Variations in the oven temperature and carrier-gas pressure influence peak retention times significantly enough so that differences from column to column and instrument to instrument make method validation a necessity for ensuring consistent results. Instrument-to-instrument variability can be brought under control by implementing a few simple calibration and set-up procedures, and validation is made more reliable as a result. The first part of this series showed that analysts should understand the effects of performing the same analysis on different gas chromatography (GC) systems on the variability of their results, in particular on retention times (1). The dependencies of peak retention times on oven temperature and inlet pressure can be large enough to cause significant deviations of 15 s or more between instruments when the individual oven temperatures differ by only 1 °C or the pressures by 1 psi. The scope of these variations depends in turn upon the chromatographic conditions, the column, and the analytes under examination. Although not a substitute for validation and suitability testing, instrument calibration can help to reduce the normal variability that will be encountered when working with multiple instruments and multiple columns.
Column Effects There are three main column variables that affect retention times: dimensional variations such as inner diameter and length; stationary phase variations both in the chemistry and the film thickness; and aging effects due to gradual contamination with sample residue as well as phase loss due to overheating. This month's column addresses some of the issues related to dimensional variations.
I vividly recall spending many long nights in the graduate school lab drawing out borosilicate glass columns on a cantankerous machine that would have made cartoonist Rube Goldberg proud (see http://www.rube-goldberg.com/). If I was lucky enough to obtain a single 10-m long piece of coiled tubing, I then was faced with the tasks of coating the column with a stationary phase that I had synthesized from scratch and installing it intact into the gas chromatograph's oven. I made no pretense of duplicating any of these handmade columns and I don't know what the tolerance levels were on their inner diameters or film thicknesses. Fortunately, this had no bearing on my work. However, it did leave me with an appreciation of the technology that goes into producing capillary GC columns.Two studies published in the 1970s recorded the state of the art of commercial glass capillary GC column production at the time (2,3). A statistical evaluation of the data published in the two papers (4) reveals retention-factor standard deviations of 5.9% for 16 methylsilicone columns, 11.4% for 7 phenylmethylsilicone columns, and 30.4% for 9 Carbowax 20M columns (2). In the second report, the authors measured the relative retention of several peak pairs and found, not surprisingly, much smaller standard deviations: 0.28-0.37% for methylsilicone and 0.32% for Carbowax 20M. Nonpolar columns prepared with twice the stationary phase film thickness had even smaller column-to-column relative retention variations (3). The variability of the column inner diameter does not affect retention factors under the isothermal conditions used, and its range was given as dc = 270 ± 20 μm (2).
Today, analysts rely upon column manufacturers to produce a consistent product from fused-silica tubing. Advances in tubing production and chemical treatment, stationary phase synthesis, column coating, and conditioning have greatly reduced the variability in column dimensions and retention as well as yielding much lower bleed levels, higher stability, and longer life. It would be very interesting to see similar data on populations of modern capillary columns that compares them with the older studies.
Gas chromatographers who want to ensure the best consistency from column to column should choose one manufacturer as their column source for each specific analytical method. There are no technical reasons to select or eliminate any particular manufacturer, but columns from the same company will be much more self-consistent than would be the case for columns from different manufacturers with the same nominal dimensions and stationary phase types. Of course, some column companies' proprietary stationary phases and column chemistry might be better suited for certain applications. Careful evaluation of multiple examples of a specific column is always a good idea before committing to any particular choice.