Evaluating MISER chromatography as a tool for characterizing HILIC column equilibration
Introduction
Hydrophilic Interaction Chromatography (HILIC) is an analytical technique used to separate polar analytes. Since its naming by Alpert in 1990 [1], HILIC has grown in popularity in many fields of study including metabolomics, biological research, food and environmental testing, and pharmaceutical analyses, with the number of published papers almost doubling in the last ten years [2]. Articles covering method development for challenging polar compounds as well as investigations of the retention mechanisms in HILIC have been published [3], [4]. The advantages of HILIC compared to reversed phase chromatography include the ability to retain highly polar analytes, different selectivity, greater sensitivity for mass spectrometry detection and lower operating pressures. One of the disadvantages of this technique is the need for greater equilibration volumes. While reversed-phase columns typically equilibrate in less than 10 column volumes (VC) [5], HILIC columns can require 20 VC or more to achieve full equilibration [6], [7]. While the notion of equilibration typically applies to gradient runs, wherein the analyst is trying to bring the column back to starting conditions as fast as possible, the same mechanisms are involved for isocratic analyses. The difference for isocratic analyses is that equilibration only takes place when transitioning from storage conditions to analysis conditions. Similar to gradient runs, reduced equilibration times would lead to an overall increase in throughput.
The equilibration of HIILC columns has been studied by several scientists including McCalley [6], Heaton [7], Rappold [8], and Shollenberger [9]. Rappold found that for targeted metabolomics using gradient elution, retention and peak shape of analytes was dependent on the amount of re-equilibration between gradient injections [8]. Peak shapes would deteriorate at low equilibration volumes, leading to poor integration and quantitation. Additionally, retention times would shift if different equilibration volumes were used. The work also indicated that when controlling equilibration between gradients, stable retention times are possible, even if full equilibration was not achieved. McCalley investigated factors that impact HILIC equilibration, including flow rate, the storage conditions of the column, and the presence of buffer in the storage solvent. The water content of the storage solvent was shown to have a strong influence on the speed of equilibration, while the presence or absence of buffer in the storage solvent was of little consequence for isocratic separations [6]. Heaton found that for their assay the selected column required 40 VC or more to achieve full equilibration, but the volume could be reduced by partially equilibrating the column [7]. This made re-equilibrating the column between gradient runs considerably faster without sacrificing retention reproducibility. Lastly, Shollenberger found that the presence of ion-exchange mechanisms slows equilibration for both isocratic and gradient runs [9].
HILIC equilibration is linked to the formation of an adsorbed aqueous layer on the surface of the stationary phase [10], [11]. This requires the bulk mobile phase to penetrate the pores of the particle and stabilize in the surface layer. Understanding the factors that affect this mechanism is critical to being able to reduce equilibration times and ultimately speed up HILIC analyses. Factors like the water content of the storage solvent and mobile phase, flow rate, and stationary phase all impact the speed at which the column equilibrates [6]. Accurate characterization of the equilibration of a column under differing conditions is crucial to understand how to reduce the equilibration needs of a column. Unfortunately, current methods for characterizing HILIC equilibration are limited. Conventional methods include flushing a column with a storage solvent and then injecting a test sample in replicate to track retention time as the column equilibrates to assay conditions. When the retention times match a target value, or stabilize, the column is considered equilibrated. While a good starting point, this approach has several limitations and an improved method of characterizing HILIC equilibration is needed.
MISER, or multiple injections in a single experimental run, is a technique for high-throughput analyses [12]. It has been utilized for applications including reaction monitoring [13], sample screening [14] and scouting ideal reaction conditions [15]. The advantage of MISER is that many injections may be made while the separation is occurring, without waiting for the initial separation to be completed. When applied to studies of column equilibration, this allows for considerably more data points per test than conventional techniques. The work presented here shows the limitations of the conventional method for characterizing equilibration, the application of MISER to study column equilibration, and comparisons to the conventional method. We also used the MISER method to characterize the equilibration under different mobile phase conditions for an ACQUITY UPLC BEH Amide column, a widely-used HILIC column that has been shown to have moderate equilibration needs.6
Section snippets
Chemicals
LC-MS grade Acetonitrile and MS grade formic acid were purchased from Fisher Scientific (Hampton, NH). Ammonium formate (≥99.995% trace metals basis), nortriptyline, maleic acid, uridine, and bretylium tosylate were purchased from Millipore-Sigma (Burlington, MA). Deionized water was produced using a Millipore Milli-Q system.
Instrumentation and columns
Tests were performed on an ACQUITY UPLC H-Class instrument from Waters Corporation (Milford, MA) consisting of a quaternary solvent manager (QSM), a sample manager with a
Choice of test probes and test descriptors
Evaluating HILIC column equilibration requires a test mixture containing a range of probes, ideally including neutral, acidic and basic compounds. This provides information on the behavior of each type of probe. As demonstrated by Shollenberger [9], the presence of ionic interactions can slow column equilibration. In the first experiment, three probes were chosen: uridine (a neutral nucleoside), bretylium (quaternary amine), and tosylate (sulfonic acid). Both bretylium and tosylate are charged
Conclusions
MISER was shown to be a useful tool for evaluating HILIC column equilibration, mitigating the limitations of the conventional technique, most notably the poor time resolution. A five-fold increase in time resolution was demonstrated and further increases may be possible. MISER was demonstrated to give reproducible equilibration volumes, with relative standard deviations ≤6%. The technique was used to characterize the equilibration of an ACQUITY BEH Amide column from acetonitrile to three
CRediT authorship contribution statement
Kenneth D. Berthelette: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing - original draft, Writing - review & editing, Visualization. Thomas H. Walter: Conceptualization, Methodology, Formal analysis, Resources, Writing - review & editing, Visualization, Supervision. Martin Gilar: Conceptualization, Methodology, Formal analysis, Writing - review & editing, Visualization. Fabrice Gritti: Conceptualization, Methodology, Formal analysis, Writing -
Declaration of Competing Interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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