Separation of acetone and butyric acid for simultaneous analysis of sugars, volatile fatty acids, acetone and alcohols by HPLC using flow programming

Abstract

This article deals with developing an efficient and economical method to quantify the major products of ABE fermentation (sugars, volatile fatty acids, acetone, ethanol and butanol) using HPLC. Other techniques based on a similar principle have poor resolution between peaks of butyric acid, acetone and ethanol. The advantage of our method was that the overlapping peaks of butyric acid, acetone and ethanol were separated and thus quantified simultaneously during analysis. Also, flow programming was used to reduce the analysis time, thereby saving energy. The maximum separation of all components was achieved using a water–sulfuric acid mixture (5 mM H₂SO₄ in water) as the mobile phase using flow programming with increasing flow rate from 0.6 ml to 0.9 ml within 21 min during analysis with a total run time of 26 min at a column temperature of 80 °C. A linear relationship over the analytical range of 10.0 to 0.01 g l⁻¹ was observed. The robustness of the developed method was evaluated using parameters such as the capacity factor, selectivity, plate number and resolution. The applicability of this approach was validated by quantifying the substrates and products of ABE fermentation using lignocellulosic waste hydrolysate as a carbon source. The results of analysis proved that the method is reproducible, accurate, precise, and sensitive.