Evaporative membrane modulation for comprehensive two-dimensional liquid chromatography
Graphical abstract
Introduction
Successful separation of complex biological and environmental samples often requires very high peak capacity, and cannot be achieved in a reasonable time using one-dimensional separation approaches. Peak capacity (nC) is related to resolution, and approximates the maximum number of peaks that can be separated under given chromatography conditions [1]. Multi-dimensional separation approaches often provide more effective separation of multicomponent samples that exceed the peak capacity of single dimension systems.
Comprehensive two-dimensional liquid chromatography (LC × LC) commonly involves the use of a switching valve with two identical sample loops. These sample loops act as alternating collection and injection loops to systematically transfer segments of the first-dimension (1D) separation column effluent to the second-dimension (2D) separation column. This methodology is referred to as passive modulation. Passive modulation has a number of limitations. First, each solute band eluted from 1D should be sampled at least three times by 2D to avoid remixing of separated solutes (undersampling), and reduced two-dimensional resolution [2]. Second, to minimise band broadening and band distortion, the smallest possible volume should be injected onto the second dimension column. Third, if the 1D mobile phase has higher elution strength than the 2D mobile phase the above stated effects are worsened [3], [4], [5]. Fourth, the large number of fast and large pressure pulses when the interface valve switches can affect the second dimension column lifetime [6]. Fifth, dilution of the analytes occurs through collection and transfer of consecutive fractions, hindering detection of low abundance analytes or forcing the user to overload the 1D column [7]. As a result of all these issues, method development is often complex. Selection of chromatographic columns and separation conditions is crucial. Micro/nano flow rates and narrow columns in 1D, and larger-bore columns for 2D are often used in an attempt to fulfil all the mentioned requirements [8], [9].
Active modulation can alleviate some of complications associated with passive modulation by modifying the volume, concentration, and/or solvent of 1D segments before they are introduced into the 2D column. Ideal active modulation is achieved when the volume of collected segments is sufficiently reduced without analyte loss such that the 2D band broadening is not significant [10], [11]. The most common active modulation approach in LC × LC is stationary-phase-assisted modulation where analytes are focused by being trapped in a packed loop interface and subsequently released [10]. However, the efficiency may slowly decrease during analysis due to lack of trap column re-equilibration in-between each 1D segment. Retention mismatch between trap and separation columns and solvent systems must be avoided and further optimization might be required [12].
The use of counter gradients added to the 1D effluent can be used to maintain a constant solvent composition and lower the solvent strength [13] to produce more reproducible baselines and separations. However, further dilution then occurs in addition to the intrinsic dilution of multidimensional LC separations. Post-1D flow splitting reduces the volume of the 1D segments as well as the amount of organic solvent that may disrupt the 2D separation [14] but the mass of low-abundance analytes is reduced further making them difficult to detect.
Thermal modulation is the main active modulation approach used to practice comprehensive two-dimensional gas chromatography, and it has also been employed in LC × LC. Here, analytes are retained on a highly retentive porous graphitic carbon packed column positioned between the 1D and 2D columns, then remobilised applying temperature ramps [15], [16]. Temperature has also been used for peak focusing before detection in a number of forms [17], [18], while a combination of solid-phase and thermal peak focusing was recently studied demonstrating great potential [19].
In-loop direct evaporation was introduced aiming to address solvent mismatch and loss of sensitivity for heart-cutting [20], [21] and LC × LC [22]. The system featured heated collection loops connected to vacuum. The interface achieved solvent evaporation as fast as the collection time, keeping the solutes in a few microliters solution that was then transferred to the second dimension column. The recovery of analytes strongly depended on their boiling point, varying from 15 to over 100%. Although some peak broadening was visible due to the vacuum-loop interface, the results showed an enhancement in chromatographic performance for solvent strength mismatch systems. The approach for LC × LC demonstrated remarkable improvement on baseline in the second dimension and increased retention for weakly retained analytes, but it also showed recoveries under 60% even for non-volatile analytes.
A combination of stationary phase assisted and evaporation assisted modulation mechanisms was recently used to exchange solvents effectively coupling normal and reversed phase separations [23]. However, featuring two sets of loop enrichment units, the elution and regeneration times limited the separation speed.
In this work, we utilise evaporation through a porous hydrophobic membrane as an alternative to direct loop evaporation with substantially higher evaporation control. In membrane evaporation systems, the solvent is removed by evaporation through a gas permeable hydrophobic membrane [24], [25], [26], [27], [28], [29], [30]. To implement an on-line evaporative concentrator as a modulator in LC × LC it is necessary to precisely control temperature to ensure constant evaporation rate and therefore maintain constant flow rate despite the changing amounts of organic solvent. This was addressed by monitoring the 1D outlet flow rate, using an interactive feedback control mechanism run by a microcontroller to adjust the intensity of a heating element such that the output flow matched the desired flow rate. The feedback control mechanism implemented was the extensively used PID, or proportional-integral-derivative control [31]. Controlling then the evaporation rate automatically, we developed and built an on-line evaporative membrane modulator that allows successful coupling of LC × LC. The benefits of this evaporative interfacing are demonstrated with the separation of phenolic acids.
Section snippets
Materials
Analytical Reagent grade gallic acid, 4-hydroxybenzoic acid, syringic acid, vanillic acid, and 98% formic acid were purchased from Sigma-Aldrich (St Louis MO, USA). Solutions were prepared in Milli-Q water from a Milli-Q Plus Ultrapure Water System (Millipore, Bedford, MA, USA). Acetic acid 100% (Merck KGaA, Darmstad, Germany) and ammonia solution 28% (Univar, Seven Hills NSW Australia) were used to prepare pH 4.3 ammonium acetate solution. HPLC grade methanol (VWS Chemicals,
Results and discussion
On-line evaporative membrane concentration benefits from the use of a closed interface to preserve the integrity of the target solution: preventing contamination and minimizing loss of analytes. It can be carried out at mild temperatures to avoid degradation and has been proven to be reproducible for diverse analytes [33]. In this work, the concept of evaporative concentration was adapted to LC × LC interfacing since solvent removal and analyte concentration is considered beneficial for active
Conclusions
Evaporative Membrane Modulation (EMM) was implemented in an LC × LC system by monitoring the 1D outlet flow rate and automatically adjusting the heating intensity to keep evaporation constant.
Event though there is a loss of peak capacity in the first dimension, the introduction of the evaporative interface preserves peak capacity of the LC × LC separation, while improving peak identification by eliminating the interference of 1D solvent in the 2D separation system. This provides a wider choice
Acknowledgments
This research was conducted by the ARC Training Centre for Portable Analytical Separation Technologies (IC140100022). MCB is the recipient of an ARC Future Fellowship. E. Fornells is a recipient of an ARC post-graduate scholarship in ASTech, University of Tasmania.
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