Column Selection for Size Exclusion …

1 HighPolarity of SolventLow42 Guideline of AvailabilityOrganic SEC(GPC)THFKF-800 seriesTHFLF seriesDMFDMFKD-800 seriesSB-800 HQ seriesDMFSB400 seriesODCB seriesUT-800 seriesAT-806 MSHFIP-800 seriesHFIP-600 seriesTHFKF-600 seriesTHFKF-400HQ serieschloroformK-800 seriesAqueous/Organic SECGF-HQ seriesH2 ODMSODMFTHFA cetonitrileChloroformTolueneMethanolKW-8 00 series, KW400 seriesSB-800 HQ series, SB400 seriesGS-HQ seriesKD seriesKF, K, LF seriesGF-HQ seriesApplicationSolventPageColumnAqueou s SEC(GFC)Analysis of biological macromolecules (proteins, peptides, nucleic acids, etc.)Analysis of general polymersAnalysis of biological macromolecules(high MW range)Water-soluble polymers (polyacrylamide, polyethylenimine, etc.)

2 Polar polymers (polyimides, polyvinylpyrrolidones etc.)Engineering resin analysis at room temperature [polyamide (nylon), polyethylene terephthalate (PET) etc.]Analysis at high temperature (polyethylene, polypropylene etc.)buffer seriesKW400 series SB-800 HQ seriesSB400 seriesH2O, bufferaqueous solution HQ seriesSB400 seriesOligosaccharide, polysaccharidesH2O,aqueous solutionKS-800 seriesGS-HQ series4444464646463248525856565254464658 58586060 HFIPLF-4045850 Column Selection for size Exclusion Chromatography (SEC)Standard analysiscolumnsColumn Selection for size Exclusion Chromatography (SEC)* See page 62 for the solvent replaceability of organic solvent SEC (GPC) packed columns .

3 High performance(solvent-saving)High performance(solvent-saving)High performance (solvent-saving)High performance(solvent-saving)High linearity of calibration curveRapid analysis(solvent-saving)High performance(solvent-saving)Rapid analysis(solvent-saving)(A)(C)(D)(B)log MW43 Interfering interactions likely to be observedMethods to reduce interactionsOrganic solvent SEC (GPC) columnsInteractions between the analyte and the packing materialsIonic InteractionAqueous SEC (GFC)Ionic InteractionPrecautions for polar polymer analysisSize Exclusion chromatography analysis of polar-polymer can be influenced by unexpected interactions in the Column .

4 These interactions will result in changing elution patterns and may cause the invalid molecular weight calculations. It is important to reduce them in order to obtain the accurate molecular weight distribution. Hydrophobic interactionThe analyte is adsorbed into the packing material. This delays the analyte elution, and thus results in underestimation of its molecular weight (Figure B, D).Ionic interaction(1) Ion ExclusionThe analyte is repelled from the packing material. This accelerates the analyte elution, and thus results in overestimation of its molecular weight (Figure A, C)(2) Ion ExchangeThe analyte is adsorbed onto the packing material.

5 This delays the analyte elution, and thus results in underestimation of its molecular weight (Figure B, D).Precautions for polar polymer analysisInteraction within and between the analyteIonic repulsion effects observed within the multivalent macromolecules causes structure expansionThis accelerates the analyte elution, and thus results in overestimation of its molecular weight (Figure A).Association between the moleculesAssociated molecule detected as a larger molecule (Figure A).Interactions between the analyte and the solventThe multivalent ion of the solvent works as a bridge to bind ionic molecules (analyte).Elution will be will salt (Example) Add LiBr to DMF Add CF3 COONa to HFIPAdd saltHydrophobic interactionHydrophobic interactionLower the polarity of the eluent(Example) Change the eluent from DMF to THFH ydrophillic interactionIncrease the polarity of the eluent(Example) Change the eluent from THF to DMFI ncrease dissociation of the analyteCationic polymer Lower the pHAnionic polymer Higher the pHLower the eluent polarity(Example)

6 Add acetonitrile or methanolExclusion limitMWExclusionvolumePermeationvolume Information44F6989000F6989103F6989104F67 00131 PROTEIN KW-803 PROTEIN KW-804 PROTEIN 504001,0001,500 21,000 21,000 16,000(guard Column )60,000170,000500,000 150,000700,000(1,000,000)* 60,000150,000500,0001,300,000 150,000600,000(1,000,000)*(20,000,000)* 4 FKW404- 4 FKW405- 4 FKW400G - 104008001,5002,000 35,000 35,000 25,000 25,000(guard Column )Base Material : SilicaUsable pH range : maximum usable concentration is 100% for methanol and acetonitrile.

