Protein precipitation in HPLC buffer

lgbell at lgbell at
Tue Jul 29 08:31:10 EST 1997

Gundars Goldsteins wrote:
> Hi!
> I am trying to purify a protein using C18 RP-HPLC. Unfortunately it
> precipitates when sample mixed with A buffer (0.1% TFA in water).
> Is there another good buffer system (pH>6) for the protein separation on
> C18 ?
> TIA,
> Gundars

One system which can work quite well is Ammonium bicarbonate (0.05M in
water for Sol A, 40% v/v A in MeCN for B) which gives pH 8-9.  This has
the advantage of being sublimable on freeze drying so is easy to
remove.  Alternatively you could try 0.1% v/v triethylamine instead of

In both cases the HPLC system should be flushed with pure water before
swithing to other the counter ion solvent system.  Also avoid leaving
the C18 column in contact with these solvent systems longer than
strictly necessercary, say flush out before leaving O/N, unless you C18
is one of those fancy polymer based ones.

Hope this helps,

Len Bell,
lgbell at

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