Extending the Usefulness of HPLC with Electrochemical Detection

Significance of the Topic

HPLC with electrochemical detection (HPLC-ECD) offers femtogram-level sensitivity but is limited to electroactive compounds.
Extending its applicability broadens its use in pharmaceuticals, neurochemistry, explosives analysis and environmental monitoring.

Objectives and Study Overview

This study examines four strategies to broaden the utility of HPLC-ECD:

• Pre-column derivatization of analytes
• On-line immobilized enzyme reactors
• Post-column photolysis
• Novel boron-doped diamond (BDD) working electrodes

Methodology and Instrumentation Used

The four approaches were implemented on a Thermo Scientific Dionex HPLC system coupled to Coulochem electrochemical detectors.

• Derivatization: OPA/β-ME reaction on autosampler prior to separation on Accucore PhenylHexyl column
• Enzyme reactor: Acetylcholinesterase and choline oxidase immobilized in SPR module, detection via Pt electrode
• Photolysis: PhredTM UV reactor (254 nm) post-column before CoulArray detection on BetaSil C18 column
• BDD electrode: Gradient separation on Restek Pinnacle C18 with in situ electrotagging and detection at high potentials

Main Results and Discussion

• Derivatization produced stable isoindole derivatives of amino acids, enabling femtomole-level detection and separation of 11 neuroactive amino acids.
• Immobilized enzymes converted acetylcholine to H2O2, achieving sensitive on-line measurement of low nanomolar ACh from microdialysis samples.
• Photolytic activation of explosive residues increased electrochemical signals by 5–100×, lowering limits of detection to tens of picograms on column.
• BDD electrodes extended the potential window to 2.5 V, generated hydroxyl radicals for electrotagging, and detected polyaromatic hydrocarbons at sub-nanogram levels.

Benefits and Practical Applications

Extended HPLC-ECD methods provide enhanced selectivity, sensitivity and scope, enabling:

• Trace analysis of neurotransmitters and amino acids in biomedical research
• Environmental monitoring of explosives and persistent organic pollutants
• Pharmaceutical impurity profiling including genotoxins at ppb levels
• Quality control assays for thiols, disulfides and polyaromatic compounds

Future Trends and Applications

Continued innovation may include automated on-line derivatization, integration of microfluidic enzyme reactors, advanced photochemical interfaces and combined sensors with BDD electrodes.
Emerging fields such as metabolomics, real-time field analysis and artificial intelligence–driven method optimization are poised to benefit.

Conclusion

The four enhancement strategies demonstrate that derivatization, bioreactors, photolysis and BDD electrodes complement conventional HPLC-ECD.
Selection of the optimal approach depends on analyte chemistry, required sensitivity and application context.

References

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2. Thermo Fisher Scientific. Analysis of Neuroactive Amino Acids Using UHPLC and Electrochemical Detection LPN 2423-01; 2010.
3. Girelli AM, Mattei E. Application of immobilized enzyme reactor in on-line high performance liquid chromatography: a review. J Chromatogr B. 2005;819:3–16.
4. Fedorowski J, LaCourse WR. A review of post-column photochemical reaction systems coupled to electrochemical detection in HPLC. Anal Chim Acta. 2010;657:1–8.
5. Granger M. Personal communication. University of Utah; 2011.
6. Plante M, Bailey B, Acworth IN. Determination of Genotoxic Besylates and Tosylates by HPLC-ECD Using A Boron-Doped Diamond Electrode. LCGC Applications Notebook. 2010.