色谱柱技术幻灯片.ppt

色谱柱技术第1页，共46页，编辑于2022年，星期二1ContentsnTechnology of TSK-GELHistoryTechnologyParticle,pore,modificationnApplicationBioseparationSEC,IEC,HIC,AFCSeparation for small compoundODS,HILIC,Mixed-mode第2页，共46页，编辑于2022年，星期二2History of TSK-GEL nTSK is originally from“Toyo Soda Kogyo”,the former company name as in Tosoh.nFirst product was GPC column for synthetic polymer analysesnOver 30 years experiences in chromatographynTSKgel SWXL are the golden standard for protein separationnPioneer for high-performance hydrophobic interaction chromatography(HIC)for protein separationnOver 50%market share for polymer analysis by gel permeation chromatography(GPC)with sophisticated GPC equipment in JapannDedicated equipments for Ion chromatography and Bio-separation nDiagnostic applicationOver 50%market share for glyco-hemoglobin analyzer with IEC column for diagnostic of diabetes in Japan第3页，共46页，编辑于2022年，星期二3HPLC Separation modenReversed-phase chromatography(RPC)nNormal-phase chromatography(NPC)Hydrophilic chromatography(HILIC)nSize exclusion chromatography(SEC)Gel permeation/filtration chromatography(GPC/GFC)nIon-exchange chromatography(IEC)Ion chromatography(IC)Ion-exclusion chromatography for ion(IEX)nHydrophobic interaction chromatography(HIC)nAffinity chromatography(AFC)nChiral separationnMixed-mode chromatography第4页，共46页，编辑于2022年，星期二4Technology on TSK-GEL and ToyopearlnMatrix/resin controlSilica,resin(methacrylate,PS/DVB),hydroxyapatite,carbonRigidity,stability,selectivitynParticle size control(2-300 micron)Analytical(TSK-GEL)to preparative(Toyopearl)Mono-disperse,narrow distributionnPore size control(5 nm-400 nm)Optimized for target molecules;peptide,protein,polynucleotide Wide-range pore distribution;polymer analysis by MultiporeHNon-porous resin;large biomoleculesnChemical modification controlEndcapping;ODS silica,3-D spacer arms(Networking)Bi-functional(In/Out);BSA-ODS/VnProductionHigh quality;Column packing and QC know-howLarge scale production,stable and continuous supply第5页，共46页，编辑于2022年，星期二5Particle Size TechnologySeamless Scale-upAnalytical to Process Same matrix(resin)(Pore size,modification)Same selectivity for elution第6页，共46页，编辑于2022年，星期二6Eluent flowPorousmaterialProteinsIonic groups+-Porousmaterial+Ionic groups-ProteinsEluent flow-Pore sizeSmallLargeSurface areaLargeSmall“Effective”surface areafor small proteinsLargeSmallfor large proteinsSmallLargePore Size Technology:Pore Size and Effective Surface Area on Matrix第7页，共46页，编辑于2022年，星期二7Pore Size Technology:BioseparationPore Size and Adsorption Capacity on Ion-ExchangerBovine Serum Albumin(DEAE-type packings,40-90 mm)Cytochrome C(SP-type packings,40-90 mm)Binding capacity was calculated from the absorbance at 280 nm of the eluted protein.