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Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

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Published on: April 26, 2016

Capillary liquid chromatography fraction collection and postcolumn reaction using segmented flow microfluidics.

Jing Nie¹, Robert T Kennedy

¹Department of Chemistry, University of Michigan, Ann Arbor, MI, USA.

Journal of Separation Science

|September 17, 2013

Summary

This study introduces a segmented flow system for collecting protein fractions from capillary liquid chromatography (cLC). This droplet technology enables efficient fraction collection and post-column derivatization for fluorescence detection.

Keywords:

Capillary liquid chromatography Postcolumn derivatization Protein separation Segmented flow microfluidics

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Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

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Curtain Flow Column: Optimization of Efficiency and Sensitivity

Curtain Flow Column: Optimization of Efficiency and Sensitivity

Published on: June 12, 2016

Area of Science:

Analytical Chemistry
Biochemistry
Chromatography

Background:

Fraction collection and manipulation from microscale columns in capillary liquid chromatography (cLC) presents significant challenges.
Segmented flow or droplet technology offers a promising solution for automated microscale sample handling.

Purpose of the Study:

To develop and evaluate a segmented flow system for fraction collection and post-column reaction in gradient cLC of proteins.
To demonstrate the feasibility of this approach for protein analysis with fluorescence detection.

Main Methods:

A system was designed where column effluent and immiscible oil were combined to create regularly segmented fractions.
Fractions were generated at 1 Hz (5 nL volumes), ensuring high throughput and preservation of chromatographic resolution.
A post-column reaction system was integrated using naphthalene dicarboxyaldehyde for fluorogenic derivatization of proteins within droplets.

Main Results:

The segmented flow system successfully collected over 30 fractions per peak for a five-protein mixture, maintaining chromatographic resolution.
Proteins were effectively derivatized within the oil-segmented droplets, enabling sensitive fluorescence detection.
The system demonstrated robust performance for gradient cLC of proteins.

Conclusions:

Segmented flow technology is a viable method for fraction collection in capillary LC, extending its application to protein analysis.
This approach facilitates automated sample handling and post-column derivatization, paving the way for streamlined protein analysis workflows.