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TOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USAPolymer Analysis:Gel Permeation and Polymer Analysis:Gel Permeation and Gel Filtration Chromatography Gel Filtration Chromatography ApplicationsApplicationsJoe MachamerGPC Marketing ManagerTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA2Outline Introduction Calibration Curves Extending Linear Range TSKgel GPC ColumnsTSKgel PWTSKgel Alpha and SuperSWTSKgel H SeriesTSKgel SuperMultipore Tosoh EcoSEC GPC SystemTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA3HPLC Separation ModesRPCNPCHILICIECAFCHICICSECReversed Phase ChromatographyNormal Phase ChromatographyHydrophilic Interaction ChromatographyIon-Exchange ChromatographyAffinity ChromatographyHydrophobic ChromatographyIon ChromatographySize Exclusion ChromatographyGPC:Organic GFC:AqueousTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA4Polymer Separation Column Types Water-Soluble Polymer Samples TSKgel PW TSKgel PWXL TSKgel PWXL-CP TSKgel SuperMultiporePW Polar Organic-Soluble Polymer Samples TSKgel Alpha TSKgel SuperAW Organic-Soluble Samples TSKgel HXL TSKgel SuperH TSKgel HHR TSKgel SuperHZ TSKgel SuperMultiporeHZTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA5SEC Separation MechanismpackingmoleculesFastSlowEluentBasis-Difference in apparent molecular size with no additional interaction between column packing material and the sample TOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA6GPC Magic TriangleColumn PolaritySolvent PolaritySample PolarityWaterDMFTHFPolystyrenePMMADextranTSKgel PWTSKgel Alpha,SuperAWTSKgel HXL,HHR,SuperH,HZTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA7GPC Magic TriangleColumn PolaritySolvent PolaritySample PolarityWaterDMFTHFPolystyrenePMMADextranTSKgel PWTSKgel Alpha,SuperAWTSKgel HXL,HHR,SuperH,HZTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA8SEC Mechanism-No Interaction Proper SEC requires sample,mobile phase,and column chemistry to have similar polarities Interactions between sample and column chemistry induces large errors in MW determination Interactions between sample and column chemistry cause polymers to appear to be smallerTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA9Exclusion VolumeElution VolumeV0ViMolecular WeightPore VolumeABCDV0VT=V0+Vi107105106104103ExclusionSelective PermeationTotal PermeationDACalibration Curve TOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA10Resolution vs.Linear Range Trade-off Shallow calibration curve means:More resolutionLess linear rangeMolecular Weight103104105106107Elution Volume Molecular Weight103104105106107Elution Volume Low Resolution,Large Linear RangeHigher Resolution,Smaller Linear RangeTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA11How To Increase Resolution Add more columns of same typeLonger Run Time Use smaller particle sizeMolecular Weight103104105106107Elution Volume One columnMolecular Weight103104105106107Elution Volume Two columnsTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA12How To Increase Linear Range Use series of columns with different pores sizes Use columns contained a mixture of beads with different pore sizesTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA13How To Increase Linear RangeLarge PoreMedium PoreSmall PoreLarge PoreMedium PoreSmall PoreIndividual gradesMixed-bed TypeConnection of conventional columnsTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA14Effect on Calibration CurveMolecular WeightElution VolumeMolecular WeightElution VolumeElution VolumeMolecular WeightElution VolumeMolecular WeightTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA15Distortion on ChromatogrammVTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA16Conventional packings/columnsMultipore type packingsTSKgel SuperMultipore TypeSchematic diagrams of SEC packingsLarge PoreMedium PoreSmall PoreLarge PoreMedium PoreSmall PoreIndividual gradesMixed-bed TypeConnection of conventional columnsTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA17Comparison of Sequential vs.Multipore ColumnsColumns:TSKgel G4000+3000+2500+2000HXL,7.8mm ID x 30cm x 4TSKgel MultiporeHXL-M x 4 Mobile phase:THF Flow rate:1.0mL/min Detection:RIInjection vol.:100L Conc.:2.0mg/mL202530354045Retention time (min)TSKgel G4000+3000+2500+2000HXLTSKgel MultiporeHXL-M x 4Phenol resinTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA18TSKgel PW Series Columns For water-soluble polymers Hydrophilic polymethacrylate stationary phase pH stability 2 to 12 Organic stability to 20%Methanol Subtypes TSKgel PW TSKgel PWXL TSKgel PWXL-CP TSKgel SuperMultiporePWTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA19TSKgel PWXLCompared to TSKgel PWSmaller Particle Size (6-13m)Higher resolutionPore size range 1000 plus mixed bedTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA20TSKgel PWXL-CPFor separation of cationic water-soluble