SEC Column Selection Guide for the Separation of Water-soluble Polymers

Transcription

1 SEC Column Selection Guide for the Separation of Water-soluble Polymers Dextrans Molecular weight (Da) 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 Elution volume (ml) Linear distribution region between Log MW & Elution volume Pullulans Molecular weight (Da) 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 Elution volume (ml) Linear distribution region between Log MW & Elution volume Polyethylene Glycols Molecular weight (Da) 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 D 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.

2 CC1 Analysis of Glucidex on Carbomix -Ca (5 µm, 8%, 4.6x3 mm) G8-G MV 3. G7. G4 G6 G5 G3 G G 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.

3 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.

4 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.

5 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 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 MV 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) Table 1. Pullulan molecular weight and retention time on SRT 3, 5 m, 7.8 x 3 mm. Sepax Technologies, Inc.

6 SL15 6 Log MW 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.

7 ZL1 PEG separation on two Zenix SEC-3 7.8x3mm columns in Tandem nriu PEG 81,4Da. PEG 5,63Da 3. PEG 4,Da 4. PEG 6,1Da 5. PEG 8,73Da 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.

8 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.

9 nriu 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 ,95 Da. 16,6 Da 3. 34,7 Da 4. 57,5 Da 5. 17, Da 6. 55,1 Da ZL

10 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 ,95 Da. 16,6 Da ,7 Da 4. 57,5 Da 5. 17, Da 6. 55,1 Da ZL was buried in the negative peak, blue trace

11 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 ,64 Da. 7,95 Da 3. 16,6 Da ,7 Da 5. 57,5 Da 6. 17, Da 7. 55,1 Da ZL

12 nriu 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 ,95 Da. 16,6 Da 3. 34,7 Da 4. 57,5 Da 5. 17, Da 6. 55,1 Da ZL

13 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 ,95 Da ,6 Da 3. 34,7 Da ,5 Da 5. 17, Da ,1 Da ZL Da

14 Sodium polystyrene sulfonate separation on SRT SEC 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 Da. 166 Da Da Da 5. 17, Da ,1 Da Longer column gives better resolution

15 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 , kd 67 kd 3 kd 15 kd 8 kd 15 kd Blank

16 ZL13 Polystyrene sulfonate MW Calibration Curve on SRT SEC MW Retention Time ()

17 SL16 UV8nm Blue Dextran on SEC mau 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

18 SL16 Blue dextran on SRT-C mau

19 Blue Dextran on SRT SEC-1 SL16 mau 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