Chromatography Forum

I am still trying to find a way to silylate disodium inosinate (IMP) and disodium guanylate (GMP). I have Handbook of Analytical derivitizations by Donald Knapp and I dug up one of the referenced articles from 1971.
http://pubs.acs.org/doi/pdf/10.1021/ja00733a039
http://www.sciencedirect.com/science/ar ... 9766902727

It says pyridine:HMDS:TMCS 2:2:1 and reflux and pyridine:BSTFA:TMCS 30:30:1 and room temp 30min or 150 degrees C should work. So I put 50 ul of solution in an autosampler tube with microinsert, I evaporated the water in a vac oven and added the reagents, capped it and did a 60 deg C incubation. I've tried it several times with BSTFA, MSTFA, TMCS and with and without pyridine. The dried white pellet does not seem to dissolve in the mixture. I saw it just floating arround.

To check the reagent after I had injected it I opened it up and added a microdrop of water with a syringe and the droplet bounced arround and shrank and the solution got cloudier so I beleive that means the reagent is active.


Any suggestions? All I have thus far is empty chromatograms with solvent peaks and some traces of silylated impurities (glycerol mid chain fatty acids).

what is purpose of your derivatization? How you are going to analyze your compounds? HPLC?

How you are going to analyze your compounds? HPLC?

Silylation would be done for GC.

With the sodium salt, I can see a chance of this working with the TMCS, as NaCl can be formed. But, if the products of the reaction are insoluable - even the NaCL, the reaction may occur only on the surface of the pellet, with a crust of reaction products on the outside. If you can get the compounds into solution before you begin the derivitization, it would help. It crosses my mind that if you could use an ammonium salt ofyour compund rather than a sodium salt, you may get rid of the problem of one insoluable raction product.

I've been looking at several silylation guides and they recommend trimethylsilyl immidazole. I am puzzeled by this as it won't do any of the amine groups. I am thinking of using a tri-sil combination.

I have also been looking at structurally similar sugar phosphates since I can't find much information on the nucleotides. I am thinking DMSO would be my best bet for solubilization as it is the strongest polar nonprotic solvent. I understand from reading that the derivatives are insolbile in DMSO so there needs to be an alkane (hexanes; isooctane? one article recommended cyclohexane) for extraction.

disodium inosinate and disodium guanylate are important flavor enhancers and I'd like to be able to do them on my 5890FID. I don't have an HPLC.

Are you considering HPLC. Here is method for something similar without derivatization or IP reagent:
Two nucleotides with different sugar fragments:
http://www.sielc.com/Separation_of_Guan ... raphy.html

Also methods for other phosphates by HPLC:
http://www.sielc.com/HPLC_Separation_of ... tions.html
http://www.sielc.com/Compound-Fructose- ... phate.html

Unfortunately I don't have an HPLC. I work for a flavors company and GC is king in flavors. Our parent company has an HPLC and we send flavors to them but I'd really like to be able to do it in house. I can do MSG via ethyl chloroformate.

I tried pyridine + TMSI and Pyridine + TMSI + MSTFA + TMCS [that ought to silylate a cheese sandwitch!] and nothing but the reagents on the FID. I am really leaning towards it being a solubility issue as it seems to just sit there on the glass after drying and do nothing. I ran the GC up to 325 it should come out arround 220-260 according to lit.

I read DMSO works quite well with sugar phosphates and silyation though the derivatives are insolbuble and need to be extracted with an alkane or the derivatives itslelf forms another layer.

I'm almost ready to give up. I tried DMSO, TMSI, MSTFA+TMCS again and moderate heat. From the GC/MS the reagents, particularly the MSTFA and TMCS are active it just doesn't seem to want to react well. My guess is the phosphate group is the problem as I can deal with alcohols and amines. The last thing I can try is a very high temperature incubation say 150deg C. Since I was doing the reaction in an autosampler tube I can't go much past 70 deg C without the cap melting even using bottom heat on a hot plate.

I also tried acidifying the phosphate on the disodium inosinate and disodium guanylate by dissolving in 0.1 N HCl, drying and then doing it and it made no difference.

Are there any easy ways to diesterfy a phosphate? I've heard about diazomethane and don't want to deal with that.

Perhaps Capillary Electrophoresis-UV would be a cheap alternative to HPLC.