Chromatography Forum

hi,
im running a size exclusion chromatography method for a protein aggregate analysis.
column: silica matrix TOSOH column
mobile phase: 50 mM ammonium bicarbonate ph 7.0
sample matrix: 10 mM sodium acetate
capture wavelength: 215 nm

what i'm seeing is.. when i inject 10 mM sodium acetate there are peak at about 10 mins and there is a large dip in the baseline after about 40-45 mins. i've confirmed these are not because of protein carry over or any artifact. these are because of the acetate buffer and also the intensity of such interference increases with increase in acetate buffer concentrations.

these peaks interfere with my analysis and are very inconsistent in shape but always there.
any ideas on what is happening and what can be done?
thanks!

Your bicarbonate buffer has a pKa of 10,3 and the recommended pH range is 9,3 to 11,3. Your acetate has a pKa of 4,76 and the recommended pH range is 3,76 to 5,76.
If you go to the manual of your column you will see that sodium phosphate is recommended as a buffer. Phsphate has a pKa of 2,15 and the recommended pH range is 1,15 to 3,15.
My recommendation is to change pH and to decrease the concentration of your Acetate. Good luck.

Alicee, at 215 nm, a solution with 10 mM acetate will have a baseline about 0.3 AU high. Since you are running isocratically, then additional peaks such as your protein's will be on top of that elevated but steady baseline. However, anything that reduces the acetate ion concentration will result in a solution with lower absorbancy and a negative peak. The protein is acquiring some acetate ions as counterions for its basic residues and those are riding through the column at the elution time of the protein. The result is that the acetate concentration is depleted at its normal elution time, which would be appreciably later than the elution time of the protein in SEC and is probably at the 40-45' time that you noted. Gerhard's suggestion to use a buffering salt like phosphate that's transparent at 215 nm would solve the problem.

Hi
So it should be against nature not trample. The 215 carbonyl group has a absorption.
What should I do? After the release of target substances to transfer the device to "a lot (big) water and wash acetate. Or "score a bolt" on acetate and to work with Phosphate BS.

Phosphate will work the best in that case. Also, Ammonium Bicarbonate is not very stable as buffer...