Column Selection for Size Exclusion Chromatography (SEC)

Transcription

1 Column Selection for Size Exclusion Chromatography (SEC) Application Solvent Column Page Biological macromolecules (proteins, peptides, nucleic acids, etc.) Buffer etc. KW-800 series KW400 series High performance (solvent-saving) Aqueous SEC (GFC) Biological macromolecules (high MW range) Water-soluble polymers (polyacrylamide, polyethylenie, etc.) Polysaccharides Buffer etc. H O, Buffer Aqueous solution etc. SB-800 HQ series LB-800 series SB-800 HQ series LB-800 series Oligosaccharide, polysaccharides H O, Aqueous solution etc. KS-800 series GS-HQ series 4 4 KF-800 series 4 General polymers THF KF-00 series KF-400HQ series Rapid analysis (solvent-saving) High performance (solvent-saving) LF series High linearity of calibration curve Chloroform K-800 series 48 Organic SEC (GPC) Polar polymers (polyimides, polyvinylpyrrolidones etc.) DMF KD-800 series 0 SB-800 HQ series 8 LB-800 series 8 Analysis at high temperature (polyethylene, polypropylene etc.) ODCB etc. HT-800 series UT-800 series AT-80MS Engineering resin analysis at room temperature [polyamide (Nylon), polyethylene terephthalate (PET) etc.] HFIP HFIP-800 series HFIP-00 series Rapid analysis (solvent-saving) High linearity of calibration curve 0 0 LF series Aqueous/Organic SEC GF-HQ series 44 Guideline for column solvent usability High Polarity of solvent H O Methanol DMSO Acetonitrile DMF THF Chloroform Toluene Low KW-800 series, KW400 series SB-800 HQ series, LB-800 series GS-HQ series GF-HQ series KD series 4 KF, K, LF series * See page for the solvent replaceability of organic solvent SEC (GPC) packed columns.

2 Precautions for Polar Polymer Analysis Size exclusion chromatography analysis of polar polymers can be influenced by unexpected interactions in the column. These interactions may change elution patterns and results in an invalid molecular weight calculation. It is important to reduce them in order to obtain the accurate molecular weight distribution. Interactions between the analyte and the packing materials Hydrophobic interaction The analyte is adsorbed into the packing material. This delays the analyte elution, and thus results in under estimation of its molecular weight (Figure B, D). Ionic interaction () Ion Exclusion The analyte is repelled from the packing material. This accelerates the analyte elution, and thus results in over estimation of its molecular weight (Figure A, C). () Ion Exchange The analyte is adsorbed onto the packing material. This delays the analyte elution, and thus results in under estimation of its molecular weight (Figure B, D). Interaction within and between the analyte Ionic repulsion effects observed within the multivalent macromolecules causes structure expansion This accelerates the analyte elution, and thus results in over estimation of its molecular weight (Figure A). Association between the molecules Associated molecule detected as a larger molecule (Figure A). Interactions between the analyte and the solvent The multivalent ion of the solvent works as a bridge to bind ionic molecules (analyte). Aqueous SEC (GFC) Interfering interactions likely to be observed Exclusion limit molecular weight log MW (C) Methods to reduce interactions Elution will be accelerated Exclusion limit volume (A) Elution will be retarded Organic solvent SEC (GPC) (B) Permeation limit volume (D) Column Selection for Size Exclusion Chromatography (SEC) Precautions for Polar Polymer Analysis Ionic Interaction Add salt Hydrophobic interaction Increase dissociation of the analyte Cationic polymer Lower the ph Anionic polymer Higher the ph Lower the eluent polarity (Example) Add acetonitrile or methanol Ionic Interaction Add salt (Example) Add LiBr to DMF Add CF COONa to HFIP Hydrophobic interaction Lower the polarity of the eluent (Example) Change the eluent from DMF to THF Hydrophillic interaction Increase the polarity of the eluent (Example) Change the eluent from THF to DMF

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7 Calibration curves for SB-800 HQ series using pullulan (eluent : H O) Calibration curves for SB-800 HQ series using PEG/PEO (eluent : DMF) Carboxymethylcellulose 7 7 Sample : Carboxymethylcellulose 0.% each, 0µL,00-,000cP Molecular weight (Pullulan) 4 SB-804 HQ SB-80 HQ SB-80. HQ SB-80 HQ SB-80 HQ SB-80 HQ SB-80M HQ Molecular weight (PEG/PEO) 4 SB-804 HQ SB-80 HQ SB-80. HQ SB-80 HQ SB-80 HQ SB-80M HQ Mn : 0,00 Mw :,940,00 Mw/Mn : cP Mn :,400 Mw :,,000 Mw/Mn : cP Mn : 4,00 Mw : 40,000 Mw/Mn : * Molecular weight was detered from the calibration curve of pullulan. Column : Shodex OHpak SB-800 HQ series Eluent : H O Column temp. : 0 C Column : Shodex OHpak SB-800 HQ series Eluent : DMF Column : Shodex OHpak SB-80M HQ x Eluent : 0.M NaCl aq. Effects of sodium nitrate in eluent on the analysis of polyallylae Gelatin For the analysis of cationic polymers, such as pollyallylae, undesired adsorption of the polymer is observed when low (0.0M) sodium nitrate eluent was used. By using higher concentration (> 0.M) salt, it suppresses the sample adsorption and enables to obtain accurate chromatograms. Sample : Polyallylae 0.%, 0µL 0. M 0.0 M Sample : 0.% each, 0μL Gelatin from bovine skin (Acid treatment, Gel strength : g) Gelatin from porcine skin (Alkali treatment, Gel strength : 90-0g) 0. M From bovine skin Mn :,400 Mw : 8,0 Mw/Mn : M From porcine skin Mn :,00 Mw : 8,700 Mw/Mn : Volume (ml) 0 0 * Molecular weight was detered from the calibration curve of pullulan. Column : Shodex OHpak SB-80M HQ x Eluent : 0.M Acetic acid + NaNO aq. Column : Shodex OHpak SB-80M HQ x Eluent : 0.M KH PO 4 aq./ 0.M Na HPO 4 aq.=0/0 Polyacrylamide Cellulose acetate SB-80 HQ Sample : Polyacrylamide, 0µL Sample : Cellulose acetate 0.% each, 0μL.0x 8 SB-807 HQ Mn~0,000 Molar Mass (g/mol).0x 7.0x Mw:,00,000 Mn~0, x Volume (ml) 40 Column : Shodex OHpak SB-807 HQ, SB-80 HQ Eluent : 0.M NaCl aq. Flow rate : 0.mL/ MALS (Multi angle light scattering) Column temp. : 0 C Column : Shodex OHpak SB-80M HQ x Eluent : 0mM LiBr in DMF

