Anion exchange column overloading. Peaks in flow through. - (May/08/2008 )
if you have some experince with ion exchange chromatography of protein you could give me answer for my question. I do anion exchange chromatography (20mM Tris-HCl, pH = 8.9; elution with 1M NaCl in the same buffer) to purify some proteins. Since my sample is difficult to dissolve I use urea (final concentration of max. 4M). The sample loop (that I use) has capacity (volume) of 2ml, but sometimes I have more than 2ml of sample, so I have to load sample for example 3-4 times. After a few minutes after the first sample injection in sample loop there is a peak (probably some unbound material/proteins), then I inject sample second time and then third time and the peaks (unbound material) are usually much higher than after the firts injection of sample. After loading whole volume of sample and when there are no peaks (in flow through) I start gradient of 1M NaCl, 0-60%, 20 column volumes. Usually I get good chromatogram with good separation and sharp peaks (280nm), however many proteins are in flow through (unbound material) - actually the biggest peak at all is comming in flow through.
My question is:
Is it normal that peak(s) in flow through is/are higher than whatever peak of bound material (peaks comming after starting gradient elution), when using anion exchange column at pH=9?
The binding capacity of column (Mono Q) is approx. 70mg, I load max. 30mg, so it should not be overloaded.
the flow through peak is often higher than the elution peaks in iec.
the bound proteins will be eluted in a spread over the range of the gradient whereas the flow through is not spread.
also, proteins will remain unbound due to the conditions of the load (concentration and pH of buffer a, buffer of the sample) and capacity of the column.
despite the capacity of the column, much of your protein binds near the top of the column. as that saturates, the protein will travel farther through the column before it binds, allowing more to pass through without binding. multiple injections will show larger flow through peaks but it would probably work out the same if you had a higher concentration rather than higher volume.
to sum up my rambling, what you are seeing is pretty much normal.
you may be able to tweak conditions to increase binding but it is probably not necessary.