How do you identify the cause of unstable retention times?
Three readers from Central Europe sent me an e-mail inquiry regarding a problem of retention time variation that was in excess
of the system suitability requirement for the method, 2% relative standard deviation (%RSD). Although the speculated source
of the problem has not been proven by replacing a suspected faulty part, enough data were presented to make this a good example
of how to track down the problem source and to speculate on the probable cause, so we joined forces for this instalment of
Go to Trusted Standard Conditions
After finding that system suitability didn't pass, the conditions were changed to standardized column test conditions. The
goal is to be able to test the liquid chromatography (LC) system independent of the method. A new 150 mm × 4.6 mm C18 column
packed with 5-µm diameter particles was installed in the system. The test conditions for this column were a mobile phase of
60:40 methanol–water at a flow rate of 1 mL/min and a column oven temperature of 25 °C. The test sample comprised a mixture
of analytes that included methyl benzoate. The LC system was a four-solvent system that uses low-pressure mixing. In this
design, the mixture of the four solvents (A, B, C, and D) is controlled by proportioning valves that feed the solvents in
pulses into a mixer (a diagram of this is shown in Figure 1[b] of reference 1). For the present experiments, the solvent inlet
tubing for the A and C channels was placed in a bottle of high performance liquid chromatography (HPLC)-grade water, and the
solvent inlet tubing for B and D was placed in a container of HPLC-grade methanol.
Figure 1: Gradient step test. Gradient ramp from 0% to 100% followed by steps back to 80%, 60%, 40%, 20%, and 0%, run on
channels A/B and C/D. Mobile-phases A and C are water, B and D are 0.1% acetone in water, run at 1 mL/min. Red dashed lines
added to highlight step size differences (horizontal) and gradient nonlinearity (diagonal). See text for details.
To test within-day and between-day variability, runs were made over three days, as summarized in the top section of Table
1; runs were made with the A and B solvents or with C and D solvents. Injections of the test mixture resulted in a peak in
every run at 1.25 min, corresponding to uracil. Additionally, a peak was seen at ~10.1 min for methyl benzoate, but the %RSD
of the retention time for the four runs of Table 1 varied from 0.30% to 2.49%; all combinations of between-day variability
ranged from 0.79% to 1.87%. From a pass–fail standpoint, only the C/D run on day 2 failed the system suitability requirement
of 2%. However, the method historically had performed with <0.5% RSD for these tests.
Table 1: Retention times for methyl benzoate (in min).
What is most worrisome here is the inconsistency of the results. For example, the A/B test on day 1 passed, but the RSD was
approximately threefold larger than on day 2. Similarly, RSD for the C/D test on day 2 was more than eightfold larger than
that on day 3. You might justify rejecting the first run on day 1 (10.36 min) because of insufficient equilibration, although
it doesn't qualify as an outlier using Dixon's Q test. With this point dropped, the RSD is reduced to 0.29%. However, you
can't apply the same logic to the C/D test on day 2, where the high and low values are approximately duplicated.
Before calling for additional help, a mobile phase of 60:40 methanol–water was hand-mixed and the retention checks for n = 6 injections were repeated twice. In both cases, the maximum difference between retention times was 0.04 min. This indicates
that the pump is operating properly and the retention times are stable when a fixed mobile phase composition is used. A problem
with the on-line mixing system is the most likely cause, so at this point, the instrument manufacturer was contacted for some
help, as described in the next section.
Gradient Step Test
The manufacturer's service technician ran the gradient step test shown in Figure 1. This is slightly different from the 10%
steps that are normally recommended in "LC Troubleshooting", but there is nothing wrong with this approach. The column is
removed and replaced with a piece of capillary tubing — approximately 1 m of 0.125-mm i.d. tubing works well. The A-reservoir
is filled with HPLC-grade water and the A and C solvent inlet lines are placed in it. The B-reservoir is filled with HPLC-grade
water containing 0.1% acetone and the B and D lines are placed in it. The ultraviolet (UV) detector is set to 265 nm, where
acetone absorbs strongly. A method is programmed with a gradient of 0–100% of the acetone solvent in 15 min at 1 mL/min, followed
by 5-min steps at 100%, 80%, 60%, 40%, 20%, and 0% using either channels A and B or C and D. This method generates the profile
shown in Figure 1. The gradient is used to test mixing linearity and the steps are used to check proportioning accuracy. The
manufacturer's acceptance criteria are that each step must be within ±2% of the set point; other sources often recommend using
±1% for this test, but in the present case either acceptance limit passes.
The step-test results were calculated by manually measuring the step heights in Figure 1 and normalizing these between the
0% and 100% values; these agree within 0.1% of the manufacturer's test measurements using the data system. You can see that
all the steps pass within the ±2% (or ±1%) criteria. However, a visual examination of the data of Table 2 shows that the results
for the intermediate steps using C/D are consistently lower than the A/B steps by 0.2% to 0.5%; you can see this highlighted
by comparing the step heights to the horizontal dashed lines in Figure 1 (offset from the tracing for clarity). This variation
may or may not have any significance.
Table 2: Step-test results from Figure 1.