Summary: HPLC Tips:
Silica dissolves at high pH and it is not recommended to
use solvents that exceed pH 7 (see SunFire columns).
C4 is used for proteins.
C8 columns are better for capturing smaller hydrophilic
peptides. C8 can be used routinely for all peptide work
and is better choice than C18 for all peptides.
C18 is used for peptides or small molecules. More
hydrophilic peptides are not captured.
GRADIENT 60/60- from 2-5% B to 60%B in 60 min
The column must be re-equilibrated to the initial high
aqueous solvent composition before another analysis can
be performed. It should be at the end of gradient. 0 min-
%B=2, 60 min-%B=60, 60.1 min-%B=2, 80min-%B=2.
Equilibration ~ 20 min.
Use HPLC grade water with 0.1%v/v acid (to improve
peak shape). In reverse phase organic phase is
acetonitrile or methanol with 0.1% v/v acid. Acids:
trifluoroacetic acid, formic acid, or acetic acid. A 0.1%
v/v solution is made by adding 1 ml of acid per liter of