hi!
may i know the simplest method to separate ethanolamines, and what is the heaviest by-product.

Here is a simple method for ethanolamines (no ion-pairing reagent) with good peak shape and base line separation:

http://allsep.com/makeChr.php?chr=Chr_041

SIELC Tech

Where do sodium, ammonium and potassium elute on your column under the conditions shown in the cgram at the link above? Few alkanolamine samples are without a least one of these cations.

Hi,
I am trying to separate the ethanolamine by LC and detection by MS.

I have tried the following:
1) Gradient of mobile phase: 0.05% TFA in water and 0.05 % TFA in MEOH. Column is C18 and C8

2) Gradient of mobile phase: 0.02M ammonium formate in water and 0.02M ammonium formate in MEOH. Column is C18 and C8

Can anyone with experience of these compound give me some advice on the condition of separation?

Thank you

My lab would derivatize and do GC or GCMS for this. We've done it many times, what levels and what matrix?

Try HILIC on a silica column instead of C18!

You can get retention on C18 if you use heptafluorobutryic acid as the ion pair reagent but this is not a good MS eluent. Better to use a polymeric cation exchanger with MSA and a suppressor.

Thank you for the replies

Rocket Scientist,

I can't use the GC/MS techniques because of maxtrix constraint. I can only use LC/MS.

A.Mouse,

I am new to LCMS technique. Your suggestion is the only option left for me. Can you please advice me a little bit more. What is HILIC? How to incorporate this column to MS detection? Can you recommend a brand or supplier?


Chris Pohl

Thank you for your suggestion but I cannot afford to buy a High performance suppressor.

Mixed mode chromatography is still another option. Simple amines method described with LC-MS compatible conditions. Ethanolamine retains about 10 min with this method. Plus you can analyze other compounds in your sample since it is reverse phase column.
Check this link
http://allsep.com/makeChr.php?chr=Chr_018

HILIC stands for HydrophILic Interaction Chromatography. You use a very polar column like silica, amino, or zwitterion together with a mobile phase of mostly acetonitrile with typically 10 to 50% water. The more water you add, the less retention you get. With your compound, I would use a silica column, because the retention will be highest (the silanols on silica help in the retention due to ion-exchange).
This technique is classically used for sugar separations. Your compound is ideal for HILIC.

I should have said also that in order to improve the ionization in MS, you may want to add a very small amount of formic acid to the acetonitrile/water mixtures.

A. Mouse

Thank you very much for your advice. I will source the supplier for the column. I will try your suggestion once I receive the column.

Zelechonok

Thank you. You have provided me with another option to try.

A.Mouse and Zelechonok

It will take me about two to three weeks to purchase the column. I will keep you update on the result once I receive the column.

A. Mouse

If you still listening. I need your advice on the column. I have two suppliers with the following columns:

1) Phenomenex Luna CN 2 X 150MM 5 micron
2) phenomenex silica 2 x 150mm 5 micron
3) Agilent Hypersil Normal Phase Column 2.1 x 100mm 5 micron

Please advice me base on your experience

Thank you and regards

Mix your matrix with carbonate buffer pH 11,0, add 20 ul chloroformate (ethyl or propyl). It will react with your target compound. Clean the sample with SPE or sipmly extract it into a nonpolar sovent. Analyze it on any C18 in gradient water/ACn (MeOH). The derivative is also detectable with UV (at about 200-210nm). This derivatization is easy to do, does not require dry reagents, it is simply to quantitate and it is reproducible. The derivative can be also analysed on GC.

Yamamoto et al. Journal of Chromatography, 194(1980) 399-403
Gyllenhall et al. Journal of Chromatography, 190 (1980) 347-357

In articles above you can learn more about derivatization procedure.

I advise you to check this out.

It is our brief description of HILIC on our specially designed column ZIC™-HILIC. We are available to support you.

http://www.sequant.com/pdf/SeQuant.ZIC-HILIC.column.pdf

Either silica column will work. I would use the Hypersil for various reasons, the most important one is that it is shorter and I can get the experiments done quicker.
Equilibrate the column with acetonitrile with 10% water and some formic acid. Run a gradient from 90% to 50% water. You should see the ethanolamine easily. But run a few blank gradients first, since it appears that you will be using older columns that have never seen HILIC before, and there may be all kinds of stuff on them that will elute when you hit the column with water.

Ethanolamine is a typical application for Ion Chromatography. You may use a cation column with direct conductivity detection (no suppression required).

See an Application Note under:
http://www.metrohm.com/docs/app/notes/pdf/c52.pdf?lcmb

Anonymous with the silica columns: If they are stored in hexane, I recommend you flush them with isopropanol before acetonitrile / water.

Thank you all for the respond.

A.mouse

I am currently using Altantis HILIC column (2.1 x 150mm, 5um). I have tried to elute the compound with the following mobile phase:

A: Acentonitrile
B: 0.1% FORMIC ACID

I equilibrate the column with 10%B and 90%A.

Run the following gradient :

Time A(%) B(%)
0 10 90
5 50 50
15 50 50
25 10 90

The ethanolamines retained at 2.5mins but no separtion. I need to separate them for confirmation. I have tried different gradient. It still did not separate. Does it means that it is not possible to separate the ethanolamines?

Can any advice.

Thanks and regards

We have developed a method for the separation of ethanolamines (mono-, di and triethanolamine) using one of our columns (Primesep 100, 4.6x150 mm, 5 um, 100A). Beautiful base line separation of all three peaks. Retention time 7-13 minutes. Mobile phase ACN-water-TFA=5/95/0.1). The method will be posted on our website tomorrow night. We will send you chromatogram upon your request. We tried HILIC without any success. We are working on the method for the reverse elution of three ethanol amines on our Primesep C column - update will follow later. Please check our website tomorrow night www.primesep.com for the separation of the ethanolamines.

