40 February / March 2021
Faster Time to Results for Ultra-Performance Liquid Chromatographic Separations of Metal-Sensitive Analytes
Thomas H. Walter, Brian J. Murphy, Moon Chul Jung†, Martin Gilar, Robert E. Birdsall and Jacob Kellett, Waters Corporation 34 Maple Street, Milford, MA 01757
† Current address: Enanta Pharmaceuticals Inc, 500 Arsenal Street, Watertown, MA Abstract
Many practitioners of HPLC think of analytical speed only in terms of the time required to carry out the separation. However, when separating analytes that interact with the metal surfaces in HPLC instruments and columns, analytical speed may be negatively impacted by the time required for conditioning. This is the case for important classes of analytes such as oligonucleotides, acidic peptides and anionic polar metabolites. In a recent advance, an innovative technology has been developed to mitigate interactions with the metal components of HPLC systems and columns, reducing the time needed to obtain accurate, reproducible results for metal-sensitive analytes.
Introduction
Analytical scientists are constantly under pressure to produce results quickly. In seeking out more efficient ways to accomplish their tasks, separation scientists look at all aspects of their instrumentation and methods. In 2004, the introduction of Ultra-Performance Liquid Chromatography (UPLC™) improved the speed with which analytical scientists could carry out LC separations [1 - 3]. The combination of columns packed with < 2 µm particles and low-dispersion high-pressure tolerant instrumentation greatly reduced separation times.
However, for some applications, the analysis time is negatively impacted by the need for extensive conditioning of the LC system and column before accurate and reproducible results can be obtained. One of the root causes of the need for conditioning is
the interaction between certain analytes and the metal surfaces of HPLC systems and columns. It has long been known that stainless steel hardware can cause poor peak shapes and low recoveries for some analytes [4 - 7]. Compounds that show this behaviour typically contain phosphate and/ or carboxylate groups, although some analytes with other electron-rich functional groups have been reported to show similar issues [4]. The severity of adsorption has been found to increase with the number of these functional groups in the analyte [6, 7]. Adsorption becomes particularly problematic when the stainless steel is corroded [8], which can occur due to exposure to mobile phases that are highly acidic and/or contain chloride salts, among other conditions [9].
One approach to mitigate these issues is to use alternative metals such as titanium or nickel-cobalt alloys (e.g. MP35N) for
Figure 1. A MaxPeak High Performance Surface impedes interactions of electron-rich analytes with metallic hardware.
components in the flow path of the HPLC system [10]. While these alternatives exhibit improved corrosion resistance, making them useful for applications that require mobile phases with high salt concentrations, they still may adsorb certain analytes [11]. To avoid metal surfaces altogether, organic polymers such as polyether ether ketone (PEEK) have been used in HPLC systems and columns. However, this engineering plastic lacks the mechanical strength necessary to
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