21
Time (min)
1
2
3
4
5
6
7
8
9
10 11
0
Run 1
Run 2
1 minute post-gradient
equilibration
(equal to approximately
0.5 column volumes)
On the other hand, reproducible chromatography is obtained when a suitable post-gradient equilibration is carried
out in between runs. This is illustrated in the figure below, where a post-gradient equilibration time of 23.5 minutes
(corresponding to 20 column volumes) leads to perfectly overlaid chromatograms.
Time (min)
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
0.0
Run 1
Run 2
23.5 minutes post-gradient
equilibration (equal to approximately
20 column volumes)
Do I Need a Buffer?
As a general guideline buffers are added to the mobile phase to reduce peak tailing and/or retention of charged analytes.
Early research indicated that the absence of buffer salts resulted in excessively long retention times and very broad peaks on
amide, cyclodextrin, cyano and amino-based columns [25].
Due to their good solubility in organic solvents, recommended buffers for HILIC are ammonium salts of acetic and formic
acids. These buffers also have the advantage of being volatile for use with mass spectrometry and charged aerosol
detection. Other salts with relatively high solubility in high organic levels are bicarbonate salts, triethylamine phosphate
and sodium perchlorate, but the last two are not MS compatible.
The following chromatogram illustrates how irreproducible chromatography, with drifting retention times is obtained when
a post-gradient equilibration of 1 minute is carried out in between runs.
Column:
Accucore 150-Amide-HILIC
, 2.6 μm
100 × 2.1 mm
Mobile Phase A:
acetonitrile
Mobile Phase B:
100 mM ammonium formate (pH 6.6)
delivered in a 5−40% B gradient over
15 minutes at 0.1 mL/min, followed by
1 minute equilibration time
Column:
Syncronis HILIC
, 5 μm
100 × 2.1 mm
Mobile Phase A:
acetonitrile
Mobile Phase B:
100 mM ammonium formate (pH 6.6)
delivered in a 5−0% B gradient over
15 minutes at 0.2 mL/min, followed by
23.5 minutes equilibration time
