The initial application of a prototype vacuum ultraviolet (VUV) absorption detector system for high performance liquid chromatography (HPLC) was validated and characterized. This detector enabled simultaneous detection and quantification of four organic acids-lactic, acetic, itaconic, and citric along with four carbohydrates-glucose, fructose, galactose, and lactose. This collection of analytes was selected based on their prevalence in fermentations, metabolic production interest, and occurrence in food processes/chemistry. Full spectrum VUV data, collected across 7 discrete wavelength bands centered at 177.3, 190.3, 203.1, 215.8, 228.3, 240.7, and 252.9 nm, yielded unique fingerprints that allowed for separation and quantification of strongly co-eluting peaks, most notably the hexose sugars. Separations were performed on an Aminex HPX-87H ion exclusion column using 5 mM H₂SO₄ as the isocratic mobile phase at 0.6 mL/min, with samples preheated to 40 °C. Across the eight analytes, Hydra limits of detection spanned 0.0228-3.59 g/L, with calibration linearity of R² = 0.941-0.998. The VUV prototype maintained linear response up to 100 g/L for all analytes (60 g/L for itaconic acid), whereas refractive index detection saturated between ∼10-60 g/L depending on the analyte. Spectral deconvolution recovered co-eluting organic acids with ∼1-4% error and the co-eluting hexose cluster within ∼11-19% error, demonstrating practical spectral selectivity for overlapped peaks. This extended dynamic range can eliminate the need for sample dilution thus greatly streamlining workflow processes and minimizing the dilution of trace components. The combination of quantitative accuracy and spectral selectivity was demonstrated though the monitoring of glucose utilization and lactic acid production during a seven-day Saccharomyces cerevisiae fermentation with minimal sample preparation. These results support the potential of LC-VUV detection as a robust, high-throughput method for metabolite analysis in high titer bioprocessing applications. However, a current limitation of the prototype is reduced sensitivity at low concentrations relative to conventional detectors, indicating that present performance is best suited to higher titer samples.

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Journal Article

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Bansal M, Dombrowski A, Nwanebi C, Harrison D, Alper HS

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