Glycomix™ strong anion-exchange phase is made of narrow-dispersed and hydrophilic polymer particles. It is a strong anion exchanger with quaternary ammonium functional groups that are chemically bonded to the hydrophilic surface of the polymer particles.
Featured Characteristics
- High capacity and high resolution
- High lot-to-lot reproducibility
- Wide pH range (2-12)
- From analytical to preparative scale
- Ideal for separation and analysis of heparin, glycans
High Lot to lot Reproducibility
With well-controlled surface chemistry and resin production, Glycomix resins exhibit high lot to lot reproducibility which leads to consistent column performance. The separation variation for Heparin from batch to batch is less than 1% for retention time and 5% for the peak area.
Figure 2. High Lot-to-lot reproducibility of three lots of Glycomix™ SAX resins.
| Conditions | |
| Column: | Glycomix™ SAX, 4.6×250mm |
| Mobile phase: | A: 0.04% NaH2PO4, pH 3.0
B: 0.04% NaH2PO4+14% NaClO4, pH 3.0 |
| Flow rate: | 0.22 mL/min |
| Gradient: | 20% - 90% B in 60 minutes |
| Wavelength: | 202 nm |
| Temperature: | 25 °C |
| Injection volume: | 10 µL |
| Pressure: | 9.5 bar |
| Sample: | 20 mg/mL Heparin sodium
1 mg/mL Dermatan sulfate (DS)
1 mg/mL Oversulfated chondroitin sulfate (OSCS) in H2O |
Lot-to-Lot peak retention time relative standard deviation.
| Peak Name | Lot A | Lot B | Lot C | RSD% |
|---|
| Chondroitin Sulfate B | 12.286 | 12.300 | 12.434 | 0.41% |
| Heparin | 21.71 | 21.723 | 21.919 | 0.44% |
| OSCS | 33.936 | 33.853 | 34.114 | 0.32% |
Long column life time
Glycomix™ SAX has long column life time due to its resin surface chemistry and stable manufacturing process. Figure below exhibits one column's performance for duplicated injections. The column shows consistent separation efficiency.
Figure 3. Chromatogram overlays of duplicated injections.
| Conditions | |
| Column: | Glycomix™ SAX, 4.6×250mm |
| Guard Column: | Glycomix™ SAX, 4.6×50mm |
| Mobile phase: | A: 0.04% NaH2PO4, pH 3.0
B: 0.04% NaH2PO4+14% NaClO4, pH 3.0 |
| Flow rate: | 0.22 mL/min |
| Gradient: | 20% - 90% B in 60 minutes |
| Wavelength: | 202 nm |
| Temperature: | 25 °C |
| Injection volume: | 10 µL |
| Pressure: | 9.5 bar |
| Sample: | 20 mg/mL Heparin sodium
1 mg/mL Dermatan sulfate (DS)
1 mg/mL Oversulfated chondroitin sulfate (OSCS) in H2O |
Sensitive separation of different Heparin loading
Glycomix™ SAX is able to separate components from commercially available standards. In Figure 4, Panel A shows the standard run with 20mg/mL Heparin, 1mg/mL DS and 1 mg/mL OSCS. Panel B is the OSCS chromatogram with 1 mg/mL concentration. In comparison to Panel B, the small peak between DS and Heparin comes from the standard OSCS sample. Column running conditions are the same as for Figure 3.
Glycomix™ SAX can be used in the quantitative analysis of Heparin and its impurities. Figure 5 shows the different heparin loadings over concentration range from 0.5 mg/ml to 10 mg/ml, 0.125 mg/ml to 4 mg/ml for OSCS and 0.25 mg/ml to 4 mg/ml for DS. The R2 values for both standards exceed 0.99 in the tested concentration range.
Figure 4. Chromatogram overlays of OSCS standard and Heparin with DS and OSCS.
Figure 5. Chromatogram overlays of different heparin injections from 0.5mg/mL to 10mg/mL concentration, DS from 0.125 mg/mL to 4.0 mg/mL and OSCS from 0.25 mg/mL to 4.0 mg/mL.
| Conditions | |
| Column: | Glycomix™ SAX, 4.6×250mm |
| Guard Column: | Glycomix™ SAX, 4.6×50mm |
| Mobile phase: | A: 0.04% NaH2PO4, pH 3.0
B: 0.04% NaH2PO4+14% NaClO4, pH 3.0 |
| Flow rate: | 0.22 mL/min |
| Gradient: | 20% - 90% B in 60 minutes |
| Wavelength: | 202 nm |
| Temperature: | 25 °C |
| Injection volume: | 10 µL |
| Pressure: | 9.5 bar |
| Sample: | 20 mg/mL Heparin sodium
1 mg/mL Dermatan sulfate (DS)
1 mg/mL Oversulfated chondroitin sulfate (OSCS) in H2O |
Column Performance Comparison
In comparison, Glycomix™ SAX performs superior to Dionex's column for heparin separation.1,2 Figure 7 shows the chromatogram of Heparin separation from Glycomix™ SAX. The resolutions are 3.8 between DS and heparin, 5.8 between heparin and OSCS. The U.S. Pharmacopeia (USP) requirements for the separation for DS/ heparin are 1.0 and 1.5 for OSCS /heparin3. GlycomixTM SAX has a much better separation resolution than USP's requirement. In Dionex's application note, Dionex IonPac AG11 guard with AS11 analytical column gives a 1.1 and 1.8 resolution for DS/heparin and heparin/OSCS respectively.
Figure 7. Heparin Sodium, Chondriotin Sulfate B, and Oversulfated Chondroitin Sulfate separation on Glycomix™ SAX.
| Conditions | |
| Column: | Glycomix™ SAX, 4.6×250mm |
| Guard Column: | Glycomix™ SAX, 4.6×50mm |
| Mobile phase: | A: 0.04% NaH2PO4, pH 3.0
B: 0.04% NaH2PO4+14% NaClO4, pH 3.0 |
| Flow rate: | 0.22 mL/min |
| Gradient: | 20% - 90% B in 60 minutes |
| Wavelength: | 202 nm |
| Temperature: | 25 °C |
| Injection volume: | 10 µL |
| Pressure: | 10 bar |
| Sample: | 20 mg/mL Heparin Sodium
0.2 mg/mL Dermatan Sulfate (DS)
0.2 mg/mL Oversulfated Chondroitin Sulfate (OSCS) |