7 * Estimated value within parenthesisBase Material : SilicaUsable pH range : maximum usable concentration is 100% for methanol and acetonitrile.* Estimated value within , , 403KW-804, 404KW405 Aqueous SEC (GFC) columns : Silica-basedStandard analysiscolumnsAqueous SEC (GFC) columns : Silica-based*Contact Shodex or our distributors near you for customized page 63 forCalibration StandardsCalibrationStandardsSilica-base d packed columns for aqueous SEC (GFC) analysisSuitable for the analysis of proteins and , particle size of the packing material enhances Column performanceThree- or four-fold higher sensitivity than KW-800 seriesKW405-4F is applicable to samples with a molecular weight above 1 millionKW400 Semi-microMicro size (mm) x LProduct CodeProduct NameParticleSize( m)Plate Number(TP/ Column )Standard columnsHigh performance semi-micro columnsExclusion Limit(Pullulan)(Protein)

8 ShippingSolventMaximumPore size ( ) Column size (mm) x LProduct CodeProduct NameParticleSize( m)Plate Number(TP/ Column ) Exclusion Limit(Pullulan)(Protein)ShippingSolventM aximumPore size ( )H2OH2OH2OH2OH2OH2OH2OH2OH2 OMolecular weight (Protein)Molecular weight range with protein (eluent : phosphate buffer)(A) ,000N=8,2004512345(B) HQGF-510 HQKW-803051015202530111315171921 min3551020304015 min50 min0510 volume (mL)Molecular weight (Protein)5791168101213 Elution volume (mL)Molecular weight (Protein)Molecular weight (Protein) HQGF-510 HQKW-803101510152020 min30 min10210310410510610745 LectinsRecovery rate of proteinsColumn : Shodex : 50mM Sodium phosphate buffer + NaCl( )Flow rate : : UV(220nm) (small cell volume) Column temp.

9 : 30 CSample : 10 L1. Blue dextran 2000 -Globulin Ovalbumin Myogrobin Uridine : A : Thyroglobulin (bovine)B : -Globulin (bovine)C : Ovalbumin (chicken)D : Nyoglobin (horse)E : CyanocobalaminSample : Whey, 5 LSample : 5 L1. Lectin from Soybean Lectin from Arachis hypogaea Lectin from Canavalia ensiformis (Con A) Lectin from Lens culinaris (LCA) Lectin from Triticum vulgaris (WGA) : Human serum each1. Fibrinogen 50 L2. 2 -Macroglobulin 50 L3. IgG 50 L4. Transferrin 50 L5. Plasminogen 50 L6. Albumin 100 L7. Antitrypsin 100 L8. Hemoglobin 100 LComparison of and whey in yogurtColumn : (A) Shodex (B) Shodex PROTEIN : 50mM Sodium phosphate buffer + NaCl( )Flow rate : (A) , (B) Detector : UV(280nm) (small cell volume) Column temp.

10 : 25 CColumn : Shodex OHpak SB-803 HQ, Asahipak GF-510 HQ, PROTEIN KW-803 Eluent : Phosphate buffer( )Flow rate : : UV(280nm) Column temp. : 30 CColumn : Shodex PROTEIN KW-803 x 2 Eluent : 50mM Phosphate buffer + NaCl( )Flow rate : : UV(280nm) Column temp. : Room : Shodex + KW403-4F Eluent : 50mM Sodium phosphate buffer + NaCl( )Flow rate : : UV(280nm) (small cell volume) Column temp. : 30 CColumn : Shodex , KW403-4F Eluent : 50mM Sodium phosphate buffer + NaCl( )Flow rate : Detector : UV(280nm) (small cell volume) Column temp. : 25 CColumn : Shodex KW400-4F series, Shodex PROTEIN KW-800 seriesEluent : 50mM Sodium phosphate buffer + NaCl( )Flow rate : (KW400) (KW-800) : (KW400) UV(280nm) (small cell volume) (KW-800) UV(280nm) (conventional type) Column temp.