6-25: Improvement in HPLC separation of acetic acid and levulinic acid in the profiling of biomass hydrolysate

HPLC with an Aminex HPX-87H column chromatography has been a standard method for quantitative analysis of biomass hydrolysate and their degradation compounds. Carbohydrates, organic acids, furan derivatives and alcohols are leading compounds in lignocellulosic bioconversion process. 5-hydroxymethylfurfural (HMF) and furfural could be separated by the Aminex HPX-87H column chromatography, however, the separation and quantification of acetic acid and levulinic acid in biomass hydrolysate has been difficult with this method. In present study, the HPLC separation of acetic acid and levulinic acid on Aminex HPX-87H column has been investigated by varying column temperature, flow rate, and sulfuric acid content in the mobile phase.

The column temperature was found critical both in resolving acetic acid and levulinic acid and in determining the retention times for acetic acid, levulinic acid, HMF and furfural. The resolution for acetic acid and levulinic acid increased dramatically from 0.42 to 1.86 when the column temperature was lowered from 60°C to 30°C. So did the capacity factors for levulinic acid that was increased from 1.20 to 1.44 as the column temperature dropped. The optimum column temperature for the separation was found at 45°C.  Variation in flow rate and sulfuric acid concentration improved not as much as the temperature does. A change to traditional separation of acetic acid and levulinic acid in biomass hydrolysate was made that increased the resolution significantly. The Aminex HPX-87 H column at 45°C should be used in the HPLC of biomass degradation compounds.