Chromatography Forum

Hello,

I'd like to test different buffer composition, specifically different concentrations of salt used for SEC-HPLC for antibody analysis.

If my starting buffer is 50 mM Sodium Phosphate 300 mM Sodium Chloride pH 7.3, is there a way to change the gradient composition so that i dont need to make 8 different salt buffers?

Also, is PBS a buffer choice for SEC? why or why not?

thank you.

In theory there is no issue using PBS for an SEC mobile phase if it gives you the separation you need, just make sure to check pH and salt concentration your column can handle.
Looking online I found this quote from phenomenex

"For some mAbs, a formulation like 1X Phosphate Buffered Saline might
be an acceptable mobile phase. However, recent work by Goyon and
colleagues showed the effects of sodium and potassium additives in
SEC, and how salt concentration, as well as buffer type and concentration, can affect separation and aggregate recovery significantly"
https://www.phenomenex.com/viewdocument ... Aggregates


for the salt concentration assuming you have multiple lines on your system you could put for example 100mM sodium phosphate on line A and 600mM sodium chloride in line B and 100% water on line C

run at 25% line A 25% line B and 50% line C and you have your original conditions
50% line A and 50% line B gives you twice the salt concentration of original conditions
continue adjusting your ratios as you like to deliver different salt concentrations and ratios, the only issue here would be pH is unknown/uncontrolled for your various conditions

good luck,
Chromavore