polymersCationic groups on the surface prevent adsorption of cationic polymerElution under low salt conditionsTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA21TSKgel PWXL-CP Cationic Polymer AnalysisTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA22TSKgel SuperMultiporePWMultipore pore morphologyExtended linear range w/o chromatogram distortionLess hydrophobic than TSKgel PWXL columnsTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA23TSKgel SuperMultiporePWTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA24PVP Analysis:TSKgel PWXL vs.TSKgel SuperMultiporePW-NTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA25TSKgel PW-type ApplicationsTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA26TSKgel Alpha and SuperAW Series Columns For Polymers of Intermediate Polarity Higher crosslinking than TSKgel PW series TSKgel SuperAW Resolution Same As Alpha,but 2x faster and 2/3 less solvent consumption Minimal shrink and swell in polar organic solventsmethanol,ACN,DMSO,IPA,THF,and HFIP Available in 5 discrete exclusion ranges plus a mixed bed columnTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA27TSKgel Alpha and SuperAW Applications Sodium chondroitin sulfate Sodium alginate Carboxymethyl cellulose Sodium polystyrene sulfonate Polyvinyl pyrrolidone Gum arabic Ethylhydroxy-ethylcellulose Vinyl alcohol/vinyl butyral copolymer Hydroxypropylcellulose Polymethyl vinyl ether Cellulose acetate N-isopropyl acrylamide Polyacrylonitrile Vinyl chloride/vinyl acetate copolymer Styrene/allylalcohol copolymer Poly(p-phenylene ether sulfone)TOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA28Solvent FlexibilityTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA29TSKgel H-Series Columns For GPC Analysis of Organic Soluble Polymers PS-DVB Matrix Eight pore sizes Range of Mixed Bed Columns Range of Multipore ColumnsTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA30TSKgel HXL and SuperHZ TSKgel HXL(7.8mm ID x 30cm)Ultra low polymer adsorption Solvent can be switched one time Extended linear range available with one Multipore column and five mixed-bed columns TSKgel SuperHZ(4.6/6.0mm ID x 30cm)Smaller particle and column size versions of TSKgel HXL Ultra low polymer adsorption Solvent can be switched one time Extended linear range available with three mixed-bed columns TOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA312.03.04.05.06.07.04567891011Elution volume(mL)Log MCharacteristics of TSKgel MultiporeHXL-MCharacteristics of linear type packing1)The range of molecular weight for measurement is wide.2)Calibration curve can be similar in a linear equation.3)The distortions of chromatogram coming from the point of inflection does not appear.More correct measurement of molecular weightLinear calibration R=0.999TOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA32TSKgel HHR and SuperH TSKgel HHR(7.8mm ID x 30cm)Solvent can be switch many times Five mixed-bed columns for extended linear range Stable to 140OC Broad Solvent Range TSKgel SuperH(6.0mm ID x 15cm)Smaller particle and column size versions of TSKgel HHR 2x shorter run times than TSKgel HHRTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA33Characteristics of Individual TSKgel HHR ColumnsThe range of molecular weight for measurement is narrow.Good separation at specific range of molecular weightTSKgel G1000HHR Separation of solvent peaksTSKgel G2000HHR OligomersTSKgel G3000HHR Low weight polymersTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA34Chromatograms with Various TSKgel HHR ColumnsColumns:TSKgel HHR,7.8mm ID x 30cm x 2Mobile phase:THF Flow rate:1.0mL/min Detection:RIInjection vol.:100L Conc.:2.0mg/mL810121416182022Epoxy resinG4000HHRG2500HHRG3000HHRG2000HHRG1000HHRRetention time minutesTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA35Characteristics of Mixed Bed TSKgel HHR ColumnsThe range of molecular weight for measurement is wide.Good for screening measurement of molecular weight for unknown polymerTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA36Comparison of Chromatograms with Individual and Mixed Type PackingColumns:TSKgel HHR,7.8mm ID x 30cm x 2 Mobile phase:THF Flow rate:1.0mL/min Detection:RIInjection vol.:100L Concentration:2.0mg/mL10121416182022Retention time (min)GMHHR-NG3000HHREpoxy resinTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA37Comparison of Calculated Molecular Weight with Various Type PackingRegular typeColumn:TSKgel SuperH5000+4000+3000+2000Mn 1.27x105 Mw 2.65x105Mixed typeColumn:TSKgel SuperHZ-M x 4Mn 1.20 x105 Mw 2.73x105Linear typeColumn:TSKgel SuperMultiporeHZ-M x 4Mn 1.28x105 Mw 2.83x105Polystyrene(NIST SRM706)234568101214161820Retention time minutesLogMTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA38TSKgel SuperMultiporeHZUltra low polymer absorptionMultipore pore morphology for linearity with out chromatogram distortionSemi-micro column sizeRun times are 50%lower than conventional