causes Column overload6. Negative peaks Possible causes absorbance of sample is less than mobile phase66
5.6 Applications of HPLC Chemistry and biochemistry research Quality control Environmental control Federal and state regulatory agencies Pharmaceutical industriesPIA /Topic 4/AY08-0967
Normal-phase HPLC (NP-HPLC) Polar stationary phase Non/less polar mobile phase Note: mixture of non/less polar solvents could be used as MP. E.g. are hexane, EA, CHCl 3, ether, dichloromethane Least polar solutes are first to elute from the column 5.2 Characteristic
