Influence of pressure fluctuation in ion exchange column on protein binding, sep - (Jul/12/2008 )
Hi all,
I would have a technical question for doing ion exchange chromatography protein purification. Sometimes I have higher volume of sample than the capacity (volume) of sample loop is, so I have to inject sample several times into the sample loop (firts time, then wait till sample is away from sample loop and then I inject second time, third time etc.). During this repetitive injection there is always a decrease of pressure (even till zero) for a few second (possibly because of some air (bubles) in tubing?). My question is, whether any decrease of pressure in column with sample in it somehow strongly influence binding of proteins to column (before applying a elution gradient)?
Or in another case, it happened to me, that I had to change a damaged tubing between pump and column, when the sample was already in column (but there was still not any elution gradient). So sample was in the column for a few minutes with zero pressure and zero flow. Again, does it strongly influnce then the binding and elution of proteins from column (if the sample is in column with zero pressure and zero flow for a few minutes)?
Do you have any experince with this?
Thanks.
vik
I would have a technical question for doing ion exchange chromatography protein purification. Sometimes I have higher volume of sample than the capacity (volume) of sample loop is, so I have to inject sample several times into the sample loop (firts time, then wait till sample is away from sample loop and then I inject second time, third time etc.). During this repetitive injection there is always a decrease of pressure (even till zero) for a few second (possibly because of some air (bubles) in tubing?). My question is, whether any decrease of pressure in column with sample in it somehow strongly influence binding of proteins to column (before applying a elution gradient)?
Or in another case, it happened to me, that I had to change a damaged tubing between pump and column, when the sample was already in column (but there was still not any elution gradient). So sample was in the column for a few minutes with zero pressure and zero flow. Again, does it strongly influnce then the binding and elution of proteins from column (if the sample is in column with zero pressure and zero flow for a few minutes)?
Do you have any experince with this?
Thanks.
vik
Hi vic,
The pressure drop shouldn't affect the binding at all. If you have a large volume to load, have you considered using the pumps to do the loading work for you? Take the "A" buffer line and put it into the sample, then load away to your heart's content. You'll probably get better returns, too, as there are always losses incurred by multiple loadings through a loop.
If you must / choose to use a loop a number of times, there are a couple of alternatives open to you. Either load 1/2 the loop volume then inject 1.5x the volume onto the column, or empty the loop (having removed it from the chromatography system) and inject into the empty loop, then refit (as you can imagine, this is for very experienced chromatographers only...). In any case, KEEP THE LOOP OVERFLOW, and reload that.
HI,
The Pressure Drop in IEX wont effect much in binding and in elution. Try the sample load using one of the buffer port, you dont need to load the sample in conecentrated (like for GFC) for IEX if your protein is more charged and if it binds to your resin very well , then there is no problem loading the sample through the the buffer port .
try it and write me if iam wrong
Regards
S
I would have a technical question for doing ion exchange chromatography protein purification. Sometimes I have higher volume of sample than the capacity (volume) of sample loop is, so I have to inject sample several times into the sample loop (firts time, then wait till sample is away from sample loop and then I inject second time, third time etc.). During this repetitive injection there is always a decrease of pressure (even till zero) for a few second (possibly because of some air (bubles) in tubing?). My question is, whether any decrease of pressure in column with sample in it somehow strongly influence binding of proteins to column (before applying a elution gradient)?
Or in another case, it happened to me, that I had to change a damaged tubing between pump and column, when the sample was already in column (but there was still not any elution gradient). So sample was in the column for a few minutes with zero pressure and zero flow. Again, does it strongly influnce then the binding and elution of proteins from column (if the sample is in column with zero pressure and zero flow for a few minutes)?
Do you have any experince with this?
Thanks.
vik
Hi vic,
The pressure drop shouldn't affect the binding at all. If you have a large volume to load, have you considered using the pumps to do the loading work for you? Take the "A" buffer line and put it into the sample, then load away to your heart's content. You'll probably get better returns, too, as there are always losses incurred by multiple loadings through a loop.
If you must / choose to use a loop a number of times, there are a couple of alternatives open to you. Either load 1/2 the loop volume then inject 1.5x the volume onto the column, or empty the loop (having removed it from the chromatography system) and inject into the empty loop, then refit (as you can imagine, this is for very experienced chromatographers only...). In any case, KEEP THE LOOP OVERFLOW, and reload that.