Asahipak - Hplc.eu

Protein separation using cation exchange columns( I ) CM-825(weak cation ion exchange)90μL injected12345( II ) SP-825(strong cation ion exchange)30μL injected1436 65( III ) SP-420N(strong cation ion exchange)non-porous type gel456Rapid analysis of proteins using UHPLC123456Sample : 5μL (13mg total protein )1.Myoglobin2. Trypsinogen3. Ribonuclease A4. α-Chymotrypsinogen A5. Cytochrome c6. LysozymeStandard analysiscolumns21230.01.0 2.0 3.0 4.0 min0 15 30 45 min 0 15 30 min 0 2 4 6 minColumn : ( I ) Shodex IEC CM-825, ( II ) Shodex IEC SP-825,( III ) Shodex IEC SP-420NEluent : (A); 20mM Sodium phosphate buffer(pH7.0) (B); (A) + 0.5M NaCl( I , II ) Linear gradient; (A) to (B), 60min ( III ) Linear gradient; (A) to (B), 10minFlow rate : ( I , II )1.0mL/min ( III )1.5mL/minDetector : UV(280nm)Column temp. : Room temp.1234HOHOCH 2·CH·NH 2COOH2. AdrenalineHO CH·CH 2·NHHO OH CH 33. NoradrenalineHO CH·CH 2·NH 2HOOH4. DopamineHOHOCH 2·CH 2·NH 2120 20 min0 10 20 30 min0 10 20 minAspThrSerGluProSample : 300μg/mL each, 10μLGlyCysAlaValMetIleLueSample : 2.5nmol each, 100μL0 1020 30 40 50 minColumn : Shodex CXpak P-421SEluent : No.1 0.12M Sodium citrate buffer(pH3.3)No.2 0.13M Sodium citrate buffer(pH3.2)No.3 0.11M Sodium citrate buffer(pH4.0)No.4 1.02M Sodium citrate buffer(pH4.9)No.5 0.2M NaOH aq./Ethanol=90/10 (Rinse solution)Low pressure gradient;No.1 (0min), No.2 (1.2min), No.3 (10.0min), No.4 (21.2min), No.5 (40min)Flow rate : 0.5mL/minDetector : VIS(570nm)post column method (ninhydrin reaction 0.35mL/min, 120˚C)Column temp. : 63˚CTyrPheLysNH 3ArgHisSample :1. Myoglobin2. Trypsinogen3. Ribonucrease A4. α-Chymotrypsinogen A5. Cytochrome c6. LysozymeCatecholamines Angiotensins Paraquat and diquat1. DOPAColumn : Shodex Asahipak ES-502C 7CEluent : 20mM Sodium malonate buffer+ 0.5M NaCl(pH6.0)Flow rate : 1.0mL/minDetector : UV(280nm)Column temp. : 30˚CStandard amino acidsSample : 10μL1. Angiotensin II (human)Asp-Arg-Val-Tyr-lle-His-Pro-Phe2. Angiotensin I (human)Asp-Arg-Val-Tyr-lle-His-Pro-Phe-His-Leu3. Angiotensin III (human)Arg-Val-Tyr-lle-His-Pro-PheColumn : Shodex Asahipak ES-502C 7CEluent : 50mM Sodium malonate buffer(pH6.0)/CH 3 CN=80/20Flow rate : 1.0mL/minDetector : UV(280nm)Column temp. : 30˚C1323Column : Shodex PIKESS SP-2BEluent : (A); 20mM Sodium phosphatebuffer(pH 7.0)(B); (A) + 0.5 M NaClLinear gradient; 100%(A) to 50%(A),2.5minFlow rate : 1.2mL/minDetector : UV(280nm)Column temp. : 25˚CSample : 20μLParaquat+ +H 3 C N N CH 3 2Cl - Diquat+ 2Br -N + NColumn : Shodex Asahipak ES-502C 7CEluent : 50mM Sodium phosphate buffer+ 150mM NaCl(pH7.0)Flow rate : 1.0mL/minDetector : UV(288nm)Column temp. : 30˚CProtein separation using hydrophobic interaction chromatography0 20 40 60 minColumn : Shodex HIC PH-814Eluent : (A); 1.8M Ammonium sulfate + (B)(B); 0.1M Phosphate buffer(pH7.0)Linear gradient; (A) to (B), 60minFlow rate : 1.0mL/minDetector : UV(280nm)Column temp. : Room temp.456Sample : 370μL1. Cytochrome c 0.03%2. Myoglobin 0.08%3. Ribonuclease A 0.16%4. Ovalbumin 0.16%5. Lysozyme 0.04%6. α-Chymotrypsinogen 0.05%27Columns for Cation Exchange Chromatography Column for Hydrophobic Interaction Chromatography