Hi -
Does anyone have experience with fatty acids? I am trying to analyze for Acetic, Lactic, Butyric, and Propionic Acids and I'm having lots of trouble. I bought an 'Ultra Aqueous' RP C18 column from Restek that is supposed to be perfect for this application but I can't get the peaks to separate. I'm using 50mM potassium phosphate at a pH of 2.5 as my mobile phase. When I inject the acids separately they give nice peaks (lactic at 3.02 min. and acetic at 3.09 min.), but when I inject a standard that contains more than one, they all appear as one big peak. I've tried injecting less sample and slowing down my pump but nothing seems to help. Restek is stumped - they are out of suggestions for me. They say that they can separate the acids just fine in-house on the same column so I don't know what to do. Thanks for any suggestions you might have.

3.02 and 3.09 minutes does not appear to be enough of a difference for the separation. Do you have an example chromatogram from Restek that you are trying to follow? What are their conditions?
What are your column dimensions and what is the flow rate?
What did you do with the column before you started to do this analysis?

Have you tried adjusting the pH of your mobile phase?

I do have an example chromatogram which is why I bought this column in the first place. I have simulated their conditions exactly with no luck. They use 99:1 50mM potassium phosphate : acetonitrile with a pH of 2.5. They run at 1.5 mL per min. and inject 20 uL of sample. They read at 210. The papers that came with the column did not say anything about conditioning. The column was stored in 85% methanol and 15% water, so it said not to add a buffer directly to that. I ran straight water through the column first for a long time, then gradually added more and more buffer. They've had me try all kinds of diff. things and nothing seems to make a difference. Everyone has told me that I have to keep the pH low, so I havn't tried it any higher than 2.5.

Michelle,

I don't have a lot of experience with this system but it's possible the phenomenon you are observing is connected to sample pH. In your analytical system maximum sample focusing will occur at low pH whereas injecting the analytes as salts will tend to be defocusing. This might explain a decrease in resolution. Are you injecting the sample as the acids or as the salts?

Regarding the other point, while the buffer won't readily dissolve in 85% methanol, the method you use was much more laborious than necessary. All you really need is a column volume or two of water to pass through the column to clear out the methanol. After that, you can switch directly to the aqueous buffer solution. There's no need to gradually increase the buffer concentration.

What were the retention times reported on the sample chromatogram? And what are the column dimensions?
Also, I do not expect to get a separation between peaks at 3.09 and 3.02 minutes.

The retention times reported on the chromatogram are Lactic: 2.1 min. and Acetic: 2.4 min. The column dimensions are: column length: 150 mm
inside diameter: 4.6 mm
particle size: 5um
pore size: 100 A

I don't expect to get a separation between peaks at 3.09 and 3.02 either, but that's where they come out at when I inject them separately and I have no idea why I'm not getting results similar to Restek's example.

I've done this separation successfully by packed column GC. However, you have to be fairly experienced in this sort of work to get the method going because of problems of adsorption of the acids in the system. When it works however, the separations would appear to be much better than the results even Restek are getting. Please ask if you are interested.

David McCalley

Michelle,

I dug a bit further into this. The two parameters that have the most influence on resolution of lactic and acidic acid are: pH (higher pH improves resolution) and ionic strength (higher ionic strength improves resolution). Of these two, pH is a more powerful effect. Based on unpublished work done here, I would say that pH 2.7 is a more optimum pH for this separation. Increasing the pH to 3.0 will further improve the resolution but at least on our column places citrate between lactate and acetate. Perhaps you should doublecheck the calibration on your pH meter. It's possible that you have the pH a bit too low (as pH 2.5 should work although as mentioned above in our hands on our column pH 2.7 is better).

Ion exclusion would be an ideal way to separate these analytes using an isocratic low pH aqueous mobile phase @ 50 deg C or greater and low UV, RI, or maybe even ELSD detection. Something like a Shodex RS Pak KC811 (300x7.8m) should do pretty well, though make sure you have your sample as clean as possible - these columns don't like proteins and hydrophobes and can foul easily if you're not careful. Luckily, these acids are amenable to SPE cleanup. I think Restek has a similar column to the Shodex and it may be cheaper, too.

Good luck!

Chris

I agree with the last proposal.Ion exclusion is a different approach for separation of organic acids.There are many other suppliers of ion exclusion columns on the market: PL,Hamilton,Jordi,Restek,Supelco,Interaction.....
Ask them for applications or look into their catalogues.Ask them for organic compatibility and how to clean the columns (some columns are very sensitive).
PETER

I am not very experienced with the different types of columns and detectors. Can you use ion exclusion columns with a UV detector? I have a UV detector and I'm not able to get anything different, so I have to make this work.
Thanks, Michelle

Michelle,

You can definitely use ion exclusion columns with a UV detector. Conductivity detection generally provides better sensitivity but as long as you choose a UV transparent eluent (sulfuric acid is generally used) you should be able to readily detect all but minor impurities in real samples.

A UV detector is certainly OK and will be reasonably sensitive when used at a low wavelengths (195 - 210 nm) for these samples. An RI will also work well here, though they require much longer equilibration time.

I agree with all above ....
these links may also be useful too for organic acid analysis. The links shows chromatograms and conditions required. Detection either by UV or conductivity.
good luck

http://www.metrohm.com/docs/app/notes/pdf/o5.pdf

http://www.metrohm.com/docs/app/notes/pdf/o15.pdf

http://www.metrohm.com/docs/app/notes/pdf/o16.pdf