*Exclusion limit:determined with PEO第8页，共46页，编辑于2022年，星期二8SuperMultiporeHZ-MTSKgel GMHHR-M0 00.10.10.20.20.30.30.40.40.50.50.0010.0010.010.010.10.11 1Pore Pore diameterdiameter mm dv/d(logr)dv/d(logr)m2/gm2/g Pore Size Technology:GPCWide-range Pore Distribution on Multi-pore Resin for GPCTSKgel GMHHR-M(Mixed-gel)TSKgel SuperMultiporeHZ-MWide range distribution of pore on SuperMultiporeHZ-M第9页，共46页，编辑于2022年，星期二9Pore Size Technology:GPCLinear Calibration Curve on TSKgel SuperMultiporeHZ-M第10页，共46页，编辑于2022年，星期二10Pore Size Technology:GPCSynthetic Polymer Separation with Multipore and Mixed-Resin TypeColumn：TSKgelG4000+3000+2500+2000HXL(7.8mmI.D.x 30cm x 4)TSKgel MultiporeHXL-M x 4 Eluent :THF Flow rate:1.0mL/min Detector:RIInjection volume:100L Concentration:2.0mg/mLG4000+3000+2500+2000HXLMultiporeHXL-M x 4Phenol resinInflection point(pseudo-peak)第11页，共46页，编辑于2022年，星期二11Pore Size Technology:BioseparationNon-Porous Resin/Parameter for Peak Broadening(A)Eddy diffusion(B)Longitudinal diffusion(C)Stagnant mobile phase mass transfer(inside pore)(D)Stationary phase mass transferNo effect on Non-Porous Resin;HETP improve resolution by IEC,HIC,RPC 第12页，共46页，编辑于2022年，星期二Pore Size Technology:BioseparationSeparation of pBR322 Hae-III Digest by Non-Porous Ion-Exchanger,TSKgel DNA-NPRColumn;TSKgel DNA-NPR (4.6 mmI.D.x 7.5 cm)Elution;(A):20 mM Tris-HCl(pH 9.0)(B):Buffer A containing 1.0 mol/l NaCl Linear gradient from 25%(B)to 45%(B)for 0.13 min,45%(B)to 50%(B)for 3.87 min,and then 50%(B)to 100%(B)for 76 miFlow rate;0.75ml/minTemperatuer;25 CDetection;UV 260nm 第13页，共46页，编辑于2022年，星期二13Surface ModificationGrafted PolymerizationNetting(Cross Linking)Functional Group:Flat Surface Graft Polymer Loose Cross LinkingAdsorption Capacity:Not So High Very High HighOperational Pressure Drop:Low High Medium Modification Technology:BioseparationChemistry for Functional Group on IEC,HIC and AFCProteinFunctionalgroup第14页，共46页，编辑于2022年，星期二14Modification Technology:ODSEndcapping of Silanol Groups on RPC Silica(TSKgel ODS-100V)Difunctional alkyllsilane reagentResidual silanolOOSiOSiOOOOOOOSiOSiOOOOOCH3SiOSiOOOSiOSiOOOSiCCCCCCCCCCCCCCCCCCH3CH3SiCCCCCCCCCCCCCCCCCCH3SiR1R2SiOOOOHSiR3R4Special endcapping procedureSilanol coverageSurface polarityHigher stability in low pH mobile phase第15页，共46页，编辑于2022年，星期二15Modification Technology:ODSSurface is Well Coverd byMinimize;ionic interaction chelating interaction第16页，共46页，编辑于2022年，星期二16Modification Technology:ODSBi-Functional(Inner/Outer)Surface for Direct Injection of Serum containing Small Drugs by Protein-Coated ODSBase ODS particles(TSKgel ODS-100V 5m)Particle size(m)5Pore size(nm)10Specific surface area(m2/g)450Pore volume(mL/g)1.1FunctionalityOctadecyl groupCarbon content*(wt%)15BSA-coated ODS particlesSurface functionalityDenatured and cross-linked BSAProteinDrugsProtein coating(outer surface)ODS phase(inner pore)Protein molecule is excluded from columnODS-100V第17页，共46页，编辑于2022年，星期二17Modification Technology:ODSDirect Separation of Serum for Therapeutic Drug Monitoring(TDM)on TSKgel BSA-ODS/VConditions;Column:TSKgel BSA-ODS/V(2 mm I.D.x 5 cm)Eluent:(A)20mmol/L HCO2NH4(pH4.5),(B)20mmol/L HCO2NH4(pH4.5)/MeOH=40/60 Gradient:0min(B 0%)-5min(B 0%)-15min(B100%)-15.5min(B100%)-16min(B 0%)Flow