8 Copovidones Sample : 0μL Poly(-vinylpyrrolidone-co-vinyl acetate) 0.% each Copolymer 7: Mn :,000 Mw : 4,400 Mw/Mn : 7.40 Copolymer :7 Mn :,400 Mw : 8,900 Mw/Mn : * Molecular weight was detered from the calibration curve of PEG/PEO. Column : Shodex OHpak SB-80M HQ x Eluent : 0mM LiBr in DMF Calibration curves for LB-800 series using pullulan (eluent : H O) Molecular weight (Pullulan) 7 4 LB-80 LB-80M Column : Shodex OHpak LB-800 series Eluent : H O Column temp. : 0 C Calibration curves for LB-800 series using PEG/PEO (eluent : DMF) Molecular weight (PEG/PEO) LB-80 LB-80M Column : Shodex OHpak LB-800 series Eluent : DMF Aqueous SEC (GFC) Columns : Polymer-based Comparison of the detection of pullulan using multiangle light scattering detector The multiangle light scattering (MALS) signal of low molecular weight substances is low with competitors columns due to the baseline noise. New product OHpak LB-800 series enables the detection by improving the baseline noise level. LB-80M (8.0mm I.D. x 00mm) Sample : Pullulan (MW :,000) 0.%, µl 0 00 LB-80M (8.0mm I.D. x 00mm) SB-80M HQ (8.0mm I.D. x 00mm) SEC column from other manufacturer (7.8mm I.D. x 00mm) SB-80M HQ (8.0mm I.D. x 00mm) Signal to noise ratio (S/N) SEC column from other manufacturer (7.8mm I.D. x 00mm) 0,40,00,000 0,800 Molecular weight (Pullulan) 0 Column : Shodex OHpak LB-80M Shodex OHpak SB-80M HQ SEC column from other manufacturer Eluent : 0.M NaNO aq. Detector : MALS (Multi angle light scattering) (90 ) Column temp. : 0 C Sodium alginate Sodium heparin Sample : Sodium alginate 0.%, 0µL Sample : Sodium heparin 0.%, 0µL.0x 8.0x 8.0x 7.0x 7 Molar Mass (g/mol).0x.0x.0x Molar Mass (g/mol).0x.0x.0x Mn : 8,790 Mw :,00 Mw/Mn : Mn :,070 Mw :,0 Mw/Mn :, Time () Time () Column : Shodex OHpak LB-80M x Eluent : 0.M NaNO aq. MALS (Multi angle light scattering) Column temp. : 0 C Column : Shodex OHpak LB-80M x Eluent : 0.M NaNO aq. MALS (Multi angle light scattering) Column temp. : 0 C 4

9 Multimode Columns Features GS-HQ SEC is the main separation mode Depends on the eluent selected, the column adds multimode features of reversed phase, HILIC, and ion exchange modes to SEC Suitable for the separation of peptides or nucleic acids with similar molecular weights Suitable for desalting samples or substituting buffer in protein analysis Standard columns Pore Size (Å) Shipping Solvent F70000 Asahipak GS-0 HQ 9, H O/CH OH=70/0 F70000 Asahipak GS-0 HQ 9, H O/CH OH=70/0 F Asahipak GS-0 HQ 7, H O/CH OH=70/0 F Asahipak GS-0 HQ 7 7, H O/CH OH=70/0 F709 Asahipak GS-G 7B H O/CH OH=70/0 Semi-micro columns * The following semi-micro columns are made to order. Base Material : Polyvinyl alcohol Usable ph range : ph- (GS-0 HQ : ph-9) Usable concentration of methanol is up to 0% (GS-0 HQ : up to 0%) Usable concentration of acetonitrile is up to 0% Pore Size (Å) F770 GS0A-4D F770 GS0A-4E F770 GS0A-4B 7 7, Preparative columns * Preparative columns are made to order. Standard Column F807 Asahipak GS-0 0F 8, GS-0 HQ F808 Asahipak GS-0 0F 8, GS-0 HQ F809 Asahipak GS-0 0F 8, GS-0 HQ F800 Asahipak GS-0 0F 8, GS-0 HQ F804 Asahipak GS-0 0G 4, GS-0 HQ F80 Asahipak GS-0 0G 4, GS-0 HQ F80 Asahipak GS-0 0G 4, GS-0 HQ F807 Asahipak GS-0 0G 4, GS-0 HQ F70 Asahipak GS-0G 7B Target molecular weight range and Exclusion limit With Pullulan (eluent : ultrapure water) Target Molecular Weight Range Exclusion Limit GS-0 GS-0 00, ,000 7,000 40,000 4 GS-0 GS-0,000 00,000, ,000 00,000,000,000 * Please use the above table as a rough indication for the column selection.

10 Calibration curves for GS-HQ series using pullulan (eluent : H O) Molecular weight (Pullulan) 7 4 GS-0 HQ GS-0 HQ GS-0 HQ GS-0 HQ Peptides GS-HQ columns can be used not only for SEC (GFC) in an aqueous system, but also for multimodal analysis where hydrophobic interaction and ionic interaction are used together as separation criteria, under certain conditions of the eluent. This results in unprecedented separation analysis. GS-0 HQ columns are excellent in the performance for separating hydrophilic peptides, in particular, acidic or basic peptides, from each other. Glu-Ala-Glu Arg-Asp Gly-His-Lys Arg-Pro-Lys-Pro MW pl Σf Sample : 0μL. Glu-Ala-Glu 0.0%. Arg-Asp 0.0%. Gly-His-Lys 0.0% 4. Arg-Pro-Lys-Pro 0.0% Multimode Columns Σf : Hydrophobic parameter pl : Isoelectric point Column : Shodex Asahipak GS-HQ series Eluent : H O Flow rate : 0.mL/ Column temp. : 0 C Column : Shodex Asahipak GS-0 HQ Eluent : 0mM Ammonium acetate buffer (ph.7) Flow rate : 0.mL/ Detector : UV (0nm) Column temp. : 0 C Analysis of purine bases in beer Purine in foods is analyzed as purine base after a step of sample preparation; homogenization, freeze drying, hydolyzation with 70% perchloric acid, and neutralization. Example below shows the analysis of purin in regular beer and beer treated with guanase (an enzyme that degrades guanine to xanthine). The following data indicate that guanine was decreased and xanthine was increased by guanase. Purine bases in beer Normal beer Guanase treated beer adenine guanine guanine hypoxanthine xanthine xanthine uric acid xanthine Column : Shodex Asahipak GS-0 HQ Eluent : 0mM Sodium phosphate buffer (ph.) Flow rate : 0.mL/ Detector : UV (0nm) Column temp. : C Data provided by Kiyoko Kaneko Ph.D., Faculty of Pharmaceutical Sciences, Teikyo University Low molecular weight water-soluble dietary fiber Umami Lignosulfonic acid By using GS-0 HQ, monosaccharides, disaccharides, and sugar alcohols elute after indigestible component faction (indicated by an arrow on the chromatogram). This separation makes the method preferable for the quantification of low molecular weight water-soluble dietary fiber. Sample : 0μg/mL each, 0μL. IMP. GMP. AMP 4. Inosine. Hypoxanthine. Guanosine 7. Guanine 8. Adenosine 9. Adenine Sample : 0μL Lignosulfonic acid sodium salt 0.% Column : Shodex Asahipak GS-0 HQ x Eluent : H O Flow rate : 0.mL/ Column temp. : 0 C Column : Shodex Asahipak GS-0 HQ Eluent : mm NaH PO 4 aq./mm Na HPO 4 aq. =00/ Detector : UV (0nm) Column : Shodex Asahipak GS-0 HQ x Eluent : 0mM Na HPO 4 aq. Flow rate : 0.mL/ Detector : UV (4nm) 4

