John W. Dolan

Too Little

The first case involves a method for the analysis of a drug extracted from a tablet. After running the system-suitability test, a duplicate injection of the reference standard is made. Then five samples are run, each injected in duplicate. The sequence of two standard injections and ten sample injections is continued, until all the samples are analyzed, ending with duplicate injections of the standard. The problem is that occasionally — perhaps 1 sample in 20 — one of the sample injections has an area that is several percent lower than its duplicate, exceeding the allowed variability of the method. When the sample is reinjected in duplicate, invariably both results match the larger of the two previous attempts at analysis. The question is what has caused this and what corrective action can be taken.

Blocked needle: Is a piece of the vial septum partially blocking the sample needle now and then? Some septum types can be more susceptible to "coring" than others. One popular type of septum has a PTFE film on the bottom and a polymer seal on top. If the needle cuts a piece of the seal out, it could be drawn into the needle and cause problems. A change to a presplit septum or simply a PTFE-film septum might be a solution if this were the problem. Sometimes damage to the sample needle will cause the tip to get roughened or sharp so that it cuts the septum instead of tearing or puncturing it; a needle replacement should eliminate this as a problem source.

Particulate matter in the sample could cause problems similar to drawing a piece of septum into the sample needle. Inspect the sample for particulates to see if this could be a problem. Each sample could be filtered or centrifuged before injection to ensure that no suspended particles remain. Other sources of particulate matter are the mobile phase (not likely) or particles released from worn pump seals. If either of these is suspected, correct the problem at its source. Addition of a 0.5-µm porosity in-line filter just upstream from the autosampler also would serve to trap any particles from the pump or mobile phase before they caused problems in the autosampler.

Bubbles: If a bubble were drawn into the needle instead of or along with sample, the sample volume would be low, giving results consistent with those observed. I would make sure that the needle and connecting tubing to the syringe mechanism was thoroughly purged and did not contain bubbles. Most autosamplers have an adjustable syringe speed to accommodate viscous samples. With viscous samples, a fill rate that is too fast can create bubbles in the syringe, which will compromise precision. If excessive sample viscosity is observed and this is suspected, a change in the syringe fill rate might help.