Questions From Afar - - Chromatography Online
Questions From Afar


LCGC North America
Volume 30, Issue 8, pp. 634-639

Retention-time drift, temperature control, and broad peaks from a new column are the topics of this month's discussion.

As many of you know, a major part of my job is teaching liquid chromatography (LC) training courses around the world. Besides getting to see some pretty fascinating places, I get to meet lots of people and have a chance to help solve some LC problems that are troubling the attendees. One thing I have found is that chromatographers everywhere tend to have the same struggles with LC problems. Sometimes I feel a bit like the detective on Garrison Keillor's A Prairie Home Companion, whose radio dramas always end with the tag line, ". . . one man is trying to find answers to life's persistent questions . . . Guy Noir, Private Eye." In this month's installment I'd like to share a few of the questions that came up in recent classes.

Retention-Time Drift

One attendee mentioned a problem related to a small, but noticeable increase in retention time that continued to grow over several weeks. In an earlier installment (1) we looked at tricks to help determine the cause of retention drift. One of the ways to differentiate between hardware (flow rate) problems and chemical (column, mobile phase, or temperature) problems was to see if the disturbance at the column dead time changed in the same proportion as the retention change. If it did, a hardware problem was indicated; otherwise it was a chemical problem. However, the current problem was observed on an LC system with a mass spectrometry (MS) detector. In contrast to ultraviolet (UV) detectors, where a solvent-front disturbance is almost always seen, unless the MS system is set to specifically look for ions at the dead time, the baseline is flat in this region. As a result, the dead-time diagnostic didn't help.

The first step taken was the easy one — replace the column. When this approach didn't help, a new batch of mobile phase was made, but it didn't solve the problem either. Next came cleaning the pump check valves and finally replacement of the pump seals. None of these changes arrested the drift.

Then one day, the user was inspecting the autosampler and noticed a reflection from a drop of liquid on the injection valve. This led him to service the valve. He discovered that there was a scratch between two of the passages in the valve rotor, creating a situation called cross-port leakage. A tiny amount of mobile phase was leaking out the waste line from the injector, effectively lowering the flow rate to the column and thus increasing the retention times. As the leak became worse over time, the loss of flow increased and retention times drifted to even longer times. Replacement of the rotor seal corrected the problem. Because the leak was small, likely only in the tens of microliters per minute, and a piece of opaque tubing was used to route the injector waste to the waste container, the leaking solvent was not noticed. Replacement of the valve rotor corrected the problem.


Figure 1: (a) Injection valve rotor showing polymeric seal (shaded) and grooves to pass liquid (clear). (b) Typical plumbing connections: 1 and 4, injection loop connections; 2, to column; 3, to pump; 5, to waste; 6, sample inlet. (c) Lines showing scratch between ports 4 and 5 (arrow). Adapted from Figure 17.3 of reference 2.
The problem can be more clearly understood with the help of the valve rotor sketches in Figure 1. The rotor usually comprises a polymer disk typically 15–30 mm across and 5-mm thick, with three semicircular grooves cut into the surface as shown in Figure 1a. In operation, a mating stationary piece (the stator) contains connections to the sample loop (ports 1 and 4), the column (port 2), pump (port 3), waste (port 5), and sample inlet (port 6) (Figure 1b). The configuration of Figure 1b is in the inject position, where the loop is in the flow path. In the load configuration (not shown), the rotor would be rotated 60 to the right and the loop would be connected to the sample inlet and waste, while the pump would feed directly to the column. The valve rotor has a very long lifetime under normal conditions — I've had numbers of 100,000 to 500,000 cycles quoted to me by instrument manufacturers — so routine replacement of the injector rotor is not something that most of us ever encounter. However, if a tiny piece of hard material gets caught in the injector, it can scratch the rotor, as illustrated in Figure 1c. Now some of the mobile phase can leak from the injection loop to waste instead of 100% of the flow going to the column. When such problems occur, they are most likely caused by a hard, insoluble particle in the sample. Centrifugation or filtration of the samples before injection should avoid this problem. I have also seen a case where a hand-cut piece of tubing was not rinsed properly, resulting in a tiny sliver of stainless steel working its way into the valve and scratching the rotor. Whenever tubing is hand-cut, whether it is stainless steel or plastic tubing, be sure to rinse it before use. The easiest way to do this is to connect the upstream end of the tubing and turn the pump on for a minute or two to flush the tubing to waste before connecting the downstream end.


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