11 Aqueous/Organic SEC Columns Features GF-HQ Polymer-based SEC columns exhibit high solvent durability Supports both aqueous and organic solvents Standard columns Pore Size (Å) Shipping Solvent F Asahipak GF- HQ 9, H O F70000 Asahipak GF- HQ 9, H O/CH OH=70/0 F70000 Asahipak GF- HQ 9,000, H O/CH OH=70/0 F70000 Asahipak GF-7 HQ,000 9, H O/CH OH=70/0 F Asahipak GF-7M HQ,000 9, H O/CH OH=70/0 F708 Asahipak GF-G 7B H O/CH OH=70/0 F7000 MSpak GF- 4B, H O F700 MSpak GF- 4D, H O F70004 MSpak GF- 4E, H O F7000 MSpak GF- D, H O GF-7M HQ is a mixed-gel column capable of analyzing samples over a wide range of molecular weight. Base Material : Polyvinyl alcohol Usable ph range : ph-9 Semi-micro columns * The following semi-micro columns are made to order. Pore Size (Å) F70000 Asahipak GF- 4D F7000 Asahipak GF- 4E F770 GFA-4D, F770 GFA-4E, F7707 GF7A-4D 9, Preparative columns * Preparative columns are made to order. Standard Columns F800 Asahipak GS- 0F 8, GF- HQ F80 Asahipak GS- 0F 8, GF- HQ F80 Asahipak GS-7 0F 8, GF-7 HQ F80 Asahipak GSM-700 0F 8, GF-7M HQ F808 Asahipak GS- 0G 4, GF- HQ F809 Asahipak GS- 0G 4, GF- HQ F8040 Asahipak GS-7 0G 4, GF-7 HQ F804 Asahipak GSM-700 0G 4, GF-7M HQ F700 Asahipak GS-G 7B Target molecular weight range and Exclusion limit With Pullulan (eluent : ultrapure water) With *PEG/PEO (eluent : DMF) 44 GF- GF- GF- GF-7 GF-7M * Please use the above table as a rough indication for the column selection. Target Molecular Weight Range 00 4, ,000,000 00,000 0,000 *(,000,000) 00 *(,000,000) Exclusion Limit 9,000 40,000 00,000 *(,000,000) *(,000,000) GF- GF- GF- GF-7 GF-7M Target Molecular Weight Range 0, ,000,000 00,000 0,000 **(,000,000) 00 **(,000,000) *( ) Estimated value * Please use the above *PEG : polyethylene glycol table as a rough indication *PEO : polyethylene oxide for the column selection. **( ) Estimated value

12 Usable solvents Solvent Water 0-0.M sodium concentration Methanol Ethanol Acetonitrile THF DMF GF- GF- GF- GF-7 GF-7M * When replacing acetonitrile, ethyl acetate or chloroform with water, replace with methanol first and then replace with water. * When replacing water with ethyl acetate or chloroform, replace with methanol first and then replace with the required eluent condition. (Note) The usable solvents for preparative columns of GF-7 HQ, GS-7 0F and 0G, are water and methanol. GSM-700 0F or 0G is recommended when other solvents are required. Sodium polystyrene sulfonates MW:4, MW:8, Polymers having both hydrophobic and hydrophilic functional groups may exhibit hydrophobic interactions with packing materials. When analyzing such polymers, the hydrophobic interaction can be suppressed by adding organic solvents to the eluent. Sample : mg/ml each, 0µL Sodiumn polystyrene sulfonate CH CH SONa n Aqueous/Organic SEC Columns Acetone Chloroform Ethylacetate DMSO 0~0% 0 MW:8, Column : Shodex Asahipak GF- HQ Eluent : 0mM LiCl aq./ch CN =0/40 Flow rate : 0.mL/ Detector : UV (4nm) Column temp. : 0 C Calibration curves for GF-HQ series using pullulan (eluent : H O) Calibration curves for GF-HQ series using PEG/PEO (eluent : DMF) Polyacrylonitrile 7 7 Sample : Polyacrylonitrile 0.%, 0µL Molecular weight (Pullulan) 4 GF- HQ GF- HQ GF- HQ GF-7 HQ GF-7M HQ Molecular weight (PEG/PEO) 4 GF- HQ GF- HQ GF- HQ GF-7M HQ GF-7 HQ Column : Shodex Asahipak GF-HQ series Eluent : H O Flow rate : 0.mL/ Column temp. : 0 C Column : Shodex Asahipak GF-HQ series Eluent : DMF Flow rate : 0.mL/ Column : Shodex Asahipak GF-7 HQ x Eluent : 0mM LiBr in DMF Flow rate : 0.mL/ Comparison of two GF columns for the separation performance of maltooligosaccharides Comparison of polystyrenes separation using various solvents GF- HQ demonstrates an improved separation of low molecular substances. The sample below shows that the peaks obtained by GF- HQ are separated with deeper notches compared to peaks obtained by GF- HQ. GF- HQ is capable of separating oligosaccharides (trisaccharides to hexasaccharides) while those oligosaccharides were eluted all together when analyzed by GF- HQ. GF- HQ x GF- HQ x ~ 4 4 Sample : 0.% each, 0μL. Maltohexaose. Maltopentaose. Maltotetraose 4. Maltotriose. Maltose. Glucose (A) THF 4 Sample : Polystyrene mg/ml each, 0µL. Mw :,090,000. Mw : 90,000. Mw : 7, Mw : 9,0. Mw :,980. Mw : 00 (B) Chloroform 4 (C) DMF Column : Shodex Asahipak GF- HQ x Shodex Asahipak GF- HQ x Eluent : H O Flow rate : 0.mL/ Column temp. : 0 C Column : Shodex Asahipak GF- HQ + GF- HQ Eluent : (A); THF, (B); Chloroform, (C); DMF Flow rate : 0.mL/ Detector : (A),(B) UV (4nm), (C) UV (70nm) Column temp. : 0 C 4

