acetate buffer gives interfering peaks in size exclusion chr

Discussions about gel permeation chromatography / gel filtration chromatography / size exclusion chromatography

5 posts Page 1 of 1
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.
Gerhard Kratz, Kratz_Gerhard@web.de
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.
PolyLC Inc.
(410) 992-5400
aalpert@polylc.com
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...
5 posts Page 1 of 1

Who is online

In total there is 1 user online :: 0 registered, 0 hidden and 1 guest (based on users active over the past 5 minutes)
Most users ever online was 1117 on Mon Jan 31, 2022 2:50 pm

Users browsing this forum: No registered users and 1 guest

Latest Blog Posts from Separation Science

Separation Science offers free learning from the experts covering methods, applications, webinars, eSeminars, videos, tutorials for users of liquid chromatography, gas chromatography, mass spectrometry, sample preparation and related analytical techniques.

Subscribe to our eNewsletter with daily, weekly or monthly updates: Food & Beverage, Environmental, (Bio)Pharmaceutical, Bioclinical, Liquid Chromatography, Gas Chromatography and Mass Spectrometry.

Liquid Chromatography

Gas Chromatography

Mass Spectrometry