From: Jim Sparrow <jsparrow@athero.med.bcm.tmc.edu>
Subject: RE: RP-HPLC at basic pH
Date: Fri, 17 Jan 97 13:34:00 C
To: Recipients of ABRF List <abrf@aecom.yu.edu>
Haydn,
We routinely run isopropanol gradients for analytical and prep. reversed
phase on C4 and C18 Vydac columns for synthetic hydrophobic peptides of up
to 70 residues. We have also used them sucessfully for tryptic digest. The
fractions can be directly lyophilized or evaporated in the speed-vac for
isolation. The gradients are usually as follows:
Time %H2O %IPA % 1.0%TFA
0.0 90 0 10
5.0 90 0 10
65 10 80 10
70 0 90 10
75 90 0 10
90 90 0 10
Flows rates are usually 1.0 ml/min for anal. and 20 ml/min. for 2.2X25 cm
prep col. The times can be shortened or extended depending on the
separation as well as the %IPA to adjust the slope. We have used them as
short as 30 min., e.i., 60 min. total time. The back pressure will be
higher than with AcCN due to the higher viscosity of IPA. Our anal col.
usually runs from 1500 psi up to 3500 psi for 10 micron as the IPA conc.
increases; of course, 5 micron will be higher. (You can compensate some for
this by using 1:1 IPA,AcCN if it is a problem.)
You may also find that ammonium or triethylammonium phosphate at pH 6.7 will
work for your hydrophobic peptides but there will be problems getting rid of
them for sequencing. However, if you have to use it here is a gradient
example that works for us repeatedly for both anal. and prep. work. In
prep. purifications the sample can be desalted on BioGel P-2 in 0.1 M
ammonium bicarbonate or 5% acetic acid depending on peptide isoelectric
point and then lyophilized. It is important when using ammonium phosphate
to switch to H2O at 50% IPA otherwise it will start to precipitate in your
pump.
Time %H2O %IPA % 0.1M NH4PO4, pH 6.7 or 3.0
0 90 0 10
5 90 0 10
65 40 50 10
70 50 50 0
75 10 90 0
80 10 90 0
85 70 30 0
90 60 30 10
95 90 0 10
110 90 0 10
Again, the gradient shape can be changed as long as the switch over to H2O
is kept as is. When working at pH 6.7 and higher, it is advisable to
include a silica saturator col. before the injector to prevent dissolution
of the anal. or prep. col.
Ammonium bicarbonate (0.05 M, pH 7.8) can also be used. This buffer can be
lyophilized but can cause problems with bubbles in check valves. Of course,
attempts to degas will lead to pH changes since CO2 is removed from the
buffer by He or vac.
Another trick for hydrophobic peptides is that your peptides can be injected
in 6M guanidine, 0.1%TFA or phosphate or as 1:1 mixture of the quanidine
sample with IPA. We have pumped samples in 200 mL of these solutions onto
2.2X25 cm Vydac columns. You may lose resolution of early eluting peaks if
you have to resort to the later procedure. You will also find that peak
sharpness is lower in IPA than in AcCN and therefore resolution can also
become a problem for some peptides.
Jim Sparrow
-------------------------------------------------------
Dr. James T. Sparrow
Dept. of Medicine
Baylor College of Medicine
One Baylor Plaza
Houston, TX 77030
Phone: 713 798-4156
FAX: 713 798-4121
email: jsparrow@bcm.tmc.edu
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