13 Organic SEC (GPC) Columns (General Analysis) : THF Features KF-800 Standard organic solvent SEC (GPC) column Supports a wide range of applications from low to high molecular weight compounds Corresponds to USP L Standard columns KF-800 series : Shipping solvent Tetrahydrofuran (THF) Pore Size (Å) F080 GPC KF-80 0 F0800 GPC KF-80 0 F080 GPC KF F0800 GPC KF F0700 GPC KF-80L 00 F08040 GPC KF-804 7,00 F07040 GPC KF-804L 7,00 F0800 GPC KF-80,000,000 F0700 GPC KF-80L,000,000 F0800 GPC KF-80,000,000 F08090 GPC KF-80M,000,000 F0700 GPC KF-80L,000,000 F08070 GPC KF-807, ,000 F07070 GPC KF-807L, ,000 F70000 GPC KF-G 4A (GPC KF-G) 8 4. F7090 GPC KF-800D solvent-peak separation column * The columns with L or M at the end of column names are mixed-gel column capable of analyzing samples over a wide range of molecular weight distribution. * See page 0 for details of the solvent-peak separation columns. * See pages 4 and for details preparative columns. * See page for applicability of SEC (GPC) columns to solvent replacement. Target molecular weight range and Exclusion limit With Polystyrene (eluent : THF) Target Molecular Weight Range Exclusion Limit Target Molecular Weight Range Exclusion Limit KF ,00 KF-80 0,000,000,000 4,000,000 KF-80 00,000,000 KF-80L 00,000,000 4,000,000 KF ,000 0,000 KF-80 0,000 *(0,000,000) *(0,000,000) KF-80,000 0,000 70,000 KF-80M,000 *(0,000,000) *(0,000,000) KF-80L 0 0,000 70,000 KF-80L 00 *(0,000,000) *(0,000,000) KF-804 7,000 00, ,000 KF ,000 *(00,000,000) *(00,000,000) KF-804L 0 00, ,000 KF-807L 00 *(00,000,000) *(00,000,000) * Please use the above table as a rough indication for the column selection. *( ) Estimated value 4

14 Calibration curves for KF-800 series using polystyrene Molecular weight (Polystyrene) 7 4 KF-804 KF-80 KF-80 KF-80 KF-80 KF-80. KF-80 KF-807 KF-80M Column : Shodex GPC KF-800 series Calibration curves for KF-800L (linear type) series using polystyrene Molecular weight (Polystyrene) 7 4 KF-80L KF-80L KF-804L KF-80L KF-807L Column : Shodex GPC KF-800L series Fat-soluble vitas Sample : 0μL. Vita A palmitate 00μg/mL. Vita E 0μg/mL. Vita D 0μg/mL 4. Vita K 0μg/mL 4 0 Column : Shodex GPC KF-80 x Detector : UV (80nm) Organic SEC (GPC) Columns (General Analysis) : THF Standard polystyrenes 4 7 Sample : 0µL. Polystyrene (Mw :,000) 0.%. Polystyrene (Mw :,0) 0.%. Polystyrene (Mw : 9,00) 0.% 4. Polystyrene (Mw : 7,) 0.%. Polystyrene (Mw :,070) 0.%. Polystyrene (Mw : 80) 0.% 7. Toluene (Mw: 9) 0.% 4 Sample : 0μL. Polystyrene (Mw : 9,8,000) 0.0%. Polystyrene (Mw :,0,000) 0.%. Polystyrene (Mw :,000) 0.% 4. Polystyrene (Mw : 7,) 0.%. Polystyrene (Mw : 80) 0.%. Toluene (Mw : 9) 0.% Column : Shodex GPC KF-80L x Detector : UV (4nm) Column : Shodex GPC KF-807L x Detector : UV (4nm) Styrene isoprene ABA block copolymer Polycarbonate resin Raw rubber Sample : Styrene isoprene ABA block copolymer Sample : Polycarbonate resin 0.%, 0µL Sample : Rubber 0.%, 00μL CH O C O C CH O n Column : Shodex GPC KF-80M x Column temp. : 0 C Column : Shodex GPC KF-80L x Column : Shodex GPC KF-80M x + KF-80 Eluent : Toluene Column temp. : Room temp. 47

15 Organic SEC (GPC) Columns (General Analysis) : Chloroform Features K-800 Standard organic solvent SEC (GPC) column Supports a wide range of applications from low to high molecular weight compounds Corresponds to USP L Standard columns K-800 series : Shipping solvent Chloroform Pore Size (Å) F08 GPC K F080 GPC K-80 0 F08 GPC K F080 GPC K F0894 GPC K-80L 00 F0840 GPC K-804 7,00 F089 GPC K-804L 7,00 F080 GPC K-80,000,000 F089 GPC K-80L,000,000 F08 GPC K-80,000,000 F0890 GPC K-80M,000,000 F0897 GPC K-80L,000,000 F0870 GPC K-807, ,000 F0898 GPC K-807L, ,000 F70040 GPC K-G 4A (GPC K-G) 8 4. F70940 GPC K-800D solvent-peak separation column * The columns with L or M at the end of column names are mixed-gel column capable of analyzing samples over a wide range of molecular weight distribution. * See page 0 for details of the solvent-peak separation columns. * See pages 4 and for details preparative columns. * See page for applicability of SEC (GPC) columns to solvent replacement. Target molecular weight range and Exclusion limit With Polystyrene (eluent : Chloroform) Target Molecular Weight Range Exclusion Limit Target Molecular Weight Range Exclusion Limit K ,00 K-80 0,000,000,000 4,000,000 K-80 00,000,000 K-80L 00,000,000 4,000,000 K ,000 0,000 K-80 0,000 *(0,000,000) *(0,000,000) K-80,000 0,000 70,000 K-80M,000 *(0,000,000) *(0,000,000) K-80L 0 0,000 70,000 K-80L 00 *(0,000,000) *(0,000,000) K-804 7,000 00, ,000 K ,000 *(00,000,000) *(00,000,000) K-804L 0 00, ,000 K-807L 00 *(00,000,000) *(00,000,000) * Please use the above table as a rough indication for the column selection. *( ) Estimated value 48

16 Epoxy resin Sample : Epikote TM 0 0.%, 0μL 0 Column : Shodex GPC K-80L x Eluent : Chloroform Detector : UV (4nm) Column temp. : Room temp. Silicon oil Low molecular polydimethylsiloxanes Sample : 0.% each, 0μL. Polydimethylsiloxane (MW:770). Polydimethylsiloxane (MW:7). Hexamethyldisiloxane (MW:) 0 Column : Shodex GPC K-80 x Eluent : Chloroform (polarity : -) 0 Bionolle TM (Polybutylene succinate/adipate) Sample : Silicone oil 0.%, 00µL Sample : Polybutylene succinate/adipate 0.%, 0µL Organic SEC (GPC) Columns (General Analysis) : Chloroform Silicone oil Column : Shodex GPC K-80M x Eluent : Toluene Column temp. : 4 C Column : Shodex GPC K-80M x + K-80 Eluent : Chloroform Recycling fractionation of lauryl stearyl thiodipropionate Sample : %, 00μL. Distearyl stearyl thiodipropionate. Lauryl stearyl thiodipropionate. Dilauryl thiodipropionate CYCLE CYCLE CYCLE CYCLE Column : Shodex GPC K-LG + K-00 Eluent : Chloroform Flow rate :.0mL/ (preparative type) Column temp. : 0 C 0 0 * See page 4 for K-00 49

