HPLC
Applications
note
RestekCorporation • (800)356-1688 • (814)353-1300 •
#59124
HPLC
Ultra C4 HPLC Column Provides High Stability At Low pH
The use of a low pH (< 2.0) mobile phase can cause acid
hydrolysis of the HPLC column stationary phase, resulting
in reduced retention and poor reproducibility. During
hydrolysis the phase is stripped from the silica backbone.
Generally, acid hydrolysis of alkyl bonded phases increases
with a decrease in the chain length of the stationary phase
ligand. Typically, stationary phase ligands as short as C4 will
be much less stable than C18 ligands. To overcome the
problem, some HPLC column manufacturers offer C4 phases
made with relatively high ligand density, which are more
stable than lower density C4 phases. However, these high
ligand density phases still exhibit gradual loss of retention
when exposed to low pH mobile phases.
The research chemists at Restek have designed the new Ultra
C4 phase to further increase stability by using both high
ligand density and a unique bonding chemistry to reduce
acid hydrolysis. The improved stability of the Ultra C4 phase
was confirmed using a controlled study comparing the Ultra
C4 to another leading C4 phase. This leading C4 phase
contains a relatively high ligand density, making it more
stable than other C4 columns (Table 1). The results of the
study are shown in Figure 1.
ExperimentalConditions
Both columns were simultaneously exposed to repeated
acetonitrile/water mobile phase gradients in the presence
of 0.1% (v/v) trifluoroacetic acid (TFA) and a temperature
of 50°C. The pH of 0.1% TFA in water is 1.9. As the mobile
phase pH is decreased towards 2, all bonded phase silicas
become less stable because the siloxane (Si-O-Si) linkage
to the stationary phase is susceptible to acid hydrolysis,
resulting in loss of stationary phase
1
. Elevated column
temperature accelerates acid hydrolysis and the resulting
loss of the stationary phase.
The hydrophobic retention of each column was measured
before exposure to hydrolysis conditions, and periodically
during the acid hydrolysis experiment. The capacity factor
(k') of phenylheptane was used to measure hydrophobic
retention, and was calculated using uracil as the void marker
(t
o
). After equilibration in a mobile phase of H
2
O:ACN
(25:75, v/v) at a flow rate of 1.25mL/min, each column was
injected with 5µL of a mixture of uracil (0.1mg/mL) and
phenylheptane (10mg/mL). UV detection was performed at
254nm.
In addition to the Ultra C4 and competitor C4 columns that
were exposed to hydrolytic conditions, an additional Ultra
C4 column was used as a control. The control column was
periodically analyzed for hydrophobic retention along with
the test columns. While the test columns were exposed to the
TFA gradients, the control column was only exposed to
H
2
O:ACN (25:75). The hydrophobic retention of the control
column varied less than 2% throughout this experiment.
The acid hydrolysis conditions of the experiment (Table 2)
were modeled after those of Kirkland, Glajch, and Farlee.
2
Both test columns were exposed simultaneously to repeated
cycles of the mobile phase gradient listed in Table 2
.
A flow
rate of 2.0mL/min was split between the two test columns
using a tee. The eluent from each column was collected and
monitored. A restrictor was added to the outlet of one of the
columns to make the flow rate as similar as possible. The
total flow through each column at the conclusion of the
experiment was comparable—3784mL for the Ultra C4
Table1
C4 Columns Evaluated
Column
Dimensions ParticleSize Pore Size Carbon%
Competitor C4 50x4.6mm 5µm 100Å
8
UltraC4
50x4.6mm 5µm 100Å
9
Table2
Acid Hydrolysis Conditions
Mobile phaseA:
0.1% TFA in deionized water
Mobile phase B:
0.1% TFA in acetonitrile
Temperature:
50°C
Gradient:
Time (min.)
%A
%B
0
100
0
20
0
100
40
0
100
40.1
100
0
60
100
0
100
80
60
40
ColumnVolumes0.1%TFA
%k', Phenylheptane
Ultra C4
Competitor C4
Figure1
Ultra C4 shows greater retention and stability
due to its unique bonding chemistry
ColumnVolumes of 0.1%TFA vs. Retention
0
1000 2000 3000 4000 5000 6000
7000 8000
