Column load and resolution- peptides and proteins

[pipettemonkey]

Manufactures of RP columns for peptides/proteins give ranges for amount of protein that can be loaded. The bottom end is amount of peptide that can be loaded without reducing resolution. The upper number is "Maximum practical sample load," and is approximately the max quantity of sample that can be purified with reasonable yield and purity on the column.

For 4.6mm ID, the range is 1-200micrograms (w/out loss of resolution), and 10mg for max practical load.

My question is this- I have a protein mixture that contains misc components, including my peptide of interest, and a whole lot of BSA. If the BSA elutes at a much higher % CH3CN than my peptide of interest, can the amount of BSA still affect my separation of components eluting near the % organic as my peptide of interest?

[Uwe Neue]

The difficulty with such numbers is that they depend on the way they are measured. My guess is that the higher values represent a total saturation capacity of the column for a given protein, while the lower numbers reflect the peak broadening encountered in high-resolution gradient runs.

In your case, the upper limit would be determined by the BSA, and the BSA loaded would displace some of the other compounds as well as the peptide. A safe first guess would be to try the assay at 10% of the "maximum practical sample load" for BSA, i.e. at 1 mg. If it still works well, you can increase it in factors of 2 until you see displacement patterns for your peaks of interest. However, my bet is that you won't be able to go that far.