17 Organic SEC (GPC) Columns (General Analysis) : DMF Features KD-800 Standard organic solvent SEC (GPC) column Supports a wide range of applications from low to high molecular weight compounds Corresponds to USP L Standard columns KD-800 series : Shipping solvent Dimethylformamide (DMF) Pore Size (Å) F08 GPC KD-80 7,000 0 F080 GPC KD-80 7,000 0 F08 GPC KD-80. 7, F080 GPC KD-80 7, F0840 GPC KD-804 7,000 7,00 F080 GPC KD-80,000,000 F080 GPC KD-80,000,000 F0890 GPC KD-80M,000,000 F0870 GPC KD-807, ,000 F7004 GPC KD-G 4A (GPC KD-G) 8 4. * The columns with L or M at the end of column names are mixed-gel column capable of analyzing samples over a wide range of molecular weight distribution. * See page for applicability of SEC (GPC) columns to solvent replacement. Target molecular weight range and Exclusion limit With *PEG/PEO (eluent : DMF) Target Molecular Weight Range Exclusion Limit Target Molecular Weight Range Exclusion Limit KD-80 0,00,00 KD-80 0,000 **(4,000,000) **(4,000,000) KD ,000 7,000 KD-80 0,000 **(40,000,000) **(40,000,000) KD-80. KD-80 KD-804 Features KF-800D K-800D 400,000,000 0,000 4,000 00,000 0,000 70,000 00,000 KD-80M KD-807,000 **(40,000,000) 0,000 **(00,000,000) * Please use the above table as a rough indication for the column selection. Solvent-peak Separation Columns for Organic SEC (GPC) **(40,000,000) **(00,000,000) *PEG : polyethylene glycol *PEO : polyethylene oxide **( ) Estimated value Use this column in combination with a linear column By shifting the elution of monomers, polymer additives, and the solvent-peak in low molecular region, it reduces interferences in the calculation of the molecular weight distribution of polymers and oligomers Solvent-peak separation columns Column Combination Shipping Solvent F7090 GPC KF-800D KF-80L, 80L, 80M, 807L THF F70940 GPC K-800D K-80L, 80L, 80M, 807L Chloroform 0

18 Calibration curves for KD-800 series using PEG/PEO Molecular weight (PEG/PEO) Molecular mass Celluloses KD-80 KD Column : Shodex GPC KD-80M Eluent : % LiCl in *DMI Flow rate : 0.mL/, MALS (Multi angle light scattering) Column temp. : 0 C * DMI,-dimethyl--imidazolidinone Potato starch KD-80 KD-80 Sample : 0μL Potato starch in DMSO 0.% * solved at 80 C KD-80 KD-80 KD-80M KD-80. KD-807 Column : Shodex GPC KD-800 series Eluent : DMF Polyvinylpyrrolidones Sample : Polyvinylpyrrolidone 0.% each (A) K-0 400µL (B) K-0 00µL (C) K-90 00µL (A) (B) (C) Column : Shodex GPC KD-80M x Eluent : mm LiBr in DMF Column temp. : 0 C Sample : 0μL Cellulose ca. 0.0% each Cellulose is known to be difficult to dissolve. A cellulose solution is prepared by repeating solvent replacement. It is reported that it requires a long time to dissolve ( to 0 days), depending on the solvent type, crystallinity and molecular weight. Data provided by Dr. Masahiko Yanagisawa, Isogai group, Graduate School of Agricultural and Life Sciences, The University of Tokyo Effects of solvent-peak separation column Sample : Poly(vinyl chloride) DOP (Dioctyl phthalate) Melae formaldehyde resin Phenoxy resin Sample : 0µL Melae-formaldehyde resin % 0 Column : Shodex GPC KD-80 x Eluent : mm LiBr in DMF Column temp. : 0 C Sample : 0µL Phenoxy resin 0.% 0 Column : Shodex GPC KD-80M x Eluent : mm LiBr in DMF Column temp. : 0 C Organic SEC (GPC) Columns (General Analysis) : DMF Solvent-peak Separation Columns for Organic SEC (GPC) KF-80L 0 Molecular weight (Polystyrene) 4 KF-80L KF-80L+KF-800D KF-80L + KF-800D Column : Shodex GPC KD-80M Eluent : mm LiBr in DMSO/DMF=7/ Column temp. : 0 C 0 Column : Shodex GPC KF-80L Shodex GPC KF-80L + KF-800D

19 Organic SEC (GPC) Columns : Rapid Analysis, High Performance Analysis Features KF-00 KF-400HQ Approximately half of the analysis time compared with standard columns The amount of solvent used is reduced to about a third Improved applicability of solvent replacement Corresponds to USP L About. times better separation performance than standard columns, obtains higher resolution About 4 times better sensitivity than that of standard columns, supports high sensitivity analysis The amount of solvent used is reduced to about a third Improved applicability of solvent replacement Corresponds to USP L Rapid analysis downsized columns KF-00 series F0809 F0809 F0809 F08094 F0809 F0809 F08097 F08098 F08099 F70000 GPC KF-0 GPC KF-0 GPC KF-0. GPC KF-0 GPC KF-04 GPC KF-0 GPC KF-0 GPC KF-0M GPC KF-07 GPC KF-G 4A (GPC KF-G) 7,000 7,000 7,000 7,000,000 7,000 7,000 8,000,000 Use of the KF-00 series with semi-micro type devices is recommended. 8 8 Pore Size (Å) ,00,000,000,000 0, High performance semi-micro columns KF-400HQ series F08 F08 F084 F08 F088 F089 F08 F70000 GPC KF-40HQ GPC KF-40HQ GPC KF-40.HQ GPC KF-40HQ GPC KF-404HQ GPC KF-40LHQ GPC KF-40LHQ GPC KF-G 4A (GPC KF-G),000,000,000,000,000,000,000 Use of the KF-400HQ series with semi-micro type devices is recommended. 8 Pore Size (Å) ,00,000, [KF-00 series and KF-400HQ series] * The columns with L or M at the end of column names are mixed-gel column capable of analyzing samples over a wide range of molecular weight distribution. * See page for applicability of SEC (GPC) columns to solvent replacement. [KF-00 series and KF-400HQ series] Shipping Solvent : Tetrahydrofuran (THF) Target molecular weight range and Exclusion limit With Polystyrene (eluent : THF) Target Molecular Weight Range Exclusion Limit Target Molecular Weight Range Exclusion Limit KF ,00 KF-40HQ 0 700,00 KF-0 00,00 4,000 KF-40HQ 00,00 4,000 KF-0. 00,000 0,000 KF-40.HQ 00,000 0,000 KF ,000 70,000 KF-40HQ 00 0,000 70,000 KF-04 7,000 00,000,000,000 KF-404HQ 7,000 00,000,000,000 KF-0 0,000,000,000 4,000,000 KF-40LHQ 00,000,000 4,000,000 KF-0 0,000 *(0,000,000) *(0,000,000) KF-40LHQ 00 *(0,000,000) *(0,000,000) KF-0M KF-07,000 *(0,000,000) 00,000 *(00,000,000) *(0,000,000) *(00,000,000) * Please use the above table as a rough indication for the column selection. *( ) Estimated value

