SEC Column Selection Guide for the Separation of Water-soluble Polymers Dextrans Molecular weight (Da) 1 6 1 5 1 4 1 3 SRT-1 SRT-15 SRT-3 SRT-5 SRT-1 SRT- 67 kd 41 kd 7 kd 15 kd 8 kd 5 kd 5 kd 1 kd 5 kd SRT-1 SRT-15 SRT-3 SRT-5 > 67 kd > 67 kd SRT-1 SRT- 1 kd 6 8 1 1 14 Elution volume (ml) Linear distribution region between Log MW & Elution volume Pullulans Molecular weight (Da) 1 6 1 5 1 4 1 3 1 SRT-1 SRT-15 SRT-3 SRT-5 SRT-1 SRT- 85 kd 366 kd 1 kd 113 kd 48.8 kd 1.7 kd 1 kd 6 kd 1.3 kd 34 D SRT-1 SRT-15 SRT-3 SRT-5 > 85 kd > 85 kd SRT-1 SRT- 6 8 1 1 14 Elution volume (ml) Linear distribution region between Log MW & Elution volume Polyethylene Glycols Molecular weight (Da) 1 6 1 5 1 4 1 3 SRT-1 SRT-15 SRT-3 SRT-5 93 kd 496 kd 71 kd 118 kd 4.7 kd 17.9 kd 6.55 kd.1 kd 68 D SRT-1 SRT-15 SRT-3 SRT-5 1 3 D 6 8 1 1 14 Elution volume (ml) Linear distribution region between Log MW & Elution volume Data generated using the following conditions: Column: SRT columns, 7.8 x 3 mm; Mobile phase: 15 mm phosphate buffer (ph 7.); Flow rate: 1. ml/; Detection: RI Column temperature: 5 o C;; Injection volume: μl Note: This column selection guide is applicable for other dimensions and series which including SRT-C, Zenix, and Zenix-C. www.sepax-tech.com www.sepax-tech.com.cn
CC1 Analysis of Glucidex on Carbomix -Ca (5 µm, 8%, 4.6x3 mm) G8-G1 5. 4. MV 3. G7. G4 G6 G5 G3 G G1 1... 4. 8. 1. 16.. 4. 8. 3. 36. 4. Min Column: Carbomix Ca-NP5 (5 µm, 8% crosslinkage, 4.6x3 mm) Mobile Phase: Water, ph 6. Flow Rate:. ml/ Temperature: 85 o C Detection: Differential Refractometer Injection Volume: µl Samples: Glucidex (1 mg/ml), 1 components from G1 (monomer) to G1 (1th mer) (Courtesy of Miyako Kawakatsu, M&S Instruments, Inc.) Keywords: Carbohydrate, Carbomix, calcium form, sugar, maltodextrins, Glucidex Sepax Technologies, Inc.
Separation of Dextran on SRT -3 SL14 Column: SRT 3 (5 m, 3 Å, 7.8 x 3 mm) Sample: 1 mg/ml in mobile phase Mobile Phase: 15 mm phosphate Buffer, ph 7. 1 kd, 5 kd, 1 kd, 5 kd, 5 kd, 8 kd, Flow rate: 1. ml/ 15 kd, 7 kd, 41 kd and 67 kd Detection: RI(3 ) Column temperature: 5 Injection volume: L Pressures: 6 psi Figure 1. Chromatogram overlays for Dextran standards on SRT -3, 5 m, 7.8 x 3 mm. Table 1. Dextran molecular weight and retention time on SRT 3, 5 m, 7.8 x 3 mm. Sepax Technologies, Inc.
SL14 Figure 3. Calibration curve of Dextran molecular weight vs. Elution volume on SRT 3. Keywords : size exclusion, SRT, Dextran, SRT 3, polysaccharide Sepax Technologies, Inc.
