Troubleshooting GC Selectivity, Resolution, and Baseline Issues

August 1, 2013
LCGC Europe

Volume 26, Issue 8

Page Number: 478

Understanding the chemistry behind GC separations can lead to faster problem identifi cation and improved troubleshooting.

An excerpt from LCGC's e-learning tutorial on GC troubleshooting at CHROMacademy.com

Understanding the chemistry behind gas chromatography (GC) separations can lead to faster problem identification and improved troubleshooting.

Fortunately, sudden selectivity changes in GC are fewer than in other forms of chromatography — it's not as if we can make up the eluent with the incorrect amount of organic or at the wrong pH after all! However, changes in selectivity — the distance between the apices of two peaks, measured as a ratio of their retention factors — do occur and the causes can usually be traced to one of a few usual suspects. The selectivity of a separation is predominantly affected by the strength of the interaction of each analyte with the two phases between which it is portioning, and in GC this interaction is affected most by the stationary-phase chemistry and the temperature of the mobile phase. If the relative peak spacing suddenly changes, one should check that the correct stationary-phase chemistry is being used and that the temperature (or temperature programme) is as it should be. If the temperature programme has been entered correctly into the instrument, one might check the oven temperature over the course of the analysis using a resistive thermocouple and digital thermometer. If the oven temperature is not following the programme to within a few degrees, one might suspect a problem with the heater and a visit by your service provider may be required. If all is well, ensure that the column equilibration time — the wait time after the oven has reached its initial temperature set point before sample injection — is sufficient to establish the correct (homogeneous) temperature throughout the length and across the diameter of the whole column. Even though capillary GC columns have relatively low thermal mass, we need to ensure that the column and carrier gas passing through it are at the correct initial temperature to begin the analysis, and this can sometimes take a surprisingly long time (>1 min for columns with thicker films). I have often seen variations in column equilibration time give rise to insidious issues during method transfer exercises and, although this parameter may seem minor, bear in mind the oft-quoted fact that a difference in temperature of 23 °C can half the retention time of an analyte. This is particularly important during splitless injection where the initial column temperature plays a vital role in determining peak shape and may influence relative analyte retention. One final check into selectivity changes would involve trimming the first 5% of the column length to remove any stationary phase whose chemistry has been modified by the adsorption of sample components or whose phase has been stripped to reveal the underlying silica, both of which may have subtle influences over the relative band spacing of analytes; this initial column section has a large influence over band spacing in GC.

As we know, resolution in chromatographic separations is influenced by efficiency (N), selectivity (α), and retention factor (k) as defined by the fundamental resolution equation. Having taken care of any issues with selectivity, we must ensure that the efficiency of the system is as it should be, because reduction in efficiency is perhaps the most common cause of loss of resolution in GC. The efficiency of the GC column will decrease gradually over time, primarily because of the loss of bonded phase through chemical and thermal degradation; this should be noted, with a minimum acceptable efficiency established for system suitability testing, especially where resolution between critical peak pairs is reliant on achieving a certain peak width or plate count. Trimming the first 1–5% of the column length can help to temporarily restore column efficiency, but note that retention times may decrease as a consequence. The other main cause of efficiency loss is poor installation of the column into the inlet and detector. Ensure that you follow your manufacturer's instructions carefully to avoid issues with sample introduction into the column or dead volumes at the detector connection.

Baseline position in GC will tend to shift with temperature but also with flow rate for certain detectors that are known to be "mass/flow" sensitive — that is, response depends not only on the amount of substance entering, but also the rate at which it enters. We typically see a shift in baseline position when thermally equilibrating the column, but a rising baseline may also be seen when using a temperature programme. This may be normal at high temperatures — for example, because of increased phase bleed — but rising baselines at lower temperatures can be mitigated by using a constant-flow mode of operation as opposed to constant pressure where the carrier flow will reduce with increased oven temperature.

Noisy baselines in GC can arise from a poorly equilibrated detector system (especially with nitrogen–phosphorus detection and electron-capture detection systems) as well as from column and septum bleed. Ensure that your column is fully thermally equilibrated by purging with carrier gas at room temperature for 10 min followed by a ramp to 10 °C above your upper method temperature, or to the isothermal temperature maximum of the column, whichever is lower. Purging with carrier gas at room temperature for a short while will initially reduce the amount of column bleed substantially that occurs during thermal equilibration. Ensure that you use low-bleed septa and that your septum purge flow is working to reduce the amount of bleed product emanating from the septum. Replace septa regularly before they core or become worn and release shards of material into the inlet liner.

The issues above are among those considered within the CHROMacademy tutorial on GC troubleshooting — follow the link shown in the lower right corner of this page to discover other causes and remedies for issues relating to selectivity, resolution, and baseline issues.