columnsSolvent consumption is 85%less than conventional columnsTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA39Pore characterization of TSKgel SuperMultiporeHZ-MSuperMultiporeHZ-MTSKgel GMHHR-M00.10.20.30.40.50.0010.010.1100.10.20.30.40.50.0010.010.11Pore diametermdv/d(logr)m2/g Pore diametermdv/d(logr)m2/g TOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA40Calibration curves of TSKgel SuperMultiporeHZ columns1.E+011.E+021.E+031.E+041.E+051.E+061.E+071.E+081.E+011.E+021.E+031.E+041.E+051.E+061.E+071.E+081.52.53.54.55.56.51.52.53.54.55.56.5Elution time(min)Log Molecular Weight(Mw)TSKgel SuperMultiporeHZ-NTSKgel SuperMultiporeHZ-MTSKgel SuperMultiporeHZ-HColumn:TSKgel SuperMultiporeHZ,4.6mm ID x 15cmMobile phase:THFFlow rate:0.35 mL/minDetection:UV254nmTemperature:25Injection.vol.:5LSamples:Std.PolystyreneTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA41Properties of TSKgel SuperMultiporeHZ columnsSuperMultiporeHZ-NSuperMultiporeHZ-MSuperMultiporeHZ-HBasePS/DVBPS/DVBPS/DVBParticle Size(m)346Exclusion Limit(MW)120,0002,000,00040,000,000Mean Pore Dia.(nm)814Separation Range(MW)300-50,000500-1,000,0001,000-10,000,000Theoretical Plates20,000TP/15cm16,000TP/15cm11,000TP/15cmColumn Size4.6mm ID x 15cm4.6mm ID x 15cm4.6mm ID x 15cmTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA42Chromatograms of various polymers on TSKgel SuperMultiporeHZ column-101030507061014182226-101030507061014182226Elution time(min)mV12345678Column:TSKgel SuperMultiporeHZ-H,4.6mm ID x 15cm x 4Mobile phase:THFFlow rate:0.35 mL/minDetection:RITemperature:40oCLoad:10L,3g/L eachSample:1.Poly isobutylene2.Acrylic resin#13.Polystyrene(SRM706)4.Polyvinylchloride5.Polyvinylbutylral6.Polycarbonate7.Epoxy resin8.Acrylic resin#2TOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA43Semi-micro vs.Conventional ColumnsConventionalSemi-microDiameter(mm)7.84.6-6.0Length(cm)3015Flow Rate(mL/min.)1.00.35Run Time(Arbitrary)10050Semi-micro columns reduce run times by 50%and flow rate by 2/3.Throughput is doubled and solvent costs drop by 1/6.TOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA44Resolution:Semi-micro vs.Conventional SystemSemi-micro system offers better resolution for semi-micro columnsTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA45EcoSEC GPC SystemTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA46Save Time and Money Low dead volume design and system layout7.5L Stroke Volume2.5L RI Cell VolumeUse of 0.2mm and 0.4mm tubingTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA470102030405060(min)TSKgel SuperHZTraditionalFlow rate:TSKgel SuperHZ(0.35mL/min)TSKgel HHR(1.0mL/min)Dimensions:4.6mm ID x 15cm*4(TSKgel SuperHZ)7.8mm ID x 30cm*4(TSKgel HHR)Reduce run times by 50%Save Time and MoneyTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA48Reduce solvent usage by 85%Save Time and MoneyTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA49EcoSEC GPC SystemOther GPC manufacturerSuperior PerformanceTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA50Room Temperature Affect On Retention TimeConventional Instrument-No Pump Temperature ControlSuperior PerformanceTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA51Room Temperature Affect On Retention Time EcoSEC GPC System-With Temperature Controlled PumpsSuperior PerformanceTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA52 Temperature Controlled Pumps Excellent Retention Time Precision#of injections:10/daySuperior PerformanceTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA53 Temperature Controlled Pumps Excellent Molecular Weight Precision#of injections:10/daySuperior PerformanceTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA54VersatilityColumn oven holds up to 8,30cm columnsTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA55Conclusions SEC requires no interaction between sample molecules and column packing Columns with shallow calibration curves provide best resolution,but less linear rangeTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA56Linear range can be extendedIndividual pore size columns in sequenceMixed bed columnMultipore column particleso No distortion in chromatogramConclusionsTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA57 TSKgel PW Columns For water-soluble polymers TSKgel Alpha and SuperAW Columns For polar-organic soluble polymers TSKgel H-Series Columns For organic-soluble polymers TSKgel SuperMultiporeHZ Columns Wide linear range with no chromatogram distortion Semi-micro dimensions Tosoh EcoSEC GPC System Optimized for semi-micro columns Ideal GPC systemConclusionsTOSOH BIOSCIENCE LLCPresented at Pittcon 2011,Atlanta,GA,USA58Available solvent of SEC columnsTSKgel Column TypeShipment SolventChangeable SolventSuperHZTetrahydrofuranBenzene,Toluene,Xylene,Chloroform,Dichloroethane,DichloromethaneHXLDimethylformamideNoneCyclohexaneNoneAcetonen-Hexane,DMF,NMP,DMACSupe
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