Derivatization of sugars for GC-MS (Part 3): Maintenance

If you have tried out any of the derivatization procedures that I outlined back in Part 1, you also saw the beautiful colors these derivatization reactions produce (Figure 1). While the colors may initially be a nice change of pace from the clear solvents and solutions you’re used to, color can be a ‘red flag’ that you will need to stay ahead of instrument maintenance. Judging by the deep red/brown of that fructose TFA-oxime (Figure 1), I just know I will need to change the liner soon.

Figure 1: Vial inserts containing TMS oximes (in ethyl acetate), TFA oximes (in ethyl acetate), and alditol acetates (in chloroform) of sugars, analyzed by GC-MS.

Starting with the syringe, make sure you are using solvents that can adequately clean the syringe and keep it from getting stuck. I like to rinse with my sample solvent (TFA/TMS-oxime = ethyl acetate; alditol acetate = chloroform) immediately before and after injection. Ethyl acetate and chloroform are polar, so it may be a good idea to wash with something non-polar as well, like hexane or isooctane. Washing with a polar and non-polar solvent after injection will help keep the syringe clean from a wider range of potential-contaminants in the sample. It is much easier to wash with two solvents after every injection, than to waste time by only using the second solvent when the syringe gets stuck.

Next up is the liner. The damage wasn’t as bad as I expected from that fructose TFA-oxime, but a liner change was certainly warranted. I cannot emphasize enough how important a liner with wool is when working with sugar derivatives. Not only will the wool help volatilize the sample, but it will also help protect your column from non-volatile contaminants. After <50 1 uL injections (from admittedly concentrated samples), a darkened ring was visible at the top of the wool of my inlet liner, and a concentrated dark spot was visible on the wool directly below the syringe needle (Figure 2). If you are like me, and don’t like having to pull out your liner to check if it needs to be changed, there are also some indirect signs the liner may need to be replaced. Are peaks heights decreasing? Is recovery of your internal standard starting to drop? Are peaks getting wider? If yes, then it’s time to check the liner.

Figure 2: Side (left) and top-down (right) views of a liner after <50 injections of concentrated derivatized sugar solutions.

Finally, the column. Excess derivatizing agents will strip away phase from the head of the column, which creates excess bleed and impedes analyte retention. I like to trim 10-20 cm (4-8 inches) from the column head when I see retention times start to shift. After trimming you can use the EZGC method translator to adjust your methods and match your previously used retention times, that way you don’t have to worry about updating your analysis software.

The syringe, liner, and column head should be your first stop for maintenance when analyzing derivatized sugars, but if your chromatograms still don’t look right after maintenance, check out some of our other blogs to learn more about instrument maintenance and when to change and clean other parts of your GC system.