Why do you run the gradient in five minutes and then hold it forever at the final composition?

Run from 10% A to 50% A in 15 minutes! Then let us see the results!

A.mouse

Sorry. I am still trying to understand LC. I will do my homework on this part. I am trying to increase my knowledge by reading as much as possible about LC/MS. Thanks for being patient.I will try your suggestion.

Allsep Tech

Can you give me your web link or e-mail address to request for the chromatogram?

contacts us at mail@allsep.com. Visit our website for more details www.primesep.com

A.Mouse

Just realise that Water Altantis HILIC column cannot run to more than 60% water.

AllsepTech
Thanks.

If you really want quick results with a simple C18 system, with water/Acetonitrile mobile phase, with no other additives,try the chloroformates. The reaction takes a few seconds and the procedure is quite simple.The derivatives are well separated from the reagent. You can evaporate the excess of reagent under nitrogen stream (if you do not want it in your sample).
I suggest this method as very straightforward solution for amines if you want them to separate on HPLC. I Also know people who use this system with MS.

Here is our method for the separation of three ethanolamines.

http://allsep.com/makeChr.php?chr=Chr_041

The method is LC/MS compatible.
Our method does not require any derivatization (no chlorformate or other derivatizing agents)-just directly inject the mixture of mono-, di- and triethanolamine in our Primesep 100 column.

I don't know why the Atlantis HILIC column should only be used up to 60% water, especially in a gradient. On the other hand, a gradient from 90% water to 50% water as suggested by A.Mouse is within the limits. Therefore I do not see the problem. I also think that HILIC is the best approach for the analysis of these very polar compounds, but I think you may need to do the separation isocratically instead of with the gradient. You could inject your sample mixture changing your solvent composition in isocratic steps from 90% to 50% in increments of 10%. This is a simple and fast way to find out what the approximately best solvent composition will be.

With all due respect to HILIC approach, the applicability of HILIC columns to the particular separation of ethanolamines must be proven first. I don't see why our Primesep method is worse than HILIC, except for the fact that is made by somebody other than “whales” of columns production. The Primesep columns are LC/MS compatible, isocratic with a very good peak shape and selectivity. We tried to achieve the same quality with HILIC today, without much success. Actually, we would be glad if scientists with more successful experience of HILIC separation of ethanolamines shared the conditions – would be interesting to compare the methods side by side.

"The Primesep method for ethanolamines is LC/MS compatible, isocratic with a very good peak shape and selectivity" - Sorry for typing it wrong....it's Friday night.

To Allseptech:

I did not say that your proposal is worse. If it separates the stuff, it is fine. However, the reason that I answered to the request by the investigator is that he has a silica column, and is using it. I gave him some advice on how to approach the problem WITHOUT needing to buy a new column.

The fact that you tried to do a HILIC separation and were not successful does not mean that this applies to the investigator as well. But it would be good if you would share, what you tried, and what did not work, so that the investigator here does not waste his time.

Best regards
Uwe

Vojtech

Do you know the reaction between triethanolamine and methyl or ethyl chlorformates? I can only find information where the chloroformates attack the NH2 OR SH group. Please advice on what is the compound I should be looking at for this kind of reaction.

Thanks and regards

Drew,
I cannot describe it completely but from experience I know it works well. For more information try to look into "Handboook of derivatives for chromatography" by Blau and Halket (Second edition). On page 47 is said that in tertiary amines the smalest group can be displacedwhen treated with a chloroformate. Especialy if it is attached to nitrogen, particulary if it is a methyl group, to form a carbamate.
Sawardeker, Sloneker, Jeanes; Anal. Chem.,m 39, 121(1967).
Another possibility is that chloroformate reacts with OH groups (?), as used with FMOC-Cl.
Regards, Vojtech

Vojtech

Thanks for the info. I will try this method for my work. Will keep your update on the outcome.

Regards
Drew

Chlorformates are reacting with primary and secondary amines and ethanolamines very fast (reaction on nitrogen atom) the hydroxyl group will react much much slower, Tartiary amines are used in reaction wit chlorformates to tie HCl generated during the reaction. In the water matrix OH group of ethanolamine will never react with chlorformate. The rate of reaction of primary amine is much faster then secondary this might cause a problem due to mixture of products. If there is a direct method for analysis I would never use derivatization, check literature for direct analysis of ethanolamines.

Thanks anon above for details. I thought the same about reaction with OH groups but I was not sure.
I used this method for scouting for triethanolamine, where I had a single peak (GC and LC).
I believe that there might be some problems if there are primary amines or other more reactive substances.
In my experience this derivatization is very simple and you can get some results fast (even if you need to resolve ethanolamines from polar and very complex matrix). I agree with anon: if there is a direct method for analysis there is no reason for derivatization.
I found some reference in: "Ion chromatography" by Weiss(second edition): p. 266 - IonPac NS1; 2 mM octanesulfonic acid/acetonitrile (92:8). Suppressed conductivity. Well resolved MEA, DEA, TEA.

Dear Drew,
Please check our method for the separation of ethanolamines, no derivatization required. Method is isocratic and LC/MS compatible. Instead of spending several days trying you can just order Primesep 100 column and be on your way to achieve your separation goal

http://allsep.com/makeChr.php?chr=Chr_041

The method from Alltech looks good. If the matrix is simple I would go for it.
Vojtech

The method from Allsep looks good. If the matrix is simple I would go for it.
Vojtech

Hi

Just to update that I have managed to separate the ethanolamines by using Primesep 100 column from Allsep.

Drew

Drew,

Thank you very much for the update. You can contact us at any time for the future method development needs.