Chromatography Forum

Please I want to development chromatographyc method for organic acids such as fumaric, mailc, citric, isocitric, glyoxilic, oxaloacetic and others.. but now I had tryed ACN 85 buffer 15 (NH4AC pH 6,8 ) but isnot good choice because the bad elution.....Do you have any sugestions, please send me...

The best option would be to email one of the Merck Sequant AB technical support people.

If your column supplier can't locate them, then try one of the people that appear on this site, such as Einar Pont.

I don't think that I would use that column for such a separation, but they should advise on what is most suitable.

Bruce Hamilton

JOCAPEN,

It is unlikely you can develop a separation for the acids you specified above using a HILIC column. You need to look at reversed-phase, ion-exchange, or reversed-phase/ion-exchange column.

What is your detection limit requirement? And what detection method are you using?

XL, I would appreciate if you elaborated on why you think HILIC is out of place here. These acids are fairly polar, I strongly doubt that some could be retained on RP.

JOCAPEN, that´s all you tried? What do you mean by bad elution?

Mueller,

To separate a certain mixture of compounds, we would need 1) sufficinet retention and 2) right selectivity. I don't doubt that a HILIC column can retain these organic acids since they are very polar. However, it is challenging to obtain right selectivity, especially for the compounds that have similar hydrophilicity. The larger number the analytes of interest, the lower the success rate. My experience on the subject tells me it is quite difficult on a HILIC column unless someone demonstrate otherwise so that I can learn something new.

Having said above, I could have conditions for separating this particular group of organic acids using a RP column. But before getting to any specific, I would like to learn more about the requirement of this application so that my 2-cent will make more sense.

I hope I explained myself clearly.

Thanks,

OK, I can imagine that it might not be possible, but the same reasoning should go into the use of RP, or especially ion exchange?
Maybe two different types of columns are required?
I would also look at separation of acids via "charge exclusion".

Mueller,

I agree with "Maybe two different types of columns are required."

RP, ion-exchange, HILIC and ion-exclusion columns complement one another. There is no much point to generalize which one is better than another. When the analytes of interest vary, the solution is usually different. Thus, it is important to define the scope of analytes along with other requirements. Although all above four types of columns provide potential solutions, there is only one way to find out.

In the past, I tried to use HILIC columns to separate hydrophlic organic acids. I had problem with selectivity as well as peak efficiency. In addition, I didn't feel right to use a high acetonitrile consumption method if I have a "greener" one.

Currently, I do have a RP method that can separate malic, isocitric, citric, fumaric, succinic, lactic and acetic acids. But without knowing the specific acids of interest, I can't be sure if this method is actually useful. Attached line provides information on Acclaim Orgainc Acid (see Figure 5): http://www.dionex.com/en-us/webdocs/259 ... OA_V22.pdf

In addition, I could develop a method using Acclaim Mixed-Mode WAX-1 column (a RP/WAX mixed-mode column, see Figure 7 in http://www.dionex.com/en-us/webdocs/488 ... 021407.pdf), which is the reason I am eager to know the "exact" scope of acids needed to be separated so that I might be able to try it in our lab.

You can use any of the approaches below:
1. Polar embedded RP with lower pH mobile phase. You need to suppress ionization of this acids to make them slightly hydrophobic. Atlantis T3 from Waters can work or any of modern RP column developed for retention of polar compounds
2. Use ion-pairing chromatography on any reversed phase (you will need basic IP reagent).
3. HILIC approach at pH 3-5, to make your compounds more polar for hydrophilic interaction. The cheapest approach is to try bare silica column with 80-90% ACN and ammonium formate/acetate (ELSD)
4. One of the mixed-mode approaches. In this case you can do anion-exclusion, anion exchange, and reversed phase anion-exchange.
Here are several columns and methods can be used for your mixture. You can use ELSD or low UV:
http://www.sielc.com/compound_059.html

Is this for fermentation samples? I ask because the acids, and your location...?

Anyhow, My last position was looking for similar acids, we did the Waters 10 minute Ethanol touchy with temperature. The heating block got turned off and the glycerol slid right into the lactic acid.

Possible get a waters column catalog, they had a few good options for wine or fuel ethanol. I ended up using mine like a reference book.

Good Luck!

You can use any of the approaches below:
1. Polar embedded RP with lower pH mobile phase. You need to suppress ionization of this acids to make them slightly hydrophobic. Atlantis T3 from Waters can work or any of modern RP column developed for retention of polar compounds
2. Use ion-pairing chromatography on any reversed phase (you will need basic IP reagent).
3. HILIC approach at pH 3-5, to make your compounds more polar for hydrophilic interaction. The cheapest approach is to try bare silica column with 80-90% ACN and ammonium formate/acetate (ELSD)
4. One of the mixed-mode approaches. In this case you can do anion-exclusion, anion exchange, and reversed phase anion-exchange.
Here are several columns and methods can be used for your mixture. You can use ELSD or low UV:
http://www.sielc.com/compound_059.html

We can now add the following to the list:

5. Multi-mode ODS

Below are organic acids separated on Scherzo SM-C18:
http://www.imtakt.com/TecInfo/TI518E.pdf

I sugest to you to use SiliaChrom HILIC. This HPLC column is great for acid and you are able to use it as either normal and reversed phase conditions.

Charles

We do organic acids including most of these on Grace Alltech Prevail Organic Acid columns with dilute acidic eluant and conductivity detector.

We are using SeQuant HILIC and one of the Primsep columns (forgot the name) for organic and inorganic acids.
We did not try Dionex column yet but while ago tested Prevail Organic acid. At one point we have tested a lot of columns for acidic counter-ions.
I looked at Scherzo and peak shape for last there acids would not be acceptable by our QC due to huge tailing. We got something similar when we used aminopropyl silica, so I guess this might be a physical mixture of two silicas -aminopropyl and RP.

For sure, you have to run your HPLC method at acidic pH. In order to have non ionized organic acid. There are few HPLC column for that kind of application. One of these is the SiliaChrom HILIC or SiliaChrom SB which is working at pH=0.8 to 7.

Charles
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SaliCycle, that "have to" is a bit whacky?