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Derivatization of sugars for GC-MS (Part 4): Automation

17 Jul 2022

The TFA and TMS oximation derivatization reaction discussed in the blogs 1-3 are well suited to automation. Many robotic autosamplers are well equipped to add, remove, and heat vial contents for derivatization reactions. While some robotic autosamplers are also equipped to handle more complex procedures like the evaporation step needed for the alditol acetate derivatization, advanced features are less common.

I used a Gerstel Multi-purpose Sampler (MPS) to demonstrate an automated TFA-oximation procedure. Between the TMS and TFA reaction, EZGC predicted we would see better separation of TFA derivatives, in a shorter amount of time. Below is a sped-up video demonstration of my automated method in action. For the demonstration, I set up three 2mL autosampler vials with 250uL glass vial inserts, containing 1-2 mg of sugar each. Vials were also equipped with a magnetic crimp-caps for transportation.

This procedure was accomplished using a “GerstelPrep” sequence (aka: PrepSequence). Within the PrepSequence I built a series of loops, with one loop for each step of the reaction, allowing multiple samples to undergo each step together (Figure 1). The PrepSequence took 3-4 hours total, including oven programs. So really, with about an hour and 10 minutes of “wait” time and about 30 minutes of oven programs, there was about 1.3-2.3 hours of active time. I was limited to using just one syringe, which slowed down my method quite a bit. This drawback is easily overcome by adding more syringe tools to your autosampler, but in my case, a large amount of time and solvent was spent rinsing the syringe between samples. However, the use of the autosampler allowed me to multi-task while samples were being processed.


Figure 1: The PrepSequence window showing how each step was programmed into a loop for each step of the derivatization reaction.

For the video demonstration above, I show only three samples, but the sample capacity is really limited by the vial capacity of your tools, which can vary with instrumentation. My autosampler is equipped with sample trays that could hold up to 162 2mL vials, but the agitator is only equipped for six 2mL vials, which limited my sample capacity to six. A magnetic piece at the bottom of the syringe tool allowed samples to be moved with the use of magnetic crimp-caps, and an adaptor piece was used in the agitator to accommodate the 2mL vials. I may have been able to save some time by shortening the distance between the trays and agitator as well. Gerstel’s MPS system is versatile though, and there are a variety of tools available to streamline routine processes like derivatization.

Figure 2 shows examples of the separation achieved on the Rtx-225 using the optimized oven program described in Part 2 of this series. Each sugar was derivatized and analyzed individually for the sake of highlighting relevant separations, but we could expect co-elution of rhamnose and arabinose isomers (peaks 4 and 5, Figure 2) if they were in the same sample. Other peaks, however, are well separated in under 7 minutes.


Figure 2: Overlaid chromatograms of sugar TFA oximes.

If you would like to see more automated derivatizations or work with derivatized sugars, let us know in the comments!