Resolution of some polar metabolites and isomers by HILIC

[song jack]

Hi,

I am trying to separate some intracellular metabolites like fumarate, malte , succinate , isoleucine and lecuine by luna NH2 column in HILIC mode. Initial phase is 85 % ACN and 15% water/ACN(95:5, 20mM ammonium acetate buffer with pH 8.5). Whatever I changed gradient or adjusted buffer pH, malate and fumarate , leucine and isoleucine couldn't get a baseline separation. It is so hard for me to make an accurate quantification of them. I am wondering if HILIC is able to resolve them since they have so similar polar characteristic. Thanks !

Jack

[grzesiek]

I have come across this article but Leu and Ile were not separated
Quantitative metabolome analysis using liquid chromatography–high-resolution mass spectrometry
Patrick Kiefer, Jean-Charles Portais, Julia A. Vorhol

[danko]

Hi Jack,

Try another column. F ex. Grace Genesis NH2, Waters Spherisorb NH2, or Agilent NH2 Zorbax, will easily outperform the column you’re using. These are just a few possibilities, but there are many other prof. columns you could try and expect nice results from.

Besides, you may be more successful if you try the same conditions but at low pH (e. g. between 4 - 5)

Best Regards

[lmh]

beware of Phenomenex-bashers (and see thread elswhere on this subject!)

OK, I can't swear whether their amino column is better or worse than other manufacturers, but if you have very poor separation between two compounds using one manufacturer's amino column, it is unlikely to become miraculously good by switching to the same chemistry from another manufacturer. The best you can hope for is a change from awful to just-about-passable.

In general, switching manufacturer is not as efficient as changing column conditions or column chemistry.

[danko]

Hi lmh,

In your eagerness to defend the utilized column, you obviously missed my second suggestion to Jack (i.e. changing the pH). So, I’m not forgetting other parameters.

Btw. the quality of the column is not as insignificant as you present it. I’ve seen differences from a shoulder with one column brand to Rs = 2 with another (everything else equal). But it takes of course some tasting and finding quality products.

Otherwise, what would be the point in improving existing and developing new stationary phases (e.g. purer silica, better coating and packing, more control with the particle size, pore size etc. etc.) if no significant gains were to be expected from the column?

With regards to the price differences between different column brands (as mentioned in other threads), I agree; some columns are more expensive than other, but that’s not a quality parameter – in my world anyway. Although, sometimes (not always) it might be a quality expectations indicator.

Best Regards

[Bryan Evans]

Hi Jack -

This is a difficult separation, but here are organic acids & amino acids on Unison UK-Amino (3um):

Branch-chain AA: http://www.imtaktusa.com/site_media/fil ... TI311E.pdf
Aliphatic AA: http://www.imtaktusa.com/site_media/fil ... TI469E.pdf
Organic acids: http://www.imtakt.com/TecInfo/TI542E.pdf

[Vlad Orlovsky]

You can try mixed-mode HILIC, where compounds are retained by cation and anion exchange in addition to HILIC:
http://www.sielc.com/application_183.html

If you don't want to switch columns, play with pH. At differnet pH, ionization and polarity of your compounds are going to be very differnet. Explore pH 2, 3, 4 and 5 (various buffers). This should be a relatively easy separation.

The draw back of ALMOST any amino propyl column is limited pH range and stability. If you hydrolize aminopropyl ligand the selectivity of your separation will change.

[lmh]

Hi Danko,

I'm not keen to defend anyone's column, but I felt the expectation of "nice results" was possibly optimistic depending on just how bad the results are at the moment. My limited experience of HILIC is that while it retains polar things, it hasn't been suddenly the solution to all our ills as we searched for a volatile solvent approach to really, really polar things. It retains, but it doesn't always resolve things, and when it fails, it may be failing because the fundamental chemistry of the separation just isn't there: the analytes are just too similar.

I'm a bit sensitive to suggestions of trying a different manufacturer as I have a drawer full of C18 lookalikes sold to me when I was young and tender by reps who promised they would be better than anyone else's for "polar metabolites", and I have corresponding lab-books full of chromatograms with peaks so close to the dead volume that they're useless. Some are a few seconds later than others (yes, Waters Atlantis was the best), but none has adequate resolution. Perhaps I should leave this unhappy part of my life in the past, but it just makes me sceptical nowadays. Hilic, for all its weaknesses, at least got me out of trying to flog the dead horse of reverse phase for polar analytes.

I'll watch this thread with interest...
Amino acids, if you don't want to derivatise, have also been done with ion pairing very successfully, but don't touch it if you've got an MS attached. The method I knew used an acidic ion pair reagent for the NH2 group, but I suppose in theory you could go for a basic one and try to get organic acids too. Dunno, haven't tried.

[danko]

Hi lmh,

I'm a bit sensitive to suggestions of trying a different manufacturer as I have a drawer full of C18 lookalikes sold to me when I was young and tender....

Believe me, I understand your sensitivity to senseless replacement of columns hoping the resolution will fall from the sky. And I’m 99.9 % in agreement with your philosophy. The last 0.1 % comes from recent experience while I was working for a former employer.
Some morons decided that if one finds the “perfectâ€

[lmh]

thanks! I envy you the insight from looking at that number of columns, but I have to sympathise with having to work in a project like that. Yuk!

[grzesiek]

"Another side-effect of the events above was a huge number of redundant columns which were disposed off over time.
So, today I say: The only thing worst than a moron is a decision making moron."

great story danko, I could write similar one

[juddc]

[quote="danko"]
.
Some morons decided that if one finds the “perfectâ€

[danko]

I can tell stories of that kind from now on and to eternity.

Here is another one – promise to make it short

One of these (they were many, because that was the only way to get promoted, or at least expect an annual bonus – and still is) savings gurus, was the boss of a good colleague of mine, who reported to him that a certain HPLC method had appalling performance records and needed a comprehensive optimization. To that he answered: OK, go on and optimize it, but remember; you MAY NOT introduce any changes. Because - as you know - the method is registered………

Best Regards

[juddc]

Thanks for the laugh as I close up shop for the evening! That's just lovely!

[grzesiek]

"OK, go on and optimize it, but remember; you MAY NOT introduce any changes"

I remeber as my boss validation report came into my hands one day. RRF was calculated in some strange way It was calculated as the slope between predicted and actual quantity of the impurity with the acceptance criteria set to 0.8 - 1.2, and you know what? It was always within the criteria set