20 Calibration curves for KF-00 series using polystyrene Molecular weight (Polystyrene) Column : Shodex GPC KF-00 series Flow rate : 0.mL/ (small cell volume) Comparison of standard, rapid analysis, and high performance type columns Standard type KF-80. x 0µL injection KF-0 KF-04 KF-0. KF-0 KF-0 KF-0 KF-07 KF-0 KF-0M ml Calibration curves for KF-400HQ series using polystyrene Molecular weight (Polystyrene) 7 4 KF-404HQ KF-40HQ KF-40LHQ KF-40HQ KF-40.HQ KF-40HQ KF-40LHQ Column : Shodex GPC KF-400HQ series Flow rate : 0.mL/ (small cell volume) Sample : EPON0 0.% By using KF-0., the analysis time is reduced to less than half of that using KF-80.. Thus KF-00 series enables rapid analysis. On the other hand, KF-40.HQ has a theoretical plate number. times more than that of the standard column, thereby improving resolution especially in the analysis of molecules that have a small to medium molecular weight. Rapid analysis and high performance type columns use less than one third of solvent per analysis compared to standard type columns do. Organic SEC (GPC) Columns : Rapid Analysis, High Performance Analysis High performance type.7ml KF-40.HQ x µl injection Rapid analysis type KF-0. x µl injection.0ml Column : Shodex GPC KF-80. x Shodex GPC KF-40.HQ x Shodex GPC KF-0. x (KF-80.) 0.mL/ (KF-40.HQ) 0.mL/ (KF-0.) (conventional type) (KF-80.) RI (small cell volume) (KF-40.HQ, KF-0.) 0 0 Styrene acrylonitrile copolymer Liquid paraffin Phthalates Sample : Styrene-Acrylonitrile (0:70) copolymer Sample : Liquid paraffin %, μl Sample : µl. Dioctyl phthalate 0.%. Dibutyl phthalate 0.%. Dipropyl phthalate 0.% 4. Diethyl phthalate 0.%. Dimethyl phthalate 0.%. Propylbenzene 0.% 7. Ethylbenzene 0.% 8. Toluene 0.% 9. Benzene 0.% Column : Shodex GPC KF-0M x Eluent : mm LiBr in DMF Flow rate : 0.mL/ (small cell volume) Column : Shodex GPC KF-40HQ Eluent : Chloroform Flow rate : 0.mL/ (small cell volume) Column : Shodex GPC KF-40HQ x Flow rate : 0.mL/ Detector : UV (4nm) (small cell volume)

21 Organic SEC (GPC) Columns : For Preparative Preparative columns * Preparative columns are made to order. KF-000 series : Shipping solvent Tetrahydrofuran (THF) Standard Columns F40 GPC KF KF-80 F40 GPC KF KF-80 F4 GPC KF KF-80. F40 GPC KF KF-80 F404 GPC KF-004 4, KF-804 F40 GPC KF-00, KF-80 F40 GPC KF-00, KF-80 F409 GPC KF-00M, KF-80M F70040 GPC KF-G 8B (GPC KF-LG) * See page 4 for GPC KF-800 series. K-000 series : Shipping solvent Chloroform Standard Columns F0 GPC K K-80 F GPC K K-80 F GPC K K-80. F0 GPC K K-80 F04 GPC K-004 4, K-804 F0 GPC K-00, K-80 F0 GPC K-00, K-80 F09 GPC K-00M, K-80M F GPC K-G 8B (GPC K-LG) * See page 48 for GPC K-800 series. 4

22 Preparative columns * Preparative columns are made to order. H-000 series : Shipping solvent Chloroform F00 F00 F0 F00 F004 F00 GPC H-00 GPC H-00 GPC H-00. GPC H-00 GPC H-004 GPC H-00,000,000,000,000,000, Standard Columns K-80 K-80 K-80. K-80 K-804 K-80 Organic SEC (GPC) Columns : For Preparative F00 GPC H-00, K-80 F009 GPC H-00M, K-80M F700 GPC H-G 8B (GPC H-G) * See page 48 for GPC K-800 series. KF-000 series : Shipping solvent Tetrahydrofuran (THF) [Customized columns] Standard Columns F80 GPC KF KF-80 F800 GPC KF KF-80 F80 GPC KF KF-80. F800 GPC KF KF-80 F8040 GPC KF KF-804 F GPC KF-G 0B (GPC KF-LLG) * See page 4 for GPC KF-800 series. K-000 series : Shipping solvent Chloroform [Customized columns] Standard Columns F90 GPC K K-80 F900 GPC K K-80 F90 GPC K K-80. F900 GPC K K-80 F9040 GPC K K-804 F GPC K-G 0B (GPC K-LLG) * See page 48 for GPC K-800 series.

23 Organic SEC (GPC) Columns : Linear Calibration Type Features LF Employs a special packing material with a wide pore size distribution (multi pore type) Highly linear calibration curve without inflection points Molecular weight distribution can be detered with high precision Enables analysis over a broad range of molecular weights Column for rapid analysis (LF-04) and column for high performance analysis (LF-404) enabling reduction in solvent use are also available Corresponds to USP L Standard columns Pore Size (Å) F04 GPC LF-804 7,000,000 F709 GPC LF-G 4. * See page for applicability of SEC (GPC) columns to solvent replacement. Rapid analysis downsized column Shipping Solvent : Tetrahydrofuran (THF) Use of the LF-04 with semi-micro type devices is recommended. Pore Size (Å) F04 GPC LF-04 9,000, F709 GPC LF-G 4. * See page for applicability of SEC (GPC) columns to solvent replacement. High performance semi-micro column Shipping Solvent : Tetrahydrofuran (THF) Use of the LF-404 with semi-micro type devices is recommended. Pore Size (Å) F04 GPC LF-404 4,000, F709 GPC LF-G 4. * See page for applicability of SEC (GPC) columns to solvent replacement. Target molecular weight range and Exclusion limit With Polystyrene (eluent : THF) Shipping Solvent : Tetrahydrofuran (THF) LF-804 LF-04 Target Molecular Weight Range 00,000,000 00,000,000 Exclusion Limit,000,000,000,000 LF ,000,000,000,000 * Please use the above table as a rough indication for the column selection. Schematic diagram of linear calibration type packing Connecting linear calibration type columns (LF series) The linear calibration type column covers a broad range of molecular weights with only one kind of packing (column). Connecting mixed-gel columns (KF-804L, etc.) Connecting different grades columns (KF KF-80 + KF-80, etc.)

