)( thgew( thgew( thgewnetorP04-4F3-4402.5-4netorP-80-803-802.5netorP () rauceoM rauceoMKW4KWKW)iill)iillKW4KW40FKWFKW405-4Fiill5678956789)iill221312105123456780411323245542315)iill05lS rauceoMnetorP( thgew rauceoMnetorP( thgew rauceoMili Aqueous SECGFCCoumns: ca-basedColumn Eluent Flow rate Detector Column temp. : 30 ˚C: Shodex PROTEIN KW-800 series: 50 mM Sodium phosphate buffer (pH7.0) + 0.3 M NaCl: 1.0 mL/min : UV (280 nm)Column Eluent Flow rate Detector Column temp. : 30 ˚C: Shodex PROTEIN LW-403 4D: 50 mM Sodium phosphate buffer (pH7.0) + 0.3 M NaCl : 0.35 mL/min : UV (280 nm) (small cell volume): Shodex PROTEIN KW-803Column : 50 mM Sodium phosphate buffer (pH7.0) + 0.3 M NaClEluent : 1.0 mL/minFlow rate Detector : UV (280 nm)Column temp. : Room temp.1071061051041031021071061051041031020.51.01.52.0Elution volume (mL)1020Elution volume (mL)107106105104103102101112131.5Sample : 100 LSystem suitability solution(prepared following USP-NF method)1. High molecular weight proteins2. Insulin glargine15202.53.0Sample : 0.1 % each50 µL1. Fibrinogen 2. α2 -Macroglobulin 50 µL50 µL3. IgG 50 µL4. Transferrin 50 µL5. Plasminogen 100 µL6. Albumin 100 µL7. Antitrypsin 8. Hemoglobin 100 µL30 minColumn Eluent Flow rate Detector Column temp. : 30 ˚CColumn Eluent Flow rate Detector Column temp. : Ambient2.02.53.03.5Elution volume (mL)4.025303540 min1520253035Sample :1. Thyroglobulin (bovine)2. γ-Globulin (bovine)3. Ovalbumin (chicken)4. Myoglobin (horse)5. Cyanocobalamin101520: Shodex KW400-4F series: 50 mM Sodium phosphate buffer (pH7.0) + 0.3 M NaCl: 0.33 mL/min: UV (280 nm) (small cell volume): Shodex PROTEIN KW-802.5 x 2: CH3COOH/CH3CN/H2O=20/30/50 (pH to 3.0 adjusted with 25 % NH3 aq.): 0.5 mL/min : UV (276 nm)SB-803 HQGF-510 HQKW-8031071061051041031024.5Sample : 40 µLWhole lipoproteins from serum of a healthy person 1.0 mg/mL1. VLDL 2. LDL 3. HDL(Sample preparation method)1. Use potassium bromide to adjust the speci■c gravity of serum from a healthy person to 1.210 g/mL. Ultracentrifuge for 24 hours.2. Dialyze the supernatant and then substitute the solvent with PBS*.3. Measure protein concentration by Lowry method and dilute the sample with PBS* to 1.0 mg/mL.40 minData provided by Ohkawa Ryunosuke,Graduate School of Health Care Sciences, Analytical Laboratory Chemistry, Tokyo Medical and Dental University 1061051041113253035 min: Shodex PROTEIN LW-803Column : 50 mM Sodium phosphate bufferEluent (pH7.0) + 0.3 M NaCl : 1.0 mL/minFlow rate Detector : UV (280 nm)Column temp. : Room temp.Column Eluent Flow rate Detector Column temp. : 30 ˚Cx10 PBS* : 80 g NaCl + 29 g Na2HPO4・12H2O + 2 g KCl + 2 g KH2PO4 in 1000 mL of H2OSeparation performances of three aqueous SEC columns (SB-803 HQ, GF-510 HQ, and KW-803) were compared. KW-803, silica-based column, showed the best separation performance for the analysis of protein standards.Column Eluent Flow rate Detector Column temp. : 30 ˚C: Shodex OHpak SB-803 HQ Shodex Asahipak GF-510 HQ Shodex PROTEIN KW-803: 0.2 M Phosphate buffer (pH6.9): 0.5 mL/min: UV (280 nm)Elution volume (mL)KW-803GF-510 HQSB-803 HQ101112131015min: Shodex PROTEIN KW-G + KW-804: 10-fold diluted x 10 PBS* with H2O: 1.0 mL/min: UV (280 nm)1715Elution time (min)1921Calibration curves for KW-800 series using proteinCalibration curve for LW-403 4D using proteinProteins in human blood serumCalibration curves for KW400 series using proteinAnalysis of impurities (high molecular weight proteins)in insulin glargine according to USP-NF methodComparing three GFC columns for the separation of common proteinsCalibration curve for LW-803 using proteinLipoproteins in serum38
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