SEC HPLC formulation buffer peak coeluting with LMWS

[dlwngus810]

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.

[LuccasLN]

Hi,

Even though this ship has probably sailed, i will write down some thoughts for the sake of providing a reply to this question. In SEC, low molecular weight species will have a high elution volume/time and may elute with whatever is also there in your sample, I have seen this happen with solvents that were just in the sample as diluents but were not there in the mobile phase for example.

If this happens, it means there is something wrong with your experiment plan and this pore size is too big for your analyte, in other words, both your analyte and every other small molecule are eluting at the largest possible time, considering ideal behavior of course.

The best practices for SEC are always comparing the size and type of your macromolecule to a calibration curve (usually manufacturers will provide those). comparing similar macromolecules is extremely important in order to eliminate the need to consider hydrodynamic radius differences into play. Always choose a molecule that shows up it's weight between about 30%~70% in the linear region of the weight cal curve - this way you always guarantee your molecule will not suffer any effect regarding either exclusiom or excessive movement freedom inside the packing!

what if you need a larger range you may ask? well for this you either couple columns in series or look for column technologies with larger ranges (always considering pros and cons of them).

i hope this helps whoever mey read this in the future!