protamine problems

[derboyl]

hello,

could anyone help me with a problem im having,
i have a protamine analyte giving a 201nm lambda max abs,
the literature say for hplc:
UV 214nm,
h20/acn/tfa (qs/8%/0.15%)
pH 2.5
waters size exlusion column,

my problem is,
im getting negative peak for protamine. this is no great surprise but im having trouble composing a mobile phase with a lower abs than my analyte,
any suggestions would be gratefully taken.
derboyl

[danko]

Hi Derboyl,

It’s TFA that absorbs too much light, especially in combination with ACN. But you knew it already – I presume. What I wonder about in this case is, what in earth’s name, is TFA’s role in Size Exclusion Chromatography? Just drop it and if you need the pH to be 2.5 (and I would expect, you do) then make 0.05 M phosphate buffer, mix it with 10 % or so ACN and you’re back in business.

Best Regards

[derboyl]

hello danko,
thanks for that,

thruthfully, i didnt realise TFA absorbed more light with ACN, i was under the impression it was transparent at lower wavelenghts, less than 200nm
so i will try your suggestion and drop the TFA.
incidently, TFA was used by the autors of the method useing size exclusion columns that im trying to replicate and develop. i was curious about its use myself.

[HW Mueller]

Just about everything absorbs below 200nm, TFA absorbs even far up.
Some years ago I tried TFA to decrease interactions of an antibody with the stat. phase in SEC. If you do a search of this forum you may find one result: I permanently decreased the separation power of the column (TSKgelSuper SW 3000). Incidentally, I still don´t know why, and that in spite of enormous efforts by local representatives of Tosoh to get info from Tosoh headquarters.