Curtain Flow Column: Optimization of Efficiency and Sensitivity

Summary

Here, we present a protocol for the operation and optimization of Active Flow Technology (AFT) column in Curtain flow (CF) mode for enhanced separation performance.

Abstract

Active Flow Technology (AFT) is a form of column technology that increases the separation performance of a HPLC column through the use of a specially purpose built multiport end-fitting(s). Curtain Flow (CF) columns belong to the AFT suite of columns, specifically the CF column is designed so that the sample is injected into the radial central region of the bed and a curtain flow of mobile phase surrounding the injection of solute prevents the radial dispersion of the sample to the wall. The column functions as an 'infinite diameter' column. The purpose of the design is to overcome the radial heterogeneity of the column bed, and at the same time maximize the sample load into the radial central region of the column bed, which serves to increase detection sensitivity. The protocol described herein outlines the system and CF column set up and the tuning process for an optimized infinite diameter 'virtual' column.

Introduction

In recent years column technology for High Performance Liquid Chromatography (HPLC) has advanced greatly; peak capacities have increased considerably thanks largely to the use of smaller particle sizes and the more efficient core shell particles. Since separations are generally more efficient, a flow-on effect has been an increase in sensitivity since peaks are now sharper and hence taller^1-8.

Nevertheless, radial bed heterogeneity is still a limiting factor in the performance of all columns, but this is not a new story since chromatographers have known this for many years. Column beds are heterogeneous in both the radial directi....

Protocol

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 instrumentation, and ensure the use of personal protective equipment (safety glasses, gloves, lab coat, full length pants, and closed-toe shoes).

Note: This protocol contains instructions on how to use a CF column on a HPLC system coupled with a UV-Vis detector. The protocol has been writt....

Results

AFT columns were developed using a specialized frit design (Figure 1) in the multiport column end-fittings to overcome the column bed heterogeneity and improve separation performance. An inter-laboratory study on the separation performance of CF chromatography columns (Figure 2) was carried out with a dual pump system set up (Figure 3B) as described in section 3 of this protocol²³. A three component test mixture was analyzed un.......

Discussion

This study involved the inter-laboratory analysis of CF chromatography columns to test the analytical performance in terms of efficiency and sensitivity. The CF column was set up with a dual pumping system as described in section '3. Dual pump system set up' to achieve a flow ratio of 40:60 (center:peripheral) on the inlet of the CF column. The 40:60 (center:peripheral) flow ratio was achieved by setting the flow rate of each pump to the value that represents 40% and 60% of the total flow rate, respectively. The .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HPLC instrument
Additional Pump			Required if 2 pump CF system set up is to be used.
Curtain 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.
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
T-piece	Any brand