24 Calibration curve for LF-804 using polystyrene Molecular weight (Polystyrene) 7 4 Xylan Column : Shodex GPC LF-804 Calibration curves for LF-04 and LF-404 using polystyrene Molecular weight (Polystyrene) 7 4 LF-404 LF Column : Shodex GPC LF-04, LF-404 Flow rate : 0.mL/ (LF-04) 0.mL/ (LF-404) (small cell volume) Polyamide (Nylon/) Polyurethane Sample : Polyurethane 0.%, 0μL 0 0 Column : Shodex GPC LF-404 x Flow rate : 0.mL/ (small cell volume) Polymethyl methacrylate Organic SEC (GPC) Columns : Linear Calibration Type Sample : Xylan 0.%, 0μL Sample : Nylon TM / 0.%, 0µL Sample : 0μL Polymethyl methacrylate Column : Shodex GPC LF-804 Eluent : 0mM H PO 4 + 0mM LiBr in (DMSO/DMF=80/0) Flow rate : 0.mL/ Column temp. : 0 C Column : Shodex GPC LF-404 Eluent : mm CF COONa in HFIP Flow rate : 0.mL/ (small cell volume) Column : Shodex GPC LF-804 x Eluent : Methyl ethyl ketone Polyamic acid Sample : Poly(pyromellitic dianhydride-co-4,4'-oxydianiline), 0μL Comparison of polystyrenes separation with different numbers of LF-404 LF-404 x 4 7 Sample : µl. Polystyrene (Mw :,00,000). Polystyrene (Mw :,000). Polystyrene (Mw :,000) 4. Polystyrene (Mw :,000). Polystyrene (Mw :,00). Polystyrene (Mw : 80) 7. Ethylbenzene LF-404 x 4 7 Column : Shodex GPC LF-404 x n Flow rate : 0.mL/ (small cell volume) Column : Shodex GPC LF-804 x Eluent : 0mM LiBr + 0mM H PO 4 in NMP Flow rate : 0.7mL/ Column temp. : 0 C LF-404 x

25 Organic SEC (GPC) Columns : High Temperature/Ultra High Temperature Analysis Features HT-800 Varied product lineup to support a wide range of molecular weights Corresponds to USP L UT-800 Dedicated to SEC analysis at high/ultra high temperatures with a maximum usable temperature of C Suitable for the analysis of ultra high molecular weight polymer containing samples Corresponds to USP L Standard columns Usable Temperature ( C) Pore Size (Å) F08700 GPC HT-80 7,000 0 ~ 0 00 F087 GPC HT-804 7,000 0 ~ 0,00 F0870 GPC HT-80 7,000 0 ~ 0,000 F0870 GPC HT-80 7,000 0 ~ 0,000 F08740 GPC HT-80M 7,000 0 ~ 0,000 F08770 GPC HT-807 4,000 0 ~ 0 8 0,000 F7094 GPC HT-G 0 ~ F0800 GPC UT-80. 4,400 0 ~ 0 00 F08 GPC UT-80M 4,400 0 ~ 0,000 F080 GPC UT-807,00 0 ~ 0 0,000 F GPC UT-G 0 ~ F0890 GPC AT-80MS,000 *Ta ~ 0, F70080 GPC AT-G *Ta ~ * The columns with M at the end of column names are mixed-gel column capable of analyzing samples over a wide range of molecular weight distribution. Shipping Solvent : Toluene *Ta : Ambient temperature Target molecular weight range and Exclusion limit With Polystyrene (eluent : THF) HT-80 HT-804 HT-80 HT-80 HT-80M HT-807 UT-80. UT-80M UT-807 Target Molecular Weight Range,000 0,000 7,000 00,000 0,000,000,000 0,000 *(0,000,000),000 *(0,000,000) 00,000 *(00,000,000) 00,000,000 *(0,000,000) 00,000 *(00,000,000) Exclusion Limit 70, ,000 4,000,000 *(0,000,000) *(0,000,000) *(00,000,000) 0,000 *(0,000,000) *(00,000,000) AT-80MS,000 *(0,000,000) *(0,000,000) * Please use the above table as a rough indication for the column selection. *( ) Estimated value 8

26 Calibration curves for HT-800 series using polystyrene Molecular weight (Polystyrene) HT-80 HT-80 HT-804 HT-80 Molecular weight (Polystyrene) Effects of gel particle size in high temperature GPC columns High temperature SEC columns are suitable for the analysis of high molecular weight polymers that are difficult to be dissolved in ambient temperature solvents; examples of such polymers are polyethylene and polypropylene. The GPC UT-800 series packed with large particle size (0 um) are recommended for the analysis of macromolecules. The large particle size prevents potential molecular shear degradation of the sample. Column : UT-80M x (Particle size : 0µm) HT-807 HT-80M Column : Shodex GPC HT-800 series Calibration curves for UT-800 series using polystyrene UT-80. UT-80M Column : AT-80MS x (Particle size : µm) UT-807 Column : Shodex GPC UT-800 series Calibration curve for AT-80MS using polystyrene Molecular weight (Polystyrene) 7 4 Column : HT-80M x (Particle size : µm) Column : Shodex GPC AT-80MS Sample :. Polystyrene (MW : 0,000,000). High density polyethylene (HDPE-A) Organic SEC (GPC) Columns : High Temperature/Ultra High Temperature Analysis Vo Shear degradation Vo 0 Vo Shear degradation 0 Column : Shodex GPC UT-80M Shodex GPC HT-80M Shodex GPC AT-80MS Eluent : 0.% BHT in o-dichlorobenzene Column temp. : 40 C High density polyethylene Low density polyethylene Sample : High density polyethylene (HDPE-B) 8 Sample : Low density polyethylene (LDPE-C) 7 Molecular weight RI Column : Shodex GPC UT-80M x Eluent : 0.% BHT in o-dichlorobenzene, MALS (Multi angle light scattering) Column temp. : 4 C Column : Shodex GPC HT-80M x Eluent : 0.% BHT in o-dichlorobenzene Column temp. : 40 C 9

27 Organic SEC (GPC) Columns : HFIP Features HFIP-800 HFIP-00 Columns exclusively for use with hexafluoroisopropanol (HFIP) Corresponds to USP L Rapid analysis, solvent saving type Corresponds to USP L Standard columns HFIP-800 series Pore Size (Å) F080 GPC HFIP-80, F0840 GPC HFIP-804,000 7,00 F080 GPC HFIP-80,000,000 F080 GPC HFIP-80,000,000 F0890 GPC HFIP-80M,000,000 F0870 GPC HFIP-807 4, ,000 F70000 GPC HFIP-G 8B (GPC HFIP-LG) * The columns with M at the end of column names are mixed-gel column capable of analyzing samples over a wide range of molecular weight distribution. Shipping Solvent : Hexafluoroisopropanol (HFIP) Rapid analysis downsized columns HFIP-00 series Use of the HFIP-00 series with semi-micro type devices is recommended. Pore Size (Å) F00 GPC HFIP-0, F040 GPC HFIP-04,000, F00 GPC HFIP-0,000, F00 GPC HFIP-0,000, F080 GPC HFIP-0M,000, F070 GPC HFIP-07, , F700 GPC HFIP-G 4A (GPC HFIP-G) 8 4. * The columns with M at the end of column names are mixed-gel column capable of analyzing samples over a wide range of molecular weight distribution. Shipping Solvent : Hexafluoroisopropanol (HFIP) Target molecular weight range and Exclusion limit With *PMMA (eluent : HFIP) Target Molecular Weight Range Exclusion Limit Target Molecular Weight Range Exclusion Limit HFIP-80,000 0,000 0,000 HFIP-0,000 0,000 0,000 HFIP-804 0,000 00,000 00,000 HFIP-04 0,000 00,000 00,000 HFIP-80 0,000 00,000,000,000 HFIP-0 0,000 00,000,000,000 HFIP-80 70,000 **(8,000,000) **(8,000,000) HFIP-0 70,000 **(8,000,000) **(8,000,000) HFIP-80M,000 **(8,000,000) **(8,000,000) HFIP-0M,000 **(8,000,000) **(8,000,000) 0 HFIP ,000 **(0,000,000) **(0,000,000) HFIP-07 70,000 **(0,000,000) * Please use the above table as a rough indication for the column selection. **(0,000,000) *PMMA : Polymethylmethacrylate **( ) Estimated value

