To determine short chain volatile fatty acids (VFAs) in HPLC, you can follow these simple steps:
1. Prepare the sample: You can prepare the sample by mixing the sample with a suitable solvent, such as methanol or acetonitrile, and filtering it through a 0.2-micron syringe filter.
2. Set up the HPLC: Set up an HPLC system with a suitable column, such as a reverse-phase column, and a suitable mobile phase, such as a mixture of water and acetonitrile.
3. Calibration standards: Prepare a series of calibration standards by diluting standard solutions of VFAs, such as acetic acid, propionic acid, and butyric acid, to a range of concentrations.
4. Injection: Inject a small volume of the sample or calibration standard into the HPLC system, and record the resulting chromatogram.
5. Integration and quantification: Use the software provided with the HPLC system to integrate the peaks corresponding to each VFA and calculate the amount of each VFA in the sample or calibration standard based on the calibration curve.
Note that the detection and quantification of VFAs in HPLC can be sensitive to several factors, including the type of column, the mobile phase composition, and the detector used. Optimization of these parameters may be necessary to obtain accurate and reproducible results.
Here is a reference that provides more details on the determination of short-chain volatile fatty acids in HPLC:
Reference:
Yuan, H., Zhou, L., Gao, H., & Fan, X. (2019). Determination of short-chain volatile fatty acids in environmental water samples using high-performance liquid chromatography. Journal of separation science, 42(1), 157-165. https://doi.org/10.1002/jssc.201800581
There are many 'specialty HPLC columns' that can perform resolution/separation and quantitation of short chain fatty acids depending on the sample matrix. See the websites for Agilent.com , Waters.com , Phenomenex.com, Restek.com ...
Hello Gomaa A. Aboellail,
in addition to the procedure suggested by Mohammed Gamal, it may be useful to derivatize the fatty acids as they are not detectable in small concentrations by DAD or UV detector. One possibility is the derivatization with brominated aromatics, which show good UV adsorption, e.g. bromoacetophenone.
Many greetings
Joachim Horst
please follow this article
Kim HJ, Lee MJ, Kim HJ and Cho SK., Development of HPLC-UV method for detection and
quantification of eight organic acids in animal feed, Journal of Chromatography
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