Derivatization of sugars for GC-MS (Part 2): EZGC libraries and optimizing methods

Researchers have been using 225 phase columns, such as the Rtx-225, to separate EtOx/TMS-, EtOx/TFA-, and alditol acetate derivatives of sugar in a wide range of research applications. To aid in those research initiatives, we have added libraries of derivatized sugars to EZGC. You can now optimize GC parameters and separation based on your Rtx-225 column dimensions without the hassle of trial-and-error runs. Considering the short life-span of these derivatives, being able to use EZGC to remove the trial-and-error phase of method development is appealing.

Accessing these EZGC libraries is a little bit different than accessing the others, but it’s still pretty easy (Figure 1). To find derivative libraries, (1) use EZGC’s “Search by Phase” feature, (2) select the Rtx-225, (3) select your library, (4) select your compounds of interest, and (5) click “Solve”.

Figure 1: Finding the derivatized sugar libraries.

The program will offer settings that optimize predicted peak resolution. You can modify these settings based on your in-house instrumentation, or to help decide what column dimensions to purchase (Figure 2). After selecting your compound(s) of interest, (1) move to the “Conditions” tab, then (2) select the “Custom” radio button to modify your settings. Once you are done with method optimization, you can (3) save the method and predicted chromatogram for future reference.

Figure 2: Adjusting column and GC parameters.

Before running any samples, I was able to look at the benefits and drawbacks of these derivatives based on the column I have available. After calculating the exact length, I knew I was using a 29.09m x 0.25mmID x 0.25µm dF, Rtx-225. For efficiency, I prefer to use a flow rate of 1.4 mL/min helium, and I know my GC is equipped with an MS detector, so I adjusted the flow and outlet pressure, then had EZGC optimize oven conditions for a single oven ramp (Figure 3).

Figure 3: Optimized run conditions for each group of sugar derivatives on a 29.09m x 0.25mmID x 0.25µm dF Rtx-225 column, with constant helium flow at 1.4 mL/min.

The TFA-oximes offer the shortest run-time and one instance of co-elution, making it an appealing derivatization method to run when using an Rtx-225. TFA derivatives, however, are prone to decomposition when left at room temperature for too long. The TMS-oximes required the longest run-time, and there were two instances of co-elution that I was unable to overcome. However, the derivatization procedure and subsequent samples were more forgiving in practice than the TFA-derivatization. The alditol acetates require a higher temperature to elute (220^oC), and probably could have been run isothermally, but to minimize solvent effects I began at 40^oC then quickly ramped up to 220^oC, achieving good resolution. However, the alditol acetate derivatization is limited in that different sugars may produce the same derivative.

Since sugars can be investigated in a wide variety of applications, it is important to consider the benefits and drawbacks of the derivatization and separation depending on your specific application. Running these EZGC comparisons prior to experiments can save valuable time (and money) in the lab, and help identify what limitations you will need to overcome on the way.