rate:0.4mL/min,Detection:UV(313 nm)第18页，共46页，编辑于2022年，星期二18Application on TSK-GELnSize-exclusion chromatographynIon-exchange chromatographynHydrophobic interaction chromatographynAffinity chromatographynReversed-phase chromatographynNormal-phase chromatography(HILIC)nIon chromatography第19页，共46页，编辑于2022年，星期二19Recent New Products in TSK-GELnSEC(smaller particle size,special pore,packing know how)TSKgel SuperSW3000TSKgel SuperMultiporeHZ-MnIEC(pore size optimization,surface modification)TSKgel BioAssist Q,S,Toyopearl GigaCap S-650MnHIC(pore size optimization)Toyopearl PPG-600M,Butyl-600MnAFC(surface modification)Toyopearl AF-Blue HC 650M,AF-Heparin HC 650MnRPC(smaller particle,base material,surface modification)TSKgel ODS-100V 3 um,ODS-100Z 3um,SuperODS for UPLCTSKgel BSA-ODS/V nHILIC(NPC)(smaller particle,surface modification)TSKgel Amide-80 3um,TSKgel VMpak(mixed mode)nIC(smaller particle,surface modification)TSKgel SuperIC-Anion AZ,TSKgel SuperIC-A/C第20页，共46页，编辑于2022年，星期二20Size-Exclusion ChromatographynFor bio-moleculesSW;silica based,proteins,peptides PW;resin based,aqueous polymernFor synthetic polymerPW;aqueous mobile phaseAlpha;aqueous/organic mobile phaseH;organic mobile phaseLinear type;Mixed-gel(M),Multi-pore type(MultiporeH)HR;Solvent replacement-resistant typenResolutionXL type(5 um)for SW,PW,HSuper type(3 um)for SW and H(Alpha)第21页，共46页，编辑于2022年，星期二21TSK-GEL for BioseparationnTSKgel SuperSW3000nTSKgel BioAssist seriesnPreparative purificationnNon-Porous Ion-ExchangernHydrophobic interaction chromatographynAffinity chromatography第22页，共46页，编辑于2022年，星期二22BioAssist SW seriesPorous hydrophilic silica base3 types for various MW proteinsParticle size;mm SEC columnsHigh resolutionHigh recoveryBioAssist 6PW Porous hydrophilic resin baseParticle size;17 umSEC for DNA and PlasmidHigh recovery BioAssist DS(Desalting)Porous hydrophilic resin baseParticle size;umDesalting and solvent replacementof protein and nucleic acidBioAssist IECPorous hydrophilic resin baseIonic group(Q or S)Particle size;7 or 13 umFor protein and nucleic acidHigh resolutionHigh capacityBioAssist Chelate(AFC)Porous hydrophilic resin baseParticle size;10 micronMetal chelate affinityFor His-tag proteinBioAssist PhenylPorous hydrophilic resin baseParticle size;10 umHIC columnProtein separationSECHICAFCIECBioseparation on TSK-GEL BioAssist Series第23页，共46页，编辑于2022年，星期二23TSK-GEL Product Line up for BioseparationTOYOPEARL PAK seriesMicro purification(ng order)TSK-GEL BioAssist series&5PW derivatives,SW,PWToyoScreen seriesTSK-GEL NPR seriesLab scale purificationSemi prep.Scale(gram order)TOYOPEARL seriesFor Lab第24页，共46页，编辑于2022年，星期二24Comparison of Resolution on Micro-SEC ColumnsColumn:TSKgel SuperSW3000 (1.0 mmI.D.x 30 cm)Shodex KW-803 1E (1.0 mmI.D.x 25 cm)Eluent:0.1mol/L Phosphate buffer +0.1mol/L Na2SO4 +0.05%NaN3Flow rate:16 uL/minDetection.:UV(280 nm)Temp.