Separation of Pullulan on SRT -3 SL15 Column: SRT 3 (5 m, 3 Å, 7.8 x 3 mm) Sample: 1 mg/ml in mobile phase Mobile Phase: 15 mm phosphate Buffer, ph 7..34 kd, 1.3 kd, 6 kd, 1 kd, 1.7 kd, Flow rate: 1. ml/ 48.8 kd 113 kd, 1 kd, 366 kd and 85 kd Detection: RI(3 ) Column temperature: 5 Injection volume: L Pressures: 6 psi 8. 7. 6. 5. MV 4. 3.. 1.. 1.. 3. 4. 5. 6. 7. 8. 9. 1. 11. 1. 13. 14. 15. 16. Minutes Figure 1. Chromatogram overlays for Pullulan standards on SRT -3, 5 m, 7.8 x 3 mm. NO Molecular weight Log MW Elution volume(ml) 1 34.5346 11.67 13 3.1574 11.341 3 6 3.778151 1.394 4 1 4 9.936 5 17 4.33646 9.3 6 488 4.6884 7.75 7 113 5.5378 6.546 8 1 5.319 6.5 9 366 5.563481 5.95 1 85 5.95796 5.839 Table 1. Pullulan molecular weight and retention time on SRT 3, 5 m, 7.8 x 3 mm. Sepax Technologies, Inc.
SL15 6 Log MW 4 6 8 1 1 Elution volume (ml) Figure 3. Calibration curve of Pullulan molecular weight vs. Elution volume on SRT 3. Keywords : size exclusion, SRT, Pullulan, SRT 3, polysaccharide Sepax Technologies, Inc.
ZL1 PEG separation on two Zenix SEC-3 7.8x3mm columns in Tandem nriu 15 1 3 4 5 1. PEG 81,4Da. PEG 5,63Da 3. PEG 4,Da 4. PEG 6,1Da 5. PEG 8,73Da 1 5-5 15 5 3 35 4 45 Columns: Zenix SEC-3 3µm, 3Å, 7.8x3mm connected in tandem Mobile Phase: 15 mm Sodium Phosphate Buffer ph=7. Flow Rate:.5 ml/ Temperature: 5 o C Detection: Refractive Index at 3 C Injection Volume: 1 µl Samples: 1. PEG 81,4Da (mg/ml). PEG 5,63Da (mg/ml) 3. PEG 4,Da (mg/ml) 4. PEG 6,1Da (mg/ml) 5. PEG 8,73Da (mg/ml) Sepax Technologies, Inc.
ZL13 Polystyrene sulfonate polymer SEC separations Columns: Zenix SEC-15 (3 m, 1.5 Å) Zenix SEC-3 (3 m, 3 Å) SRT SEC-5 (5 m, 5 Å) SRT SEC-1 (5 m, 1 Å) Samples: 164, 795, 166, 347, 575, 17, 551 Da 15, 8, 15, 3, 67, 1, and,35 kda With shorter column 7.8 x 15 mm and higher flow rate at 1.5 ml/, the separation can be achieved under 5 utes. Different pore size columns in tandem will expand the MW separation range. www.sepax-tech.com
nriu 35 3 5 15 Sodium polystyrene sulfonate separation on Zenix SEC-15, 783 Column: Zenix SEC-15 (3 m, 15 Å, 7.8 x 3 mm) Mobile phase: 15mM Phosphate Buffer, ph 7 Flow rate: 1. ml/; Detector: RI; Column temperature: RT Injection volume: 5 µg, Sample: See chromatogram, total run time 15 1. 7,95 Da. 16,6 Da 3. 34,7 Da 4. 57,5 Da 5. 17, Da 6. 55,1 Da 6 5 4 3 ZL13 1 5-5 1-1 4 6 8 1 1 14 www.sepax-tech.com
Sodium polystyrene sulfonate separation on Zenix SEC-3, 7815, RI nriu Column: Zenix SEC-3 (3 m, 3 Å, 7.8 x 15 mm) Mobile phase: 15mM Phosphate Buffer, ph 7 Flow rate: 1.5 ml/; Detector: RI; Column temperature: RT Injection volume: 5 µg, Sample: See chromatogram, total run time 5 3 1. 7,95 Da. 16,6 Da 5 3. 34,7 Da 4. 57,5 Da 5. 17, Da 6. 55,1 Da 15 6 5 4 3 1 ZL13 1 5 164 was buried in the negative peak, blue trace. -5.5 1 1.5.5 3 3.5 4 4.5 www.sepax-tech.com
