By Anonymous on Wednesday, November 26, 2003 - 02:22 am:

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

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By Rocket Scientist on Wednesday, November 26, 2003 - 06:40 am:

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

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By A.Mouse on Wednesday, November 26, 2003 - 12:46 pm:

Try HILIC on a silica column instead of C18!

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By Chris Pohl on Wednesday, November 26, 2003 - 06:31 pm:

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.

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By Anonymous on Thursday, November 27, 2003 - 01:00 am:

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.

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By Zelechonok on Thursday, November 27, 2003 - 07:05 am:

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

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By A.Mouse on Thursday, November 27, 2003 - 08:50 am:

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.

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By A.Mouse on Thursday, November 27, 2003 - 08:52 am:

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.

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By Anonymous on Thursday, November 27, 2003 - 05:09 pm:

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.

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By Anonymous on Thursday, November 27, 2003 - 11:31 pm:

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

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By vojtech on Friday, November 28, 2003 - 03:12 am:

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.

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By Vojtech on Friday, November 28, 2003 - 03:25 am:

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.

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By Einar Pontén - SeQuant AB on Friday, November 28, 2003 - 11:55 am:

I advise you to check this out.

It is our brief description of HILIC on our specially designed column ZICâ„¢-HILIC. We are available to support you.

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

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By A.Mouse on Friday, November 28, 2003 - 04:43 pm:

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.

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By Markus Laeubli, Metrohm on Sunday, November 30, 2003 - 11:53 pm:

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

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By MG on Monday, December 1, 2003 - 06:53 am:

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

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By Anonymous on Thursday, December 4, 2003 - 05:21 pm:

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

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By AllsepTech on Thursday, December 4, 2003 - 05:36 pm:

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.

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By A.Mouse on Thursday, December 4, 2003 - 06:57 pm:

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!

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By Anonymous on Thursday, December 4, 2003 - 08:15 pm:

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?

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By AllsepTech on Thursday, December 4, 2003 - 09:32 pm:

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

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By Anonymous on Friday, December 5, 2003 - 12:57 am:

A.Mouse

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

AllsepTech
Thanks.

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By Vojtech on Friday, December 5, 2003 - 06:14 am:

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.

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By AllsepTech on Friday, December 5, 2003 - 08:01 am:

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.

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By Uwe Neue on Friday, December 5, 2003 - 03:35 pm:

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.

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By AllsepTech on Friday, December 5, 2003 - 08:30 pm:

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 “whalesâ€