Determination of Glycols and Alcohols in Fermentation Broths Using Ion-Exclusion Chromatography and Pulsed Amperometric Detection

Importance of the Topic

The accurate measurement of glycols and alcohols in fermentation broths and beverage samples is critical for process control, quality assurance and product safety in biotechnology, brewing and pharmaceutical industries. Monitoring these neutral, low–molecular-weight compounds provides insights into microbial metabolism, fermentation yield, and flavor profiles, enabling optimization of culture conditions and ensuring regulatory compliance.

Aims and Study Overview

This study demonstrates a robust analytical approach using ion-exclusion chromatography coupled with pulsed amperometric detection (PAD) and a disposable platinum electrode for simultaneous quantification of glycols (ethylene glycol, propylene glycol, glycerol) and alcohols (methanol, ethanol, isopropanol) in complex matrices. The work evaluates method linearity, sensitivity, reproducibility, robustness over time and real-sample applicability including fermentation broths and commercial beers and wines.

Methodology and Instrumentation

• Ion-exclusion column: Dionex IonPac ICE-AS1 (4×250 mm) at 30°C with 100 mM methanesulfonic acid eluent, flow 0.2 mL/min.
• Pulsed amperometric detection: three-step waveform (+0.30 V, +1.15 V, –0.30 V vs Ag/AgCl), disposable platinum working electrode, pH-Ag/AgCl reference.
• System: Thermo Scientific Dionex ICS-3000 RFIC with autosampler, degassed Type I water, helium blanket for eluent, Chromeleon software for data acquisition and pH monitoring via virtual channel.

Main Findings and Discussion

The method exhibited high sensitivity with detection limits of 1.6–4.5 μM for target analytes. Calibration curves were linear or quadratic over 25–6400 μM (r2 >0.999). Noise remained below 10 pC, and disposable electrodes stabilized within 1 hour, maintaining >80% response over two weeks. Retention times for glycerol, propylene glycol and ethanol were stable over 250 injections (RSD <0.4%), with peak area drifts under 5% over 125 hours, recoverable after eluent refresh. Alditols and high-molecular carbohydrates eluted in exclusion volume while glycols and alcohols resolved by steric partition. Ethylene and propylene glycol showed a 0.4-minute separation, enabling quantification despite bimodal peaks.

In yeast fermentation broths (Saccharomyces cerevisiae), ethanol profiles rose exponentially, and glycerol emergence was detected after 20–24 h of incubation. Spike recovery in 300-fold diluted broths yielded 95–101% for glycerol, propylene glycol and ethanol. In commercial beverage samples (beer and wine), 400-fold dilutions produced recoveries between 99% and 101% across analytes.

Benefits and Practical Applications

• Direct determination of glycols and alcohols without derivatization or complex sample cleanup.
• Disposable electrodes reduce maintenance and extend throughput.
• Regulatory compliance: avoids perchloric acid eluents by using methanesulfonic acid.
• Real-time pH monitoring via virtual channels enhances data quality.
• Applicable to fermentation process monitoring, beverage quality control, and pharmaceutical production.

Future Trends and Applications

Integration of fully automated reagent-free IC systems with inline sampling and data analytics will support continuous monitoring of fermentation and bioprocessing. Advances in electrode materials and waveform optimization may further lower detection limits and broaden the range of detectable neutral compounds. Combining IC-PAD with high-resolution mass spectrometry could enable rapid identification of novel metabolic by-products and contaminants.

Conclusion

This application note validates ion-exclusion chromatography with pulsed amperometric detection on a disposable platinum electrode as a sensitive, reproducible, and maintenance-friendly technique for quantifying glycols and alcohols in complex fermentation and beverage matrices. The method’s robustness, high recoveries and regulatory compliance make it a valuable tool for industrial and research laboratories.

References

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6. Thermo Scientific Application Note 123. Determination of Inorganic Anions and Organic Acids in Fermentation Broths.
7. Thermo Scientific Application Note 150. Determination of Amino Acids in Cell Cultures and Fermentation Broths.
8. Thermo Scientific Application Note 117. Quantification of Carbohydrates and Glycols in Pharmaceuticals.