28 Calibration curves for HFIP-800 series using PMMA Molecular weight (PMMA) 7 4 HFIP-80 HFIP-80 HFIP-80 HFIP-804 HFIP-807 HFIP-80M Calibration curves for HFIP-00 series using PMMA Molecular weight (PMMA) 7 4 HFIP-04 HFIP-0 HFIP-0 HFIP-07 HFIP-0 HFIP-0M Polyethylene terephthalate (PET) Sample : Polyethylene terephthalate 0.%, 0µL Mw=9,00 0 Organic SEC (GPC) Columns : HFIP Column : Shodex GPC HFIP-800 series Eluent : HFIP Column : Shodex GPC HFIP-00 series Eluent : HFIP Flow rate : 0.mL/ (HFIP-0, 04) 0.mL/ (HFIP-0, 0, 0M, 07) Detctor : RI (small cell volume) Column : Shodex GPC HFIP-0M x Eluent : mm CF COONa in HFIP Flow rate : 0.mL/ (small cell volume) Polyamide (effects of salt addition) Polybutylene terephthalate (PBT) Sample : Polycaprolactum (Nylon ) Sample : Polybutylene terephthalate 0.0%, 00µL HFIP mm CFCOONa in HFIP In SEC analysis using HFIP, some samples may yield abnormal peaks as a result of ionic interaction. In this case, ionic interaction can be suppressed by adding sodium trifluoroacetate to HFIP Column : Shodex GPC HFIP-80M x Eluent : HFIP (solid line), mm CF COONa in HFIP (broken line) Column : Shodex GPC HFIP-80 + HFIP-80 Eluent : mm CF COONa in HFIP Polyamides (Nylon and Nylon ) Polyacetal Sample : Nylon 0.%, 0µL Sample : Nylon 0.%, 0µL Sample : Polyacetal 0.%, 0µL Mw=9,00 Mw=8,0 Mw=7, Column : Shodex GPC HFIP-0M x Eluent : mm CF COONa in HFIP Flow rate : 0.mL/ (small cell volume) Column : Shodex GPC HFIP-0M x Eluent : mm CF COONa in HFIP Flow rate : 0.mL/ (small cell volume)

29 Applicability of SEC (GPC) Columns to Solvent Replacement Shipping Solvent : THF Shipping Solvent : DMF Solvent KF-80 KF-80 KF-80. KF-80L KF-804L KF-80 KF-804 KF-80 KF-80 KF-807 KF-80M KF-80L KF-80L KF-807L KF-0 KF-0 KF-0. KF-0 KF-04 KF-0 KF-0 KF-07 KF-0M LF-804 LF-04 LF-404 KD-80 KD-80 KD-80. KD-80 KD-804 KD-80 KD-80 KD-807 KD-80M Shipping Solvent : Chloroform Shipping Solvent : THF K-80 K-80 K-80 K-80. K-80L K-804L K-804 K-80 K-80 K-807 K-80M K-80L K-80L K-807L KF-40HQ KF-40HQ KF-40.HQ KF-40HQ KF-404HQ KF-40LHQ KF-40LHQ Tetrahydrofuran (THF) Chloroform Carbon tetrachloride Benzene Toluene p-xylene o-dichlorobenzene (ODCB) Trichlorobenzene (TCB) Dioxane Diethyl ether Ethyl acetate Acetone Methyl ethyl ketone Dimethylformamide (DMF) Dimethylacetamide (DMAc) Hexafluoroisopropanol (HFIP) m-cresol o-chlorophenol Quinolin N-Methylpyrrolidone (NMP) Dimethylsulfoxide (DMSO) 0% m-cresol/chloroform 0% o-chlorophenol/chloroform 0% HFIP/Chloroform Hexane Acetonitrile Methanol Water : Solvent replacement possible : Solvent replacement possible, but this may cause column performance to deteriorate slightly : Usable at 40 C or higher : Solvent replacement not possible

30 Calibration Standards for SEC [Polystyrene (PS)] Features For organic solvent SEC (GPC) Less branched polystyrene with anionic polymerization Easily soluble in tetrahydrofuran (THF), chloroform, toluene, and o-dichlorobenzene (ODCB) Kit type F80 F80 F8007 SL- Std. No. Mp Mw/Mn S-9 S- S-9.9 S-.9 S-4.8 S-.7 S-.8 S-. S-0.8 S-0. 9,00,00 9,970,940 4,80,780,80, STANDARD SL- STANDARD SM- STANDARD SH S-700 S-90 S- S- S-4 S-47 S-8 S-.9 S-.0 S-.,700,000,90,000,000,000 4,000 47,00 8,00,940,980,0 Contents 0.g x kinds 0.g x kinds 0.g x 7 kinds Std. No. Mp Mw/Mn [ Polymethylmethacrylate (PMMA)] Features Contents F80407 STANDARD M-7 0.g x 7 kinds Features S-870 S-90 S-990 S-0 S-80 S-99 S-9 MW Range 80 9,00,0,700,000 9,000,870,000 Std. No. Mp Mw/Mn,870,000,90,000,990,000,0,000,80,000 99,000 9,000 For organic solvent SEC (GPC) Narrow molecular weight distribution range Easily soluble in hexafluoroisopropanol (HFIP) and dimethylformamide (DMF) Kit type [Pullulan] SM- SH-7 MW Range,880,00,000 (Note) Molecular weights (Mp, Mw/Mn) of each kit may vary depending on production lots Structural formula of S series M-0 M-9 M- M-9 M-8 M-.8 M-.9 C 4H 9 CH CH (Note) Molecular weights (Mp, Mw/Mn) of each kit may vary depending on production lots. Std. No. Mp Mw/Mn,00,000 9,000,000 8,800 7,800,80,880 n H Applicability of SEC (GPC) Columns to Solvent Replacement Calibration Standards for SEC For aqueous SEC (GFC) Unbranched pullulan standard High solubility in water eliates the possibility of recrystallization Kit type Contents MW Range Structural formula of P series F STANDARD P-8 Single type 0.g x 8 kinds,0 4,000 O CH H H H O OH OH H H OH O H CH OH H H CH OH H H O H O OH H O OH H H OH H OH n F F F F84000 F F F84000 F STD P-800 STD P-400 STD P-00 STD P-0 STD P-0 STD P-0 STD P- STD P- Contents 0.g 0.g 0.g 0.g 0.g 0.g 0.g 0.g Mp 4,000 7,000 94,000 7,000 47,0,0 9,00,0 Mw/Mn Std. No. Mp Mw/Mn STD P-800 STD P-400 STD P-00 STD P-0 STD P-0 STD P-0 STD P- STD P- 4,000 7,000 94,000 7,000 47,0,0 9,00, (Note) Molecular weights (Mp, Mw/Mn) of each kit may vary depending on production lots.