Transferring a Method for Analysis of DNPH-Derivatized Aldehydes and Ketones from HPLC to UHPLC

Significance of the Topic

Aldehydes and ketones are ubiquitous carbonyl pollutants originating from natural and industrial sources and pose significant health risks, including respiratory irritation and carcinogenic effects. Reliable quantification of these compounds is vital for environmental monitoring and public health protection.

Objectives and Study Overview

To demonstrate the transfer of a conventional HPLC assay for 13 DNPH-derivatized aldehydes and ketones to UHPLC using Agilent 1290 Infinity II LC, aiming to drastically reduce analysis time and solvent usage without sacrificing chromatographic performance.

Methodology

The study employed DNPH derivatization followed by reversed-phase liquid chromatography. A standard HPLC method using a 4.6×150 mm, 5 µm C18 column with a water–acetone gradient was compared against UHPLC methods with a 2.1×50 mm, 1.8 µm column at flow rates of 0.25 mL/min and 1.25 mL/min. Chromatographic conditions were optimized for speed, maintaining detection at 360 nm and column temperature at 45 °C.

Used Instrumentation

• Agilent 1290 Infinity II High-Speed Pump (G7120A)
• Agilent 1290 Infinity II Multisampler (G7167B)
• Agilent 1290 Infinity II Multicolumn Thermostat (G7116B)
• Agilent 1290 Infinity II Diode Array Detector (G7117B) with 10 mm cell
• Agilent OpenLab CDS ChemStation Edition C.01.07

Main Results and Discussion

Transitioning to a 1.8 µm UHPLC column reduced run time by 60 % (9 min) and solvent consumption by over 60 %, while the accelerated 1.25 mL/min method cut analysis time by 90 % (<2.5 min). Both UHPLC approaches maintained retention time RSDs below 0.15 %, area RSDs below 0.65 %, and resolution comparable to HPLC. Correlation coefficients exceeded 0.9998, and LOD/LOQ improved markedly under UHPLC conditions.

Benefits and Practical Applications

• High-throughput environmental monitoring with rapid sample turnaround
• Reduced solvent costs and waste generation
• Enhanced sensitivity for trace-level quantitation
• Compatibility with existing HPLC methods for seamless method transfer

Future Trends and Potential Applications

Further miniaturization and increased pressure capabilities may enable sub-minute separations and integration with on-line mass spectrometry for comprehensive speciation. Portable UHPLC platforms could facilitate field-based air quality assessments and real-time monitoring.

Conclusion

The UHPLC method transfer demonstrated significant reductions in analysis time and solvent consumption without compromising precision, linearity, or detection limits, highlighting the advantages of sub-2 µm column technology for environmental carbonyl analysis.

Reference

1. Levart A, Veber M. Determination of aldehydes and ketones in air samples using cryotrapping sampling. Chemosphere. 2001;44(4):701–708.
2. Uchiyama S, Inaba Y, Kunugita N. Derivatization of carbonyl compounds with DNPH and their determination by HPLC. J Chromatogr B. 2011;879(17-18):1282–1289.
3. Zwiener C, Glauner T, Frimmel FH. Optimization for carbonyl determination in disinfected water by DNPH derivatization and LC-ESI-MS/MS. Anal Bioanal Chem. 2002;372(5-6):615–621.