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What problems in chromatogram may reveal, and what to do
Retention times in capillary gas chromatography (GC) can change from run to run or even within the run, and the underlying causes generally rest with carrier flow stability, poor temperature control, or the quality of the stationary phase within the column. Some minor retention-time variability from batch to batch are perhaps most common and can be mitigated through the use of retention factor (k) in calculations or for qualitative peak identification. Slight differences in carrier gas flow rate may arise from incorrect instrument parameters that would typically include the nature of the carrier gas or the column dimensions — which will "fool" the system into applying an incorrect pressure to generate what it thinks is the correct carrier flow or linear velocity. Remember, GC electronic pressure controls calculate flow from the applied pressure, the nature of the carrier gas, and the column dimensions. One should also take care to select the correct operating mode in gradient temperature-programmed GC, because retention-time differences can be pronounced when operating in either constant-pressure or constant-flow modes. Differences in split flow or column oven temperature profile may also give rise to retention changes between batches of samples. Changes in k values may be due to deterioration of the stationary-phase quality (loss of bonded phase, especially at the column inlet) via thermal or chemical decomposition, which will cause the analyte retention time to vary relative to the elution time of an unretained component. In this case one should consider removing up to 5% of the total column length from the inlet end to eliminate badly degraded stationary phases. The act of trimming the column will in itself require a change in the column length setting in the instrument (most instruments will calculate column length based on the elution time of an unretained peak and the nominal internal diameter); although the absolute retention time will change, the retention factor value should remain constant.
Problems of retention-time variability between subsequent injections are perhaps more serious. One of the more insidious causes relates to the "equilibration" time between gradient temperature program runs. Although the GC oven may be at the correct temperature for the next injection, the column has a small but significant thermal mass and enough time should be allowed for the column and carrier to reach and stabilize at the initial gradient temperature before the next injection — otherwise small retention time variability will occur from run to run. Usually, 30 s to 1 min is sufficient for equilibration after the oven temperature has reached its set point. Injection-to-injection retention-time instability can also be caused by faults with the GC oven temperature controller or the electronic pressure control module, and these problems will require attention from your service provider. Small retention time shifts may also be caused by irreproducible split flows (suspect a blockage in the split vent line) or from small leaks created during the injection phase caused by a split or cored septum — a further clue here may be a shift in baseline position before and after the injection phase.
The high efficiency of GC separations is crucial for successful analysis, especially with complex samples or when fast analysis is required. Factors causing reduced efficiency include compromised stationary phase, leaks, and dead volumes within the system as well as poor selection of analysis conditions. Wall-coated capillary GC columns deteriorate with time because of contamination and phase bleed and, as such, a gradual reduction in efficiency is expected. This process may be accelerated when operating at high temperatures for prolonged periods, when using aggressive solvents (acetonitrile or tetrahydrofuran), or when dirty samples are injected repeatedly. Trimming the column inlet is recommended to restore peak, shape, and efficiency as discussed above. Dead (unswept) volumes in the system may be created through poor installation of the column into the inlet and more especially the detector (that is, because of incorrect column insertion depths or distances). Follow vendors' column installation instructions carefully. If the split (purge) is not initiated at an optimum time post injection during splitless injection, peaks within the chromatogram will appear broad. The use of constant-flow mode during temperature programming is recommended to avoid loss of efficiency in later eluted peaks because the linear velocity of the carrier slows appreciably with the increase in oven temperature.
Peak tailing in capillary GC is most commonly associated with secondary retention of the analyte by active sites (silanol species predominantly) within the GC system. If analytes are polar, ensure the system is deactivated by purchasing presilylated liners (with deactivated packing if required) and use the most modern, inert, stationary phases. Regularly clean and deactivate the GC inlet and ensure that the column inlet is regularly trimmed to remove active sites. Peak splitting problems can arise from improperly cut GC column ends, improperly installed columns (incorrect insertion distances into the inlet or detector), or a blocked detector jet. Ensure that column cuts are at 90° to the column wall and "clean" with no jagged edges — use a magnifying glass or microscope to assess the quality of the column cut. Peak shoulders can occur when the stationary-phase polarity does not match the polarity of either the analyte or the sample solvent, especially during splitless injection.
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