RE: base-deactivated HPLC

Bruce A. Stanley (bstanley@psu.edu)
Fri, 17 Sep 1999 09:14:06 -0400

The problems of positively charged solutes sticking to incompletely
endcapped silanol groups (leaving a negative charge on the matrix)
usually is evidenced by peak tailing, but I suppose sticking to the
column "indefinitely" could occur as well. Possible solutions: If you
wish to stick to C18 type separations, I have mentioned POROS columns
from Perseptive Biosystems to the ABRF list before - the matrix used
is not silica-based (and not), and therefore there are no negative
charges to interact undesirably with cations in the solute. While not
technically C18, the separations are very similar (except 5-10 times
faster), and the matrix can easily tolerate very low and very high pH
buffers, unlike the silica-based columns. Another solution is
newer-generation silica based columns which achieve 100% endcapping
of the silica matrix, therefore leaving no negative charges; however,
I expect that these columns shouldn't be run at the pH 2 you mention,
nor should any other silica-based columns be run at very low pH,
since the silica matrix itself begins to leach out (Someone correct
me if this is not true for newer silica-based columns...).

If the two peptides you mentioned are all you have in your mixture to
separate, however, a simple ion-exchange column should work fine.
Such HPLC columns are available with the POROS matrix, as well as
with silica-based matrix.

>-----Original Message-----
>Hello all,
>
>I'm trying to use reversed-phase HPLC to seperate and quantify an acidic
>(A) peptide and a basic (B) peptide. The A peptide is only soluble > pH
>6ish, but at this pH the B peptide sticks irretrievably to the column
>(Vydac TP218, C18).
>
>I presume it's getting stuck to silanol groups, as it is very basic,
>definitely soluble, and elutes fine at pH2. I also suspect that although
>changing my buffer system (Phosphate/MeCN) may help, it won't completely
>negate the problem, meaning that I'm still not getting quantitative
>results.
>
>>From reading the archives, I suspect a base-deactivated column is what I
>need. I can't find anything specifically labelled such in our catalogues -
>are there other terms for the same thing, or is it fairly specialised?
>Does it cost loads more?
>
>Alternatively, is there a way to solve my silanol problem without buying a
>new column? Or is there a completely different way to seperate these very
>differently charged peptides (PI's about 3 & 10) quantitively?
>
>Thanks!
>
>Jenny Shipway
>Centre for Biomolecular Drug Design and Drug Development
>University of Sussex

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