Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection

Summary

Here, we present a protocol for the operation and tuning of parallel segmented flow chromatography columns to enable multiplexed detection.

Abstract

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms of efficiency and sensitivity and to enable multiplexed detection. This form of AFT uses a parallel segmented flow (PSF) column. A PSF column outlet end-fitting consists of 2 or 4 ports, which can be multiplexed to connect up to 4 detectors. The PSF column not only allows a platform for multiplexed detection but also the combination of both destructive and non-destructive detectors, without additional dead volume tubing, simultaneously. The amount of flow through each port can also be adjusted through pressure management to suit the requirements of a specific detector(s). To achieve multiplexed detection using a PSF column there are a number of parameters which can be controlled to ensure optimal separation performance and quality of results; that is tube dimensions for each port, choice of port for each type of detector and flow adjustment. This protocol is intended to show how to use and tune a PSF column functioning in a multiplexed mode of detection.

Introduction

Active Flow Technology Columns

Active flow technology (AFT) chromatography columns were recently developed to overcome inefficiency in separations associated with flow heterogeneity^1-6 and also to enable multiplexed detection. In this particular communication we detail the operational process of the parallel segmented flow (PSF) column with multiplexed detection. The key functional advantages of the PSF column are: (1) the flow from the radial central region of the column bed is isolated from the peripheral or wall region of flow, (2) the volume of mobile phase that must be processed by a detection source is redu....

Protocol

Note: This protocol contains instructions on how to use a PSF column on a HPLC system coupled with multiple detectors for multiplexed detection. The protocol has been written assuming the reader has basic knowledge and experience in chromatography and various HPLC detection methods.

Caution: Please refer to material safety data sheets (MSDS) for all materials and reagents before use (i.e., MSDS for methanol). Ensure the use of all appropriate safety practices when handling solvents and High Performance Liquid Chromatography (HPLC) eluent. Ensure appropriate use of engineering controls of HPLC, analytical balance and detector instru....

Results

A multiplexed HPLC analysis was carried out using an AFT column in PSF mode (Figure 1) and set up as illustrated in Figure 2. This type of setup allowed a coffee sample to be analyzed simultaneously using UV-Vis, DPPH^• and MS in Total Ion Count (TIC) mode. The compounds of the coffee sample that responded to DPPH^• could then be easily matched up to the UV-Vis and MS - TIC responses based on the alignment of retention time as illustrated in Figure 3,.......

Discussion

This study involves the characterization and profiling of coffee using HPLC with multiplexed detection employing a parallel segmented flow (PSF) column. Multiplexed HPLC using PSF columns enables the characterization and identification of key chemical entities by reducing the data complexity of the sample whilst obtaining a greater degree of molecule-specific information within a fraction of the time it takes using conventional multi-detection processes. The PSF column not only allows a platform for multiplexed detection.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HPLC instrument			Multiple detectors of choice for multiplexed detection. Detectors of choice may require additional instrumentation, e.g., pump.
Parallel Segmented Flow HPLC column	Thermo Fisher Scientific	Not Defined	Soon to be commercialized
Methanol	Any brand		HPLC Grade
PEEK tubing	Any brand		Various lengths and i.d.
Column stoppers	Any brand		For blocking unused peripheral ports.
PEEK tube cutter	Any brand
Analytical Scale Balance	Any brand
Stop watch	Any brand
Eluent collection vessels	Any brand		1-2 ml sample vials can be used as eluent collection vessels