Dear Soha,

Size exclusion columns (silica and other types) in general give highly reproducible results. So the irreproducibility should not be because of the column unless the column has stopped working properly.

Are you sure that your sample preparations are reproducible? You should also note that some proteins stick to the columns under some conditions which might lead to irreproducible results. So make sure that you are recovering all the sample you are injecting. In case of sticking of protein to column, you might as well try changing pH, salt concentration etc.

Best,

Jogender

thanx Jogender....sometimes i make my last washing with water and leave it for days...might this deteriorate my column; coz of bacterial contamination.. i am using different buffer concentration; and you know how problematic buffers are, also what is the best method to get red of protein; i am using sls. dmso and phosphate buffer at pH=3; but always repeating the same experiment gives very different result.

best regard

soha

Soha,

Leaving column in just water for long is not really good for column. Store in 20 % ethanol for short-term storage. If you want to store for long, then use water which contains 0.05 % sodium azide.

If you want to clean your column, then use 6 M guanidine hydrochloride solution. But after cleaning the column, you should check that nothing is sticking to the column. For that inject a calculated amount of your sample in the column and then collect the peaks and check if you get the same amount of sample back.

If you find that your sample is sticking to the column, then change buffer. May be you can add some salt (if that is ok with your sample). Anyway you should make sure that nothing is sticking to the column. I hope you'll get reproducible results after these precautions.

Best,

Jogender

Well Jogender is very right. In fact, for each colum there is a specific life span, whereby you can run a certain number of samples, after which the column does not give good results. Please check if your column is not very old (may have completed its running life).

After passing a sample through the column, one should run at least a blank (just buffer) to flush out any contamination or residues in the column.

The column should never be stored in water and proper preservation liquid should be used.

I hope this will help

Hello Soha, 

Although the column is best to be stored in 20% ethanol, storing in water for one night should not be the reason for bacterial growth and inconsistent results. Depending on your system you can also keep the column under low flow rate of water over night (e.g. I used an Akta purifier and kept the column under 0.1 mL/min flow). 

As other researchers have mentioned, the column might not be the problem but once you ruled out other sources it would be best to check the quality of the column. For example injections of blue dextran and 2% acetone shows void volume and total volume of the packed bed and peak symmetry is an indication of quality of packing (compare these with manufacturer recommended values). As mentioned by others using a buffer with suitable conductivity is important to avoid non-specific interaction of protein with the column (check the manufacturer recommended range). 

Hope this helps

hellow everyone,

thanks for replying; i wonder  Pegah Saramirad if dextran blue in 2% acetone could be used as a standard to check if my coloumn is okay; i never hear or read about it; they only use a standard of proteins available in the same company; phenomenix; in fact i tried to wash my coloumn with 0.5% SLS, but now i am experiencing a problem in backpressure elevation, in fact i am confused if it was of buffer or protein; i used denaturant and now i am using hot water to get red of any possible buffer. the problem is such a coloumn is highly expensive.

best regard for you all collageous

Soha

Hello acetone and blue dextran are two different tests. I would strat with acetone test as blue destran sometimes tend to get stuck on SEC matrix. Inject 2-5% (v/v) acetone in water/buffer on your column and elute with water/buffer. I used water as acetone did not interact with the matrix. If the peak is symmetric that's a good sign. If you see peak tailing the column is not clean and that explains back pressure. If the peak is leading (i.e you are detecting part of acetone earlier that the total volume, the column could be over packed and you have channeling in the column which can happen for various reasons. 

silica gel type columns can not be stored at all in water, instead of that it should be stored in organic water content. At least 20% of organic should be used in storage conditions. 

Its obvious that the washing step for column is not sufficient because u have protein stuck. Try to use 1% of acetic acetic as a first step to wash out proteins and buffer. if it doesn't work well, try to use TFA 0.1%. the final step of the previous one is to wash the column with water. 

isopropanol along with water as a final hope could also be used. Gradually increase it against water over time till it reaches 100% isopropanol. Check the maximum tolerant pressure of ur machine because as u know isopropanol is a viscous liquid.

Best regards,

Zaiter 

thanks ahmad, i dont think i can use TFA or acetic acid, instead phosphate buffer pH=3 is commonly recommended, and for isopropanol the company never recommend using it, maybe methanol or acetonitril gradient.