How to derivatize in water ?

[lola129]

Hello everyone,

I need some help for my work...

I have a very polar compound to analyse (spinochrome and echinochrome; pigments from sea urchins).They are only soluble in water, alcohol, DMF and DMSO (they don't in pyridine, acetonitrile, ethyl acetate, acetone, ether, dichloromethane,...) but I would like to analyse them in gas chromatography and for that, I must derivatize them.

I have tried BSTFA-TMCS 1% in DMF and DMSO but it seem these solvent denature my molecules... I can't use water or alcohol because BSTFA react with -OH group...

Do you know how derivatize a polar compound with water as solvent ?

Thanks a lot !

[Peter Apps]

Welcome to the forum

A water soluble, hydrophillic, coloured molecule is an excellent candidate for HPLC with UV-visible detection, but rather a poor candidate for GC. So why do you want to use GC ?

Peter

[Steve Reimer]

Or even HPLC with fluorescence detection.

[X]

Evaporate your standard or extract to dryness, and derivatize with BSTFA/1% TMCS ONLY, do not add any solvents, as once derivatized, the solubility of the derivatives in solvent or just derivatizing agent will increase.

Hello everyone,

I need some help for my work...

I have a very polar compound to analyse (spinochrome and echinochrome; pigments from sea urchins).They are only soluble in water, alcohol, DMF and DMSO (they don't in pyridine, acetonitrile, ethyl acetate, acetone, ether, dichloromethane,...) but I would like to analyse them in gas chromatography and for that, I must derivatize them.

I have tried BSTFA-TMCS 1% in DMF and DMSO but it seem these solvent denature my molecules... I can't use water or alcohol because BSTFA react with -OH group...

Do you know how derivatize a polar compound with water as solvent ?

Thanks a lot !

[lola129]

Thank you for your responses, it's very kind to you to help me...

In fact, I have already try to only use BSTFA-TMCS but my molecules aren't soluble in it, too...

And I would like use GC-MS because we want to have an high resolution and to have the MSMS fragmentation to identify potentially new structures... =/

[X]

How much standard did you use? I would suggest 5 ug first. The other thing you can try is to use a more polar GC column, and inject standard in methanol without derivatization.

Thank you for your responses, it's very kind to you to help me...

In fact, I have already try to only use BSTFA-TMCS but my molecules aren't soluble in it, too...

And I would like use GC-MS because we want to have an high resolution and to have the MSMS fragmentation to identify potentially new structures... =/

[Don_Hilton]

Given that these are phenolic, diazomethane should be able to derivatize them in water. I would be careful with evaporating them to dryness, as polyphenolic compounds are easily oxidized.

Because phenolic compunds are mildly acidic, adding a bit of dry pyridine - or using it for the solvent with MSTFA or BSTFA may help. But this does require removing water first.

For trying MSTFA or BSTFA, you can try 1% methanol in DMF and see if that will get the compound into solution. A small volume of this mixture ( a few microliters) and some MSTFA may get the derivitization to go for you. I've doen this when I had an issue with a polyhydroxy compound binding to the glass -- and failing to derivatize.

[James_Ball]

If you are trying to derivatize the -OH groups by methylation, you could try using Iodomethane with tetrabutylammoniumhydroxide. I used this to derivatize coal -OH groups when I was in college.

[MSCHemist]

Sorry to be a bit late to the thread but I believe alkyl chloroformates would works very well for your compounds. They derivitize thiols, amines, carboxylic acids, and phenolic (usually not aliphatic) hydroxyl groups.

The procedure is simle. You mix sample, pyridine, some NaOH to .1N, the corresponding alcohol for the chloroformate and finally the chloroformate, immediately vortex, wait a few minutes, add a second dose, vortex and wait a few minutes then add NaHCO3 and chloroform (possibly isooctane for less polar derivatives) and extract the organic layer.

Here is my procedure carried out in an autosampler tube.
150ul aqueous sample
30ul of norvaline ITSD
20ul 4N NaOH
135ul of an ethanol/pyridine (4:1) mix
100ul of ethyl chloroformate in chlorofrom (2:8)
-immediately vortex vigorously capped then loosen cap for gas to escape
-wait 3 minutes
100ul ECF/Chloroform
-immediately vortex vigorously capped then loosen cap for gas to escape
-wait 3 minutes
Add 200ul of sat NaHCO3
-vortex allow gas to escape
-wait a few minutes
Transfer bottom organic layer to microinsert and new autosampler tube.

Inject on a GC with a 1701 or db-5 column FID or MSD.

