Sample prep for SCFA from fecal samples

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

2 posts Page 1 of 1
Water alliance 2695
Bio-Rad Animex HPX-87H column
Waters SH-G guard column
Waters W2996 PDA detector

Hi everyone,

I am new to this forum and I hope my question is not too basic. I am looking to improve my current method for short chain fatty acid analysis in particular the sample preparation. My company has been using the same method for over 6 years, but I am not convinced it is working. The matrix is liquid manure and sometimes solid fecal material.

Problems:

I suspect poor recovery from samples
Last component RT is 60 min
Major tailing in standards after 45 min
Large, 5-8min gaps between peaks

Current method:

isocratic with 5mM H2SO4 mobile phase
35 C column temp
0.6 ml/min flow rate
PDA wavelength 224

10g sample centrifuged at 5000 rpm for 5 minutes
1 ml of supernatant is diluted in 9 ml mobile phase, vortexed, and filtered through a 0.2 um filter

Proposed new method:

isocratic with 10% acetonitrile in 5mM H2SO4
65 C column temp
0.6 ml/min
PDA wavelength 210

sample prep ?

Unfortunately I am not a chemist and since no one knows how to use the HPLC at my work, I am on my own. I've found literature for fecal sample prep for GC, but not HPLC. Any help would be appreciated!

Best advice I can give you is "change one thing at a time!".

If this were my problem, I'd begin by working with standards and address the run time/tailing issue first.
- Begin by increasing the temperature
- They try adding ACN.
With each change, once you have established what things look like with standards, inject an extract to check for interferences.

The time between peaks, by itself, is not terribly relevant. What counts is the resolution (the difference in retention times divided by the average baseline width); ideally, that should be 2.0 or more.

Once you have your chromatography set up, you can begin to explore the extraction issue. Again, if this were my problem, I'd go one step at a time:
- Is there an extraction time issue? Extract for varying periods of time and see what happens.
- Is there a pH issue? A pH 2.5 extraction buffer is below the pKa of most of your fatty acids, which means they are likely to be in the free acid form, and therefore less soluble in water. Spike a sample and study the recovery. You may have to go up in pH (at the price of shortening the guard cartridge lifetime) or go to a more elaborate sample prep extracting into a non-polar solvent and then back-extracting at higher pH.

Sample workup for GC and LC should be similar up to the point where you would derivatize for GC.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
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