I have a problem with my Sephacryl S-300 HR column. I recently purchased the resin to pack with a 70-cm long glass column (homemade), with a 1.75 cm diameter. The bed volume is about 150 ml and the column was packed with a peristaltic pump using the flow rate described in the insert. The problem is when I tried to run a 2-ml mixture between blue dextran (BD) and potassium dichromate (PD) (3 mg/ml each and ran at 0.58 ml/min), both BD and PD seemed to diffuse throughout the column as they traveled to the end of the column. They don't seem to separate well. Something must be wrong here. BD should leave the column way before PD but that wasn't the case here. Could anyone help me troubleshooting this problem, please? I have these questions below:
1. Does Sephacryl resin require a buffer to pack and run? My sample is quite stable in water and this is why I use it instead of a buffer. And so the packing and running were done using milli-q water. Would this be the cause of the problem?
2. I don't think the resin was bad since I just bought it from the company and I don't think BD and PD was the culprit either. I am also sure that the loading technique was not the problem either. I think the problem is the column was not properly packed. Could anyone recommend me about packing and dealing with Sephacryl resin?
Thank you very much in advance
Hi Decha,
1. I do not think that water is a problem for the packing. However, salt is generally used to prevent interactions between macromolecules and the resin, so this might be your problem with BD (If so, it would elute later than predicted).
2. OK for the resin. If I were you, I would first check which marker elutes at the wrong volume: PD should elute in the total volume (around 150 ml) and BD in the void volume (in general about 1/3 of total, so 50 ml). If one is OK and the other wrong, I would try another marker for the wrong one. If both are wrong, or if they diffuse too much (that is if you recover the dye in more than 15-20 ml, it might be the packing. The trick with the packing is to prevent irregularities in the resin bed. To do this, the ideal way is to have a reservoir above the column so that you can pour all the resin that will be necessary for your 150 ml (probably around 300 ml) in one shot. If your column is homemade, you probably don't have a reservoir. So flow a little water through your column first to wet the frit, remove bubbles and close the bottom tap keeping 2 cm water at the bottom of the column (to prevent the resin from clogging the bottom frit). Then thoroughly resuspend the resin in your bottle to get a homogeneous slurry. Slowly pour your slurry down the column (ideally with a pipet, moving it in small circles so that the slurry drops do not fall all on the same spot). Completely fill the column. Open the bottom and start a slow flow-rate. You should see the horizontal separation between the packed bed (more opaque) and the slurry (more transluscent) appear rapidly and move slowly upwards. At the same time, there should be a separation at the top between the slurry and the liquid. Keep adding slurry slowly, always by pipetting in slow circles close to the surface until the packed bed reaches the desired height, always avoiding to perturb the surface of the bed. After that, lower the top adaptor close to the bed, run two column volumes water through the column at the standard flow rate, and one at the maximum flow rate. Lower the top adaptor until it slightly enters the packed bed, tighten the O-ring. The column should now be ready for use. Good luck!
Olivier
Olivier is right. I did it all that way with my Sephacryl S 200 column. All the gel slurry must be in one reservoir during packing. Pack in water, but afterwards equilibrate in the buffer for elution, at least 3 volumes of the column. Then add blue dextran alone, and PD alone. My blue dextran was awfully wide band, I still remember it, wide peak in the absorbance either, but then thyroglobulin, serum albumin and insulin which I used for the calibration gave reasonably narrow peaks. After all, I wanted to separate only three proteins with different mol. masses, one in the void volume, the second one eluted in the middle of the Vo and Vt and the third was very small. If you dont need a great resolution, and it seems that you dont, since you use 1,75 cm wide column bed, leave it that way and try your sample if it is available.
if you have conductivity reading, try comparing NaCl
(by conductivity) with BD. PD is not very conventional
substance to run on these columns.
Chromates are very reactive (does manufacturer list them as
compatible with you resin?) and may form high molecular weight
species, kind of defeating the purpose..
Also 0.58 ml/min sound very slow for your column geometry.
You should be able to work at 4 ml/min, it could save some time.
(don't forget to set pressure alarm)
Hi everyone,
Thank you very much for all your recommendations. After I asked my questions, I actually did one thing before repacking my column: reran the column with Tris buffer and 150 mM NaCl. What I found was all the yellow tint color from PD came off. Then, I used BD and phenol red to run a column in this buffer. BD came out first (at about 60 ml, which is a void volume) but I noticed quite a long tail (don't remember how many fractions) and about 120 ml later, the peak of phenol red showed up. So I think
the packing might be fine. The BD and PD were still ok. without some salt, PD binds to the resin and BD also doesn't behave well. Adding some salts prevents non-specific interactions between the resin and those molecules.
I guess I'll leave the column the way it is for now and try to run with the actual samples. (repacking a column is probably a pain!). Anyway, thank you everyone, especially Oliver for your recommendations. I will take your advice if I do need to repack my column. Thanks again!
Regards,
Decha S
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