Added you may need to try 2 or so differnt mixes
The method of Qiu uses 0.1 N NaHCO3 at the beginning then a neutralization with 7N NaOH then a second dose of ECF. My method above works well for amino acids and citric acid cycle acids but has a low yeild of carnosic acid. The Qiu method works well for most amino acids and TCA acids except Glutamate yeild is 1/3 of my procedure above.

[Peter Apps]

Simple phenols can be derivatized with acetic anhydride in aqueous base (sodium carbonate if memory serves). I don't know if it would work with your molecules, but it might be worth a try.

Peter

[MSCHemist]

One of my first methods was a derivitization of avermectins (moxidectin, abamectin, doramectin, and ivermectin) with a mix of methyl immidazole and trifluoroactic anhydride. The samples were in acetonitrile and the reaction was indeed water sensitive in fact the methylimmidazole would keep taking in water and had to be discarded in the summer even when nitrogen blanketed and stored in a desicator.

[mrussell]

Hello everyone,

I need some help for my work...

I have a very polar compound to analyse (spinochrome and echinochrome; pigments from sea urchins).They are only soluble in water, alcohol, DMF and DMSO (they don't in pyridine, acetonitrile, ethyl acetate, acetone, ether, dichloromethane,...) but I would like to analyse them in gas chromatography and for that, I must derivatize them.

I have tried BSTFA-TMCS 1% in DMF and DMSO but it seem these solvent denature my molecules... I can't use water or alcohol because BSTFA react with -OH group...

Do you know how derivatize a polar compound with water as solvent ?

Thanks a lot !

Try DNPH (dinitrophyenylhydrazine) . Its used for instance in EPA and one of the OSHA formaldehyde method. It is derivatizes aldehydes and ketones. Some methods give detailed instructions for recrystalizing and drying the DNPH. DONT DO THIS. DNPH is explosive when dry and is therefore sold with 30-50% water content,so its safe to handle. Recrystalizing such things should be done only by those with experience handling those kinds of compounds. Just use it wet,and its fine. Another posibility is PFBHA(O-(2, 3, 4, 5, 6-pentafluorobenzyl)hydroxylamine hydrochloride).

DNPH is often used in aqueos solutions for analysis of ketones and aldehydes. DNPH has a strong orange color and derivatives can be analyzed using GC-FID,GC-NPD or LC-UV/VIS.

I know that PFBHA is used for an alysis of a 2,3 butadione and acetoin in ethyl alchohol. I dont know off the top of my head if it works in water (it works with about 5% water in the solution),but if not you might get away with ethyl alchohol as a solvent.I dont know how it goes on LC,but it can be analyzed via GC-FID or GC-ECD.

Why do you even need to derivatize it. It should run as is. Especially on LC-UV/VIS.

[Peter Apps]

Mrussell

All very interesting, but how does it help with derivatizing phenols for GC-MS analysis ?

Peter

[carlo.annaratone]

I would like to give a warning before you use the methylating agents: I think they could be the best derivatization reagent for your sample, but they tend to methylate the user as well. If you are new to GC and derivatization, I'd do it in the presence of someone more expert the first time, and use all of the possible safety measures. Another caveat is that the drying step might oxidize much of your stuff before it gets derivatized.

As for the chloroformates, I used them to derivatize sulfur amino acids, so I am aware that they are very mild and tolerate well water, but I know that sometimes they have problems with molecules with many vicinal hydroxils. I do not know if this applies to phenols as well (e.g. I think there are problems derivatizing sugars)

[MSCHemist]

They generally don't touch aliphatic hydroxyls but they do phenolic hydroxys.

The main thing with the chloroformates is that the reaction mixture can affect the yeild especially with carboxylic acids. That is because the reaction with carboxylic acids is 2 steps, initially an anhydride is formed and that anhydride is unstable and gets attacked by the corresponding alkyl alchol to form the ester. The trouble is there can be competiting reactions. They can form cyclic anhydrides. Petr Husek has a good paper on it regarding the hydroxy acids. Fumaric makes a misutre of diethyl maleic acid and diethyl fumaric acid, citric there is some dhydration to cis aconitic acid tri ethyl ester as well as the main triethyl citrate.

The pH seems to be a key factor. I currently have a quandry between carnosic acid and glutamic acid. Glutamic acid sees best yeild when the aqeuous layer is highly alkaline at least 0.1N OH-. Carnosic acid reacts poorly like that, it reacts best in 0.1N NaHCO3 and then a second dose after adding some 7N NaOH to bring it up to pH 9-10

http://www.ncbi.nlm.nih.gov/pubmed/19573517
http://link.springer.com/article/10.136 ... ccess=true
https://www.yumpu.com/en/document/view/ ... ing-solid-