Chromatography Forum

Hi everyone,
I have to analyze the lyophilized protein. When I run the injection of dissolved freeze-dried cake with C18 column I see new peaks absent in solution before freeze-drying. Mobile phase: A: 0.1% triflouroacetic acid (TFA) , B: 0.1% TFA water with gradient, detector UV 210.
In addition to protein in freeze-dried cake are sodium chloride, sodium and potassium phosphates, and polysorbate 70. Those peaks are present in freeze-dried product without protein e.g. they are not protein derivates. I thought that 210 is a specific wave for peptide bond.

May be anybody could help me to solve this problem
Thank you very much

Amaryllis,
I am assuming your detection Wavelength is 210 nm. At 210 nm, almost everything absorbs. If the peaks are in a blank preparation, without protein, I would probably think that the Polysorbate is the source of your extra peaks.

Hi Amaryllis,

I can indeed confirm that the peaks in the blank are coming form the polysorbate. I observed this while dealing with the same type of analysis last year. It influenced our results as those peaks elute at the retention time of our product.

Not all PS brands did show this "problem"


Philippe