mau Sodium polystyrene sulfonate separation on Zenix SEC-3, 7815 Column: Zenix SEC-3 (3 m, 3 Å, 7.8 x 15 mm) Mobile phase: 15mM Phosphate Buffer, ph 7 Flow rate: 1.5 ml/; Detector: UV 14 nm; Column temperature: RT Injection volume: 5 µg, Sample: See chromatogram, total run time 5 3 1. 1,64 Da. 7,95 Da 3. 16,6 Da 5 4. 34,7 Da 5. 57,5 Da 6. 17, Da 7. 55,1 Da 15 1 7 6 5 4 3 1 ZL13 5 1 3 4 www.sepax-tech.com
nriu 175 15 15 1 75 5 5 Sodium polystyrene sulfonate separation on SRT SEC-5, 7815 Column: SRT SEC-5 (5 m, 5 Å, 7.8 x 15 mm) Mobile phase: 15mM Phosphate Buffer, ph 7 Flow rate: 1.5 ml/; Detector: RI; Column temperature: RT Injection volume: 5 µg, Sample: See chromatogram, total run time 5 1. 7,95 Da. 16,6 Da 3. 34,7 Da 4. 57,5 Da 5. 17, Da 6. 55,1 Da 6 5 4 3 1 ZL13.5 1 1.5.5 3 3.5 4 4.5 www.sepax-tech.com
Zenix SEC-3 and SRT SEC-5 in tandem Column: Zenix SEC-3 (3 m, 3 Å, 7.8 x 15 mm), SRT SEC-5 (5 m, 5 Å, 7.8 x 15 mm) Mobile phase: 15mM Phosphate Buffer, ph 7 Flow rate: 1.5 ml/; Detector: RI; Column temperature: RT Injection volume: 5 µg, Sample: See chromatogram, total run time 1 nriu 175 1. 7,95 Da 15. 16,6 Da 3. 34,7 Da 15 4. 57,5 Da 5. 17, Da 1 6. 55,1 Da 75 5 6 5 4 3 1 ZL13 5 164 Da -5 1 3 4 5 6 7 8 9 www.sepax-tech.com
Sodium polystyrene sulfonate separation on SRT SEC-5 783 Column: SRT SEC-5 (5 m, 5 Å, 7.8 x 3 mm) Mobile phase: 15mM Phosphate Buffer, ph 7 Flow rate: 1. ml/; Detector: RI; Column temperature: RT Injection volume: 5 µg, Sample: See chromatogram, total run time 15 ZL13 nriu 16 1. 795 Da. 166 Da 14 3. 347 Da 1 4. 575 Da 5. 17, Da 1 6. 55,1 Da 6 5 4 3 8 6 4 Longer column gives better resolution. 1 4 6 8 1 1 14 www.sepax-tech.com
Polystyrene sulfonate separation on SRT SEC-1 Column: SRT SEC-1 ( 5 µm, 1 Å, 7.8 x 3 mm) Mobile phase:. M NaCl +.1% NaN 3 Flow rate:.5 ml/, Detector: RI ( 35 ), Column temperature: 35 Injection volume: 1 µl.1 mg/ml, Pressure: 3 bar Sample: Blank, 15, 8, 15, 3, 67, 1, and,35 kd Sodium polystyrene sulfonate ZL13 nriu 1,35 kd 8 6 4 1, kd 67 kd 3 kd 15 kd 8 kd 15 kd Blank 1 1 14 16 18 4 www.sepax-tech.com
ZL13 Polystyrene sulfonate MW Calibration Curve on SRT SEC-1 1 1 MW 1 1 1 1 14 16 18 Retention Time () www.sepax-tech.com
SL16 UV8nm Blue Dextran on SEC mau 4 3 1 1 1. SRT-C 3, 463, 15 mm phosphate buffer,.35ml/, 3 mg injection. SRT-5, 783, 5 mm phoshpate buffer with % IPA, 1mL/, 5 mg injection 3. SRT-5, 783, 15 mm phosphate buffer, 1mL/, 5 mg injection 3 4 6 8 1 1 14 www.sepax-tech.com 1
SL16 Blue dextran on SRT-C 3 463 mau 3 5.498 5 15 1 5 4 5 6 7 8 9 1 11 www.sepax-tech.com
Blue Dextran on SRT SEC-1 SL16 mau 1 1 8 6 Column: SRT SEC-1, 783 Mobile phase: 15 mm phoshpate:1%ipa=8: Detection: 8 nm Flow rate: 1 ml/ Injection: 5 ml 5 mg/ml blue dextran in water 4 4 6 8 1 1 14 16 www.sepax-tech.com 3