Chromatography Forum

Hi,

I am testing IgG antibodies, and notice that the formulation buffer peak is eluting at the same time as LMWS peaks, essentially masking them so i cant really quantify LMWS %.

Is there any way to solve this problem?

Mobile Phase/Diluent is PBS pH 7.2, column is 300A 4.6x 300 mm

With isocratic for 16 minutes at 0.3 mL/min(recommended column flow rate)

The formulation buffer that antibody is in contains EDTA, PBS, and ammonium acetate.

Thank you.

Hello,

Edit: What I wrote in my comment was incorrect! Vmu and Multi have a much better understanding and will provide significantly better advice than I have

I am testing IgG antibodies, and notice that the formulation buffer peak is eluting at the same time as LMWS peaks, essentially masking them so i cant really quantify LMWS %.

1. How do you know that the peak you consider LMWS actually corresponds to LMWS rather than to a buffer component?
2. Try to use different column with better selectivity in the LMW range.

I could be misinterpreting your statement

You don't understand. This is size-exclusion chromatography with no retentive interactions of the compounds of interest. Everything is eluted between the volume close to the interparticle volume V0 in the column and the dead volume, which is the total volume of the eluent in the column Vt (= interparticle volume + intraparticle pore volume).
The analyte distribution coefficient K is given by
K = (VR - V0)/(Vt - V0).
K varies from 0 to 1.

As stated, the poster is using SEC HPLC, so conventional K prime issues do not apply here. If the sample peak is eluting with the buffer (and the buffer has been correctly chosen for the column), then THE COLUMN CHOICE IS WRONG. Select a column with the appropriate Mw range for your actual sample. You may need more than one. No way to know as no information was provided, but please speak to someone who is knowledgeable in HPLC SEC method development before proceeding.