FAQ
Sephadex G-10, G-15, G-25, G-50, G-75, and G-100 are based on dextran of different molecular weight ranges. The lower the G-value, the higher the degree of cross-linking of the dextran polymers, and the lower the molecular weight fractionation range. Additionally, with increasing degree of cross-linking, the swelling of the medium decreases.
Coarse and Medium grades are preferred for group separations at process scale where high flow rates and low operating pressures are required. The Fine and Superfine grades are for routine laboratory work including small-scale preparative separations (e.g. HiPrep 26/10 Desalting [Fine], HiTrap Desalting [Superfine]). The smaller particle sizes of the Fine and Superfine grades give shorter diffusion distances and allow for highly efficient separations at high flow rates. DNA grade is tested and certified to ensure reproducibility and high recovery with DNA to ensure no nonspecific binding.
Medium | Grade | Fractionation range, peptides and globular proteins (Mr) |
---|---|---|
Sephadex G-10 | - 700 | |
Sephadex G-15 | - 1 500 | |
Sephadex G-25 | Superfine Fine Medium Coarse |
1 000 - 5 000 1 000 - 5 000 1 000 - 5 000 1 000 - 5 000 |
Sephadex G-50 | Superfine Fine Medium Coarse |
1 000 - 30 000 1 000 - 30 000 1 000 - 30 000 1 000 - 30 000 |
Sephadex G-75 |
Superfine |
3 000 - 80 000 3 000 - 70 000 |
Sephadex G-100 |
Superfine |
4 000 - 150 000 4 000 - 100 000 |
* in distilled water
Packing By Gravity Flow
- Pour the entire slurry into the column in one portion; be careful not to trap air bubbles.
- Start the gravity flow to initiate packing.
Packing Using a Flow Adapter
Good packing is essential to obtain good resolution. Ideally, the column should be packed at the highest pressure possible without deforming the beads.
For softer resins, like Sephadex, care must be taken to avoid compressing the resin. Do not increase the pressure beyond the values given in the table below.
With wider columns, slightly reduced maximum operating pressures must be used. Flow rate is inversely proportional to bed height: increasing the bed height will decrease the flow rate, but does not affect the maximum operating pressure.
- Using a reservoir if necessary, pour the entire slurry into the column in one portion.
- Start the pump to initiate packing.
- Once all the resin has settled into the column, remove the reservoir.
- Insert an adapter and pack at the maximum operating pressure of the resin.
- Once the resin is thoroughly packed into the column, adjust the flow adapter to the surface of the resin.
- Wash the column with an additional 2 to 3 column volumes of the buffer to be used in the separation. This stabilizes and equilibrates the bed.
- Readjust the flow adapter to the surface of the resin bed.
Chromatography medium | Grade | Approx. maximum operating pressure (bar) |
---|---|---|
Sephadex G-10 | All | 0.6 |
Sephadex G-15 | All | 0.6 |
Sephadex G-25 | All | 0.8 |
Sephadex G-50 | All | 0.1 |
Sephadex G-75 | All | 0.16 |
Sephadex G-100 | All | 0.096 |
Product | PSDdry (Within specification)1 | PSDwet2 |
---|---|---|
µm | µm | |
Sephadex G-10 | 40-1203 | 52-173 (20-180) |
Sephadex G-15 | 40-1203 | 58-184 (20-160) |
Sephadex G-25 SF | 20-504 | 33-89 (10-110) |
Sephadex G-25 F | 20-804 | 33-140 (10-180) |
Sephadex G-25 M | 50-1505 | 84-262 (20-280) |
Sephadex G-25 C | 100-3005 | 166-531 (90-550) |
Sephadex G-50 SF | 25-504 | 51-106 (20-130) |
Sephadex G-50 F | 20-804 | 41-171 (20-240) |
Sephadex G-50 M | 50-1505 | 103-320 (40-370) |
Sephadex G-75 | 40-1204 | 92-293 (40-320) |
Sephadex G-75 SF | 20-504 | 46-122 (20-150) |
Sephadex G-100 | 40-1204 | 94-329 (40-360) |
Sephadex G-100 SF | 20-504 | 49-134 (20-170) |
1 Within sieving limits, guide for which Size Exclusion resin to choose depending on application
2 Calculated wet PSD range based on water regain. Range within which particles can be found is presented within parentheses and should be used as guide for choosing filter.
3 ≥ 85% of the volume of particles are found within the range shown
4 ≥ 80% of the volume of particles are found within the range shown
5 ≥ 90% of the volume of particles are found within the range shown
Sephadex is compatible with commonly used aqueous solutions in the pH range 2 to 13. It is stable to all commonly used buffers, solutions of urea (8 M), guanidine hydrochloride (6 M), and all non-ionic and
ionic detergents. Lower alcohols (methanol, ethanol, propanol) may be used in the buffer or the sample, but we recommend that the concentration be kept below 25 v/v%. Prolonged exposure (hours) to pH values below 2 or above 13, or to oxidizing agents should be avoided.
Sephadex is supplied as a dry powder and must be allowed to swell in excess buffer before use (see steps below). After swelling, adjust with buffer to form a thick slurry from which air bubbles are removed under vacuum. Approximately 75% settled medium is suitable. Fine particles can be decanted. Filter all buffers through a 0.22 µm Whatman filter to help prevent microbial growth.
- Weigh out the appropriate amount of dry Sephadex for the required bed volume of your column (see table). For group separations, use up to a 10 cm bed height. For example, if using a 26 mm i.d. column, a 10 cm bed height equals 53 ml of medium. Sample volumes can be up to 30% of the column volume. Pack a quantity of medium up to five-fold the volume of the sample. If packing at the maximum pressure for the medium, choose the low end of the bed range from the table for calculating the amount of dry Sephadex.
- Calculate the total volume of the column. Add 1.3 times this volume of your swelling buffer to the dry beads. Swelling times for the different types of Sephadex are given in the table. The process is accelerated by using a boiling water bath, which also serves to deaerate the buffer.
- Stir - excessive stirring should be avoided as it may break the beads. DO NOT USE MAGNETIC STIRRERS!
- After swelling is complete, decant the supernatant.
- Add buffer to make a 75% suspension (i.e., a ratio of 75% settled gel to 25% buffer, as measured by height in a beaker or other container).
- Degas the suspension before packing.
Resin | Grade | Approx. mL of bed volume/1 g resin* |
Swelling time, 20° C (h) |
Swelling time, 90° C (h) |
---|---|---|---|---|
Sephadex G-10 | All | 2-3 | 3 | 1 |
Sephadex G-15 | All | 3-4 | 3 | 1 |
Sephadex G-25 | All | 5-6 | 3 | 1 |
Sephadex G-50 | Superfine | 121 | 3 | 1 |
Sephadex G-50 | Fine | 13-16 | 3 | 1 |
Sephadex G-50 | Medium | 14-15 | 3 | 1 |
Sephadex G-75 | All | 191 | 24 | 3 |
Sephadex G-100 | All | 211 | 72 | 5 |
* Swelling in swollen bed in 0,02M NaCl, should be used as guide to column/vessel size.
1 Representative swelling presented, statistical foundation not available