Determination of dihydroxyacetone and glycerol in fermentation broth by pyrolytic methylation/gas chromatography

A method to determine dihydroxyacetone (DHA) and glycerol in fermentation broth was developed on the basis of pyrolytic methylation–gas chromatography (GC) using a vertical microfurnace pyrolyzer. With the on-line pyrolytic methylation in the presence of an organic alkali, tetramethylammonium hydroxide ((CH3)4NOH, TMAH), DHA and glycerol were converted into their corresponding methyl ethers. Thus, fermentation broth samples containing DHA and glycerol could be well determined simultaneously by this pyrolytic methylation–GC without using any pretreatment procedures. Important factors such as reaction temperature, the amount of TMAH to DHA and glycerol were investigated in order to obtain the optimum methylation conditions. Thus, obtained results showed that 0.4 μl 25% TMAH aqueous solution at 400 °C enabled the highly sensitive determination of DHA and glycerol contained in 0.2 μl aqueous samples. The achieved calibration curves exhibit good linearity with regression coefficients of 0.996 and 0.998 for DHA and gelycerol at the concentration range from 1 to 100 g/l, respectively. The precision was quite high with the R.S.D. within 5.0% for DHA at 30 g/l and glycerol at 15 g/l concentration levels, and the limit of detection reached 0.016 g/l. The potential of the proposed method was assessed by applying it to determination of both the analytes in fermentation broth during the bioconversion of glycerol to DHA by Acetobacteria sp. The results proved that this on-line pyrolytic methylation–GC technique was a rapid, convenient and highly sensitive method for monitoring the bioprocesses of DHA.