Why do my silylations always fail?

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

15 posts Page 1 of 1
Hi all

I have tried silylation many times and have never once had a successful reaction. I've tried it on inosinate and guanylate, acrylamide, now arginine. In each case when I inject I can see a huge intact silylation reagent peak (confirmed with the MSD) and some much smaller hydrolysis breakdown peaks some traces of silylated contaminants like glycerol or palmitic acid and a whole lot of nothing.

I've uses MSTFA, BSTFA, +TMCS and TMSI.
I've trield acetonitrile, pyridine and DMSO as solvents.
I typically heat to 70 deg C for 30 minutes to overnight.
I do the reaction in an autosampler tube.

same results. How can a confirmed active reagent and anayte go into the same vial and not react?
I've never tried assaying for any of those.

But are you taking the GC temperature up high enough, and long enough, to elute these? What kind of column? These derivatized molecules will not be small.
I use a db-5 column and take it from 70 to 310 deg C over an hour (280 inlet). under these condition arginine as ornithine-triTMS is suposed to elute arround 23 minutes in.

It's just weird that I keep trying to use TMS derivatization which is reported to work and the MSD is confirming that there is active intact reagent even after the reaction period and I get almost nothing.

I am aware silyation is water sensitive but if significant ammounts of water were present I'd assume I'd see much larger hydrolysis breakdown product peaks rather than active reagent peaks.

Acrylamide on the other hand is quite a small derivative and still nothing.
can you describe which reagents do you use? your exact protocol?
Yesterday I tried Arginine. I tried putting a few grains of pure arginine in an autosampler vial, adding 100 ul of acetonitrile and 100 ul of MSTFA with 1%TMCS (with a syringe through the septa cap bottle it came in), capping it, and putting it in an oven with a metal rack at 70 deg C for 2 hours.

I also took 100ul of an aqueous solution of arginine in a 2ml autosampler vial, threw it in a vac oven for 30 minutes to evaporate the water, then put 100 ul of CH3CN and either MSTFA with TMCS or TMSI, capped it, and heated it to 70 deg for 2 hours.

I also tried DMSO which is the most polar nonprotic solvent available.

I then injected in into a GC/MS 10:1 split, deactivated liner with wool, 280 inlet 70deg c +5 deg C to 310 hold 5 minutes.

I saw huge intact MSTFA and TMSI peaks. a bunch of small garbage and silylated impurities like palmitic acid and not even a trace of my product which I have the EI spectra for and I even programmed a SIM method to look for.

I did the same thing a few weeks ago for acrylamide with the same result.

Before that Inosinate and Guanylate.

It just drives me crazy. I have active reagent and analyte in a vial, they are supposed to react, and they don't react. Are they that water sensitive like a grignard reagent?
I thought I'd try again today. I put some acetonitrile in a 20ml vial with NaOH to strip any residual water.
Those derivatization chemicals will simply react with excess water, so not typically an issue if you have excess reagent. Our samples here typically can contain up to 80% water (consumer products, duh !) and we don't "dry" before derivatizing.

We use high temperature capillaries for big stuff here, metal capillaries and thin film, especially designed to do big molecules, like at temperatures up to 400 C.

And make sure that your inlet temperature is high enough too.
All I have here are db-1's and 5's that can go up to 325 deg C plus the MS transferline I keep at 280. According to the lit that should be sufficient for the compounds I am trying.

If not water I'm not sure what else can be stopping the reaction.
I got it working. The only thing I did different is I took some of my acetonitrile and put it in a 20ml screw/septa bottle with a bunch of NaOH pellets. I have mostly the ornithine with 1 TMS on each amino group and 1 on the carboxylate but also some with 2 TMS groups on the N5 side chain.

I tried the AA18 std and saw everything except histidine and the same arginine showed up as the ornithine TMS. Tyrosine was a mix of TMS on the phenol and no phenol.

It is kind of a pain in the neck I think I am going to try my idea on the other treat of treating the arginine wth aqeous hydrazine to convert it to ornithine and then do derivatization with ethyl chloroformate. The only issue is getting rid of the hydrazine hydrate.
Why do you add acetonitrile? Normalyy I would dry whatever in my vial, and then add to the dry residue BSTFA + Pyridine to derivatize. I think the reaction will ahve higher yields
The procedure for TMS amino acids recommends adding acetonitrile.

I actually did Inosinate and guanylate as well today (using pyridine as recommended by lit). Inosinate worked fairly well on the db-5 column but guanylate was a big blob almost like a ghost peak. That's probably the extra amino group.

So it all came down to water.
tms TRIFLATE is I believe the most reactive commercial silating agents. 100% conversion in most cases with TEA without heating. running the reactions in the prescence of activated molecular sieves to savange water is a helpful tip to increase yield also.

look at the book Greens protecting group for a good description of all the conditions that can remove a silyl group. There is a chart in the back the tells you what the TMS groups are stable to and what they are not. You may be removing them in your work up or before you get them into the instrument.
Thanks I don't really do any workup. I just mix pyridine or acetonitrile for amino acids, microwave or heat it and inject. As long as it is not exposed to protic solvents ie water alcohols it should be fine. I mainly use silylations for qualitative analysis. It esterfies a large variety of normally nonvolatiles and many of them are in the NIST MS libraries. Then once I see what it there I use quantitative methods. I am not a fan of silylation for quantitation. The reagents are fairly expensive and prone to inconsistant yeilds on many compounds as well as multiple peaks for primary amines such as glycine where there can be one or two TMS groups added to the amino group.
I have several publications using trimethylsilyl derivatizations; these demonstrated excellent reproducibility, precision, and accuracy.
It can vary. For instance Glycine and many other compounds with primary amines have a variance where there can be one or two trimethyl silyl groups put onto the amine. For acids and alcohols though that is not an issue so the results may be much more consistent.
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