Chromatography Forum

Hi!

I'd have a question concerning the separation of lactic acid from acetaldehyde to be found in aqueous solutions with up to 20% salts.
Is there any C-8 or C-18 which could succeed in separating them? Is there any other technique to do it without derivatization of the two compounds?

Thank you in advance.

Use an organic acid column and dilute sulfuric acid eluent, either conductivity or about 210nm UV detector for lactic acid.

I know a Rezex ROA column from Phenomenex, but up to my knowledge it works on ion exchange so it wouldn't work for my aqueous samples with 20% ionic species. Do you know a column to separate acetaldehyde from lactic acid but support solutions with ionic species in the concentration range mentioned above?

Actually the Rezex ROA is an organic acid analysis column of the type
suggested by CPG. It is not ion exchange but ion exlusion. Your salts will mostly come out early and your lactic acid and acetaldehyde come out later. 20% salt shouldn't be a problem unless the anion coelutes with your acid and aldehyde.

Mardexis, I have asked the guys from Phenomenex about the high salt concentration and they said that RezexROA-OrganicAcid doesn't support such high salt concentrations without altering the separation capacity of the column. I tried to find out what exactly ion exclusion is and I´ve found a paper(M.Novic, P.R. Haddad J.Chromatogr.A 1118(2006) 19-28) which presents detailed information on the subject. I only know the notion "Ion exclusion" from you but it seems to me that with so many anions on the resin surface there should also be some ion exchange with samples of high salt concentrations...eventually hindering the retention of the analytes...
Should there be another option?

Lactic acid is nicely retained on a Atlantis dC18 (or Atlantis T3) which can be used with 100% aqueous mobile phases. Th acetaldehyde will be retained longer. Please see: http://www.waters.com/webassets/cms/lib ... 0472en.pdf

Acclaim Organic Acid column (http://www.dionex.com/en-us/columns-acc ... s6953.html) should work well for your application. I injected a mixture of 8 organic acids (inluding lactic and acetic acids) dissolved in saturated NaCl solution, and still got good separation similar to the one shown in the link. Acetaldehyde is more hydrophobic than lactic, thus separation won't be a problem. Acclaim Organic Acid column is a reversed-phase column and is compatible with 100% aqueous. Send me a email so that I can send you the result for organic acid separation in concentrated NaCl sample.

Still not successful in separating lactic acid from acetaldehyde. I have also tried a dC18 Waters Atlantis: It separates lactic acid from acrylic acid nicely but lactic acid and acetaldehyde always come together.

XL, are you sure that acetaldehyde and lactic acid come separately? I cannot contact you as I can't find your email adress...

If lactic acid and acetaldehyde coelute, you need to increase the pH until the lactic acid is partially ionized and will retain less. The acetaldehyde won't move, when you do that. There should also be enough of a retention window for the lactic acid to do so.

I thought it shouldn't be difficult but I don't have firsthand experience. There is only one way to find out. Can you send me the list of analytes you want to separate for this application and I will see what I can do? My email is: xiaodong.liu@dionex.com

Uwe, I have tried pH 5 on the Atlantis dC18... The peaks don't look so well, although the separation could have succeeded because of the different reaction towards H+ of lactic acid and acetaldehyde. I wonder what could be the cause for these peak forms...Maybe the test column is too old?
For the chromatogram at pH=5 I have used sulfuric acid instead of phosphoric acid.
I attach the chromatograms achieved at pH=2 and pH=5. Please note that the second major peak is always a third substance: Acrylic Acid.

For pH=2:

For pH=5:

The sulfuric acid does not have any buffer capacity at pH 5. That is why it does not work.

So your peaks overlap with a phosphate buffer at pH2. Lactic acid has a pK at 3.83. To get it partially ionized and move a bit away from the acetaldehyde. I would first try a pH between 3 and 3.5 with phosphate. At a high enough concentration, such as about 50 mM, you will still have a reasonable buffer capacity and you may ionize it enough to move it away from the acetaldehyde. I would do this first before anything else.

For the chromatogram at pH=2 I have used phosphoric acid... Yet, I have used 50mM phosphate buffer at pH=3,5 and there was still no separation.
I give up...The ion exclusion column works fine and separates acetaldehyde and lactic acid well... I will use as much ion exchange resin as needed to remove the 20% salt...
Now, just for the sake of the issue itself: although the chromatogram looks bad, there seems to be some separation at pH=5, right? So is there a possibility to make them look like peaks?

Thank you ver much for the given help!

Some thoughts: If the acetaldehyde should still be acetaldehyde it ~ wouln´t change shape nor rt if only the pH of the mobile phase was changed. Even though I think it is not that easy to get a pH = 5 with H2SO4, if you really had something near this pH you could have gotten exclusion of the acids which was partially lifted by the coinjected salt, hence the "broad peaks". This would almost require that your acetaldehyde was acetic acid instead.

Did you do any of this with individual standards (separatly injected) which had no salt in them?
What is this strange baseline?

I do not give up that quickly...

You are using a refractomer. So you have plenty of options. The first one would be to use pH ~5, but with a proper buffer: an acetate buffer prepared from an acetate and acetic acid to a pH of around 4.75. You will get good looking peaks, instead of the ugly mess that you have. We will then see where all the other peaks are eluting, but your acids will elute earlier. I do not know by how much. We will see. If they elute too early, we can use acetic acid alone, instead of preparing the buffer. This will end up with a lower pH. If the retention declines for all peaks, we can add some salt, but let us first see what the other conditions give us.