：25 CInj.Volume:0.2 uLSamples:Standard samples 1.Tyroglobulin(1.0 g/L)2.Gamma-Globulin(2.0 g/L)3.Ovalbumin(2.0 g/L)4.Ribonuclease A (3.0 g/L)5.p-Aminobenzoic acid (0.02g/L)SuperSW3000Shodex KW-803E第25页，共46页，编辑于2022年，星期二25Sensitivity on Column Size on TSKgel SuperSW3000Column:TSKgel SuperSW3000 (1.0 mmI.D.x 30 cm)Eluent:0.1mol/L Phosphate buffer +0.1mol/L Na2SO4 +0.05%NaN3Flow rate:16 uL/minDetection.:UV(280 nm)Cell vol.:2 uL(4.6 mmI.D.)35nL(1.0,2.0 mmI.D.)Temp.：25 CInj.Volume:0.2 uLSamples:Standard samples 1.Tyroglobulin(1.0 g/L)2.Gamma-Globulin(2.0 g/L)3.Ovalbumin(2.0 g/L)4.Ribonuclease A (3.0 g/L)5.p-Aminobenzoic acid (0.02g/L)第26页，共46页，编辑于2022年，星期二26TSK-GEL for Small CompoundsnReversed-phase chromatographynNormal-phase chromatographynMixed-mode chromatographynIon chromatography第27页，共46页，编辑于2022年，星期二27Properties of TSK-GEL RPC columns第28页，共46页，编辑于2022年，星期二28ODS Columns Mapping(Relationship between Hydrophobicity and Surface Porality)第29页，共46页，编辑于2022年，星期二29A)pH 7.0B)pH 4.5Conditions Column;TSKgel ODS-100V TSKgel ODS-100Z (4.6mm I.D.15cm)Eluent ;A)50mmol/L P.B.(pH 7.0)/MeOH(30/70)B)50mmol/L NaH2PO4/MeOH(50/50)Flow ;1.0mL/min Temp.;40 Inj.Vol.;10L Sample ;A)Desipramine B)NortriptylineComparison of Peak Shape(Asymmetry Factor)for Basic Compounds第30页，共46页，编辑于2022年，星期二30 Column :TSKgel ODS-100V(4.6mm I.D.15cm)Inj.Vol.:10L Eluent :10mmol/L Phosphate Buffer(pH 6.8)Detect :UV(260nm)Flow Rate:1.0mL/min Temp.:40 Sample :1.Cytocine,2.Uracil,3.Uridine,4.Adenine,5.GuanosineTSKgel ODS-100V is durable to dewetting,and applicable to 100%aqueous mobile phase without decrease of retention.Separation of Hydrophilic Compounds on TSKgel ODS-100V with 100%Aqueous Mobile PhaseAfter 30 min from flow-stop1 23 4第31页，共46页，编辑于2022年，星期二ConditionsColumn:TSKgel ODS-100V 3m (2.0mmI.D.10cm)Eluent:A)H2O/CH3CN(40/60)B)H2O/MeOH(30/70)Flow rate:A)0.50 mL/min B)0.20mL/minDetection:UV 254 nmTemp.:25Inj.vol.:1LSample:1.Uracil 2.Caffeine 3.Phenol 4.Methyl benzoate 5.Benzene 6.TolueneLow viscosity in acetonitrile enables high-through put analysis.High-throughput Separation on 3 micron ODS0.5 mL/min0.2 mL/min第32页，共46页，编辑于2022年，星期二32uracil void volume markertoluene hydrophobic retentionethylbenzene methylene selectivityquinizarin activity towards chelating reagentsamitriptyline activity towards basesStandards Reference Material 870 for RPC(National Institute Standards&Technology)第33页，共46页，编辑于2022年，星期二33A)A)1.1.2.2.3.3.4.4.5.5.B)B)C)C)D)D)ConditionsColumn:A)TSKgelODS-100V3m(4.6mmI.D.15cm)Inj.Vol.:10LB)InertsilODS-33m(4.6mmI.D.15cm)Detect:UV254nmC)L-Column3m(4.6mmI.D.15cm)Temp.:40D)CAPCELLPAKMG3m(4.6mmI.D.15cm)Eluent:20mmol/LP.B.(pH7.0)/MeOH(20/80)FlowRate:1.0mL/minSample:1.Uracil,2.Toluene,3.Ethylbenzene,4.Qumizarine,5.AmitriptylineQunizarineQunizarineTP=20973Asf=1.09TP=20973Asf=1.09AmitriptylienAmitriptylienTP=18201Asf=1.15TP=18201Asf=1.15QunizarineQunizarineTP=22119Asf=1.32TP=22119Asf=1.32AmitriptylienAmitriptylienTP=21501Asf=1.27TP=21501Asf=1.27QunizarineQunizarineTP=14489Asf=6.76TP=14489Asf=6.76AmitriptylienAmitriptylienTP=20270Asf=2.10TP=20270Asf=2.10QunizarineQunizarineTP=15987Asf=1.95TP=15987Asf=1.95AmitriptylienAmitriptylienTP=15898Asf=1.26TP=15898Asf=1.26444TSKgelInertsilL-ColumnCapcellpakChromatogram of SRM870(1)第34页，共46页，编辑于2022年，星期二LC/MS of beta-Lactoglobulin/Trypsin Digest by RPCon TSKgel ODS-100V 3 umInstrument:QTRAP (Applied Biosystems)Ion source :ESIPolarity:PositiveColumn:TSKgel ODS-100V 3m(2.0mmI.D.x 15cmL)Eluent:A;0.1%TFA in H2O B;0.1%TFA in CH3CNGradient:0min(B 10%)15min(B 70%)17min(B 70%)Flow rate:0.2mL/minInjectin vol.:2LSample:-Lactoglobulin tryptic digest1234567891110 1213151614171819208m/z1;331.02;409.03;347.44;857.85;916.96;673.67;623.68;573.59;934.010;818.911;247.012;970.013;1066.014;696.715;815.016;937.917;1106.418;1158.019;1192.020;555.6Application to Peptide-MS-Fingerprint(PMF)method.第35页，共46页，编辑于2022年，星期二35LC/MS of beta-Lactoglobulin/Trypsin Digest by HILICon TSKgel Amide-80 5 umInstrument:QTRAP (Applied Biosystems)Ion source :ESIPolarity:PositiveColumn:TSKgel Amide-80 5m(2.0mmI.D.x 15cmL)Eluent:A;0.1%TFA in H2O B;0.1%TFA in CH3CNGradient:0min(B 95%)15min(B 50%)17min(B 50%)Flow rate:0.2mL/minInjectin vol.:1LSample:-Lactoglobulin tryptic digest12345678911101213151614171819208m/z1;331.02;409.03;347.44;857.85;916.96;673.67;623.68;573.59;934.010;818.911;247.012;970.013;1066.014;696.715;815.016;937.917;1106.418;1158.019;1192.020;555.620HILIC/NPC has different selectivity of peptides from RPC by ODS column.第36页，共46页，编辑于2022年，星期二36LC/MS of beta-Lactoglobulin/Trypsin Digest by SECon TSKgel SuperSW2000 semi-micro columnInstrument:QTRAP (Applied Biosystems)Ion source :ESIPolarity:PositiveColumn:TSKgel SuperSW20000(Specially prepared)(2.0mmI.D.x 25cmL)Eluent:0.1%TFA in(H2O/CH3CN=55/45)Flow rate:0.065mL/minInjectin vol.:1LSample:-Lactoglobulin tryptic digestm/z1;331.02;409.03;347.44;857.85;916.96;673.67;623.68;573.59;934.010;818.911;247.012;970.013;1066.014;696.715;815.016;937.917;1106.418;1158.019;1192.020;555.6第37页，共46页，编辑于2022年，星期二37Hydrophilic Interaction Chromatography(HILIC)nA mode of normal phase chromatographynInteracts with hydrophilic compounds which RPC/ODS column cannot retain.nDifferent selectivity from RPCnTarget moleculesLow MW hydrophilic compoundPeptideOligonucleotideOligosaccharide第38页，共46页，编辑于2022年，星期二38TSKgel Amide-80Column AColumn BColumn CTP:13,600TP:6,700TP:9,700TP:4,700Comparison of Commercial HILIC columnsConditionsColumn;1 mm ID x 15 cmEluent:CH3CN/H2O=75/25Flow rate:20L/minTemp.:25CDetection:UV 254nmSample:Uracil(35mg/L,0.5）System:Agilent 1100 Cap-LC第39页，共46页，编辑于2022年，星期二39Pyridylamino-Saccharides Separation by HILIC on TSKgel Amide-80 3 um and 5 um5 um3 umPA saccharides are obtained from TAKARA Biochemicals.The courtesy of Dr.Miyamoto,Osaka Medical Center for Cancer and Cardiovascular Diseases.第40页，共46页，编辑于2022年，星期二40Mixed-Mode Separation on TSKgel VMpak-25COO-COO-COO-COO-COO-ACBHydrophobic moietyIonic interaction PartitionIonic compoundHydrophobic/acidic compoundHydrophobic/basic compoundMixed-Mode column should be applied to compounds,which RPC nor HILIC column cannot separate,nor better resolution or recovery.PartitionRep