The topic of this month's instalment of "LC Troubleshooting" was prompted by a manuscript I recently reviewed and a question
I received from a reader of this column. Both inputs related to the variability of retention times observed in liquid chromatography
(LC) methods. Variable retention is a topic that has been touched on many times over the history of this column, sometimes
just in passing and other times in depth. Yet, it seems to be a problem that keeps recurring, so I think it is worth considering
again.
Sensitivity to Mobile Phase Composition
One case related to extreme sensitivity of the retention time to mobile phase composition. The method comprised a reversed-phase
separation of two isomers with molecular weights of approximately 400 Da, no ionizable functional groups and a mobile phase
of acetonitrile and water. Normally, the two isomers were separated by approximately 1 min, giving a resolution value of >2.
Under isocratic conditions, a 1% change in acetonitrile caused a 2 min shift in retention times. When a shallow gradient was
used (5% change in B over 10 min), a 1% shift in the starting and ending percentage of acetonitrile resulted in a 1 min shift
in retention times. It is easily seen that with either method, a small error in mobile phase composition could change retention
enough to cause peak misidentification if peaks were only identified by retention time.
Figure 1
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A similar problem: I encountered a similar problem in my laboratory more than 10 years ago and shared it in this column.1 Because the symptoms were almost the same as those reported here, it is worth reviewing the problem and its solution. Our
separation was of a 1000 Da peptide on a 250 mm × 4.6 mm, 5 μm reversed-phase column operated at 1.5 mL/min and 35 °C. A gradient
of 19–24% acetonitrile–0.1% trifluoroacetic acid in water was run over 30 min. Even with freshly serviced pumps (new check
valves and pump seals), retention varied by approximately 1 min over three consecutive runs, as illustrated in Figure 1(a).
The source of the problem was related to the ability of the LC system to blend solvents on-line. Note that the gradient programme
for this method called for a small gradient range, 5%, over a long time, 30 min, or ≈0.17%/min. Most LC pump manufacturers
specify a proportioning accuracy of ±0.5–1%. It is easy to see that we were asking for a gradient that required better control
of the mobile phase mixture than the system specification.
The solution — premixing: The solution to this problem was quite simple. We premixed the mobile phases so that the A-reservoir contained 10% acetonitrile
and 90% 0.1% trifluoroacetic acid in water; the B-reservoir was filled with 30% acetonitrile and 70% of the trifluoroacetic
acid mixture. The system controller was reprogrammed to deliver a gradient of 40–65% B over 30 min for an effective gradient
of 18–23% acetonitrile–trifluoroacetic acid over 30 min (compared to the original 19–24% gradient). Under these conditions,
the retention range for three consecutive injections was <0.1 min [see Figure 1(b)]. From the standpoint of the LC system,
the gradient was 25% over 30 min, or ≈0.8%/min, a five-fold reduction in the programmed gradient slope. Clearly, the system
was able to produce this level of performance quite nicely.
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