Temperature-responsive comprehensive two-dimensional liquid chromatography coupled to high resolution mass spectrometry for the elucidation of the oxidative degradation processes of chemicals of environmental concern.

This study explores the possibilities offered by temperature-responsive liquid chromatography (TRLC) based comprehensive 2-dimensional liquid chromatography in combination with reversed-phase liquid chromatography (RPLC) for the analysis of degradation products formed upon oxidative treatment of persistent organic pollutants, in this case exemplified through carbamazepine (CBZ). The TRLC×RPLC combination offers the possibility to overcome peak overlap and incomplete separation encountered in 1D approaches, while the transfer of the purely aqueous mobile phase leads to refocusing of all analytes on the second dimension column. Consequently, this allows for about method-development free and hence, easier LC×LC. The study focuses on the oxidative degradation of CBZ, a compound of environmental concern due to its persistence in water bodies. The TRLC×RPLC combination effectively separates and identifies CBZ and its degradation products, while offering improved selectivity over the individual TRLC or RPLC separations. This allows gathering more understanding of the degradation cascade and allows real-time monitoring of the appearance and disappearance of various degradation products. The compatibility with high-resolution mass spectrometry is last shown, enabling identification of 21 CBZ-related products, nine of which were not previously reported in CBZ degradation studies. The approach's simplicity, optimization-free aspects, and ease of use make it a promising tool for the analysis of degradation pathways in environmental contaminants.

Facilitating structural elucidation of small environmental solutes in RPLC-HRMS by retention index prediction.

Implementing effective environmental management strategies requires a comprehensive understanding of the chemical composition of environmental pollutants, particularly in complex mixtures. Utilizing innovative analytical techniques, such as high-resolution mass spectrometry and predictive retention index models, can provide valuable insights into the molecular structures of environmental contaminants. Liquid Chromatography-High-Resolution Mass Spectrometry is a powerful tool for the identification of isomeric structures in complex samples. However, there are some limitations that can prevent accurate isomeric structure identification, particularly in cases where the isomers have similar mass and fragmentation patterns. Liquid chromatographic retention, determined by the size, shape, and polarity of the analyte and its interactions with the stationary phase, contains valuable 3D structural information that is vastly underutilized. Therefore, a predictive retention index model is developed which is transferrable to LC-HRMS systems and can assist in the structural elucidation of unknowns. The approach is currently restricted to carbon, hydrogen, and oxygen-based molecules <500 g mol-1. The methodology facilitates the acceptance of accurate structural formulas and the exclusion of erroneous hypothetical structural representations by leveraging retention time estimations, thereby providing a permissible tolerance range for a given elemental composition and experimental retention time. This approach serves as a proof of concept for the development of a Quantitative Structure-Retention Relationship model using a generic gradient LC approach. The use of a widely used reversed-phase (U)HPLC column and a relatively large set of training (101) and test compounds (14) demonstrates the feasibility and potential applicability of this approach for predicting the retention behaviour of compounds in complex mixtures. By providing a standard operating procedure, this approach can be easily replicated and applied to various analytical challenges, further supporting its potential for broader implementation.

Hyphenating temperature gradient elution with refractive index detection through temperature-responsive liquid chromatography.

Refractive index detection (RID) is attractive because it allows approaching the benefits of universal detection with liquid chromatography, by which ideally standard independent calibration and hence compound independent quantification becomes possible. Nevertheless, the implementation of RID has remained limited as it offers poor detection sensitivity while only being compatible with isocratic mobile phases. The implementation of compositional solvent gradients has remained prohibitively challenging in commercial HPLC-RID systems due to the resulting drastic alterations in refractive index and extreme baseline drift. While the refractive index is also highly dependent on temperature, more leeway appears possible to mitigate the problem, particularly when the used temperature gradients can be limited. Temperature-responsive liquid chromatography (TRLC) allows obtaining isocratic reversed phase type of separations, whereby retention is modulated via temperature changes âˆ¼ 15 °C-20 °C above and below the polymer conversion temperature. Elution profiles, reminiscent of what can be obtained with solvent gradients in conventional RPLC, can then be obtained by enacting downwards temperature gradients on the columns. This work comprises a proof-of-principle to illustrate the possibilities of combining thermal gradient TRLC with RID. The observed baseline drift appeared thereby very minor (<5 nRIU min-1), and hence easily controllable. Short chain fatty acids are used as representative compounds to assess this new approach. Overlapping calibration lines are accordingly obtained for all fatty acids between butyric and decanoic acid.

Molecular weight distribution characterization of reactive higher ethyleneamines using size-exclusion chromatography with conventional calibration.

Ethyleneamines have been produced and commercialized for decades in the chemical industry for a diverse range of applications. The presence of amine functional groups provides them opportunity to adsorb onto surfaces which can make them a very challenging sample matrix to analyze using separation techniques. In the present report, a new aqueous SEC-RI method, which enables MWD characterization of higher ethyleneamines, is described. The sample preparation was based on the dilute-and-shoot methodology. A surface-modified SEC column with positively charged groups attached to the stationary phase was used. The mobile phase composition (salt concentration, pH) was optimized to suppress interaction between the ethyleneamines and the packing material. Very symmetrical peak shapes were achieved for low MW monodisperse ethyleneamines despite their high primary amine content. MWD calculations were conducted using conventional narrow standard calibration with partial linear extrapolation of the calibration curve. The narrow standards were of the same chemistry as the samples of interest. Consequently, the standard components display a consistent behaviour towards the column packing as the sample components which makes the present method more robust and the interpretation of the quantitative results more convenient. Effect on the measured MW averages and MW distribution due to various experimental parameters (e.g., system variability, mobile phase preparation, sample concentration) were investigated showing good repeatability (RSD < 2%) for Mn, Mw, and Mz.

Design and performance evaluation of a microfluidic ion-suppression module for anion-exchange chromatography.

A microfluidic membrane suppressor has been constructed to suppress ions of alkaline mobile-phases via an acid-base reaction across a sulfonated poly(tetrafluoroethylene)-based membrane and was evaluated for anion-exchange separations using conductivity detection. The membrane was clamped between two chip substrates, accommodating rectangular microchannels for the eluent and regenerant flow, respectively. Additionally, a clamp-on chip holder has been constructed which allows the alignment and stacking of different chip modules. The response and efficacy of the microfluidic chip suppressor was assessed for a wide range of eluent (KOH) concentrations, using 127 and 183µm thick membranes, while optimizing the flow rate and concentration of the regenerant solution (H2SO4). The optimal operating eluent flow rate was determined at 5µL/min, corresponding to the optimal van-Deemter flow velocity of commercially-available column technology, i.e. a 0.4mm i.d.×250mm long column packed with 7.5µm anion-exchange particles. When equilibrated at 10mM KOH, a 99% decrease in conductivity signal could be obtained within 5min when applying 10mM H2SO4 regenerant at 75µL/min. A background signal as low as 1.2µS/cm was obtained, which equals the performance of a commercially-available electrolytic hollow-fiber suppressor. When increasing the temperature of the membrane suppressor from 15 to 20°C, ion suppression was significantly improved allowing the application of 75mM KOH. The applicability of the chip suppressor has been demonstrated with an isocratic baseline separation of a mixture of seven inorganic ions, yielding plate numbers between 5300 and 10,600 and with a gradient separation of a complex ion mixture.

The axial rearrangement mixer: working principles and in-depth investigation.

In the current paper, an axial rearrangement mixer is studied. The mixer aims to flatten out occasional composition fluctuations originating from the pump. In a first phase, dispersion in a single mixer channel is investigated using pulses introduced by a dedicated injection pulse generator. The mixing in the single channel is studied for different flow rates and a model is presented based on the experimental data. Next, the resulting signal for the whole mixer is studied.

Performance evaluation of ion-exchange chromatography in capillary format.

The performance of a recently introduced capillary ion-exchange chromatography system was explored. Experiments were conducted in isocratic mode with a commercial capillary anion-exchange column (id = 0.4 mm, L = 15 cm) using a five-anion standard mixture. The achieved results were compared to the performance of a standard bore ion-exchange system (id = 4 mm, L = 15 cm), which was considered as a reference. The first-generation capillary columns exhibited a minimal reduced plate-height value below two witnessing a good packing quality and system performance. However, compared to the standard bore system the capillary system displayed an increased apparent C-term which could be due to a difference in packing morphology and/or possible external band-broadening contributions. For fast separations, the standard bore system outperformed the capillary system, while for complex separations both systems performed nearly equally well. In addition, the retention characteristics of the capillary system were investigated. To illustrate the suitability of the capillary system, the analysis of real-world water samples originating from two local Belgian rivers was demonstrated.

Exploring the possibilities of cryogenic cooling in liquid chromatography for biological applications: a proof of principle.

The possibilities to use cryogenic cooling to trap components in liquid chromatography was investigated. In a first step, van 't Hoff plots were measured with a reversed-phase column using the temperature control unit of a conventional high performance liquid chromatography (HPLC) system to gain insight in the retention behavior of proteins at low temperatures. It was estimated that retention factors in the range of k = 10(4) could be achieved at T = -20 °C for lysozyme, indicating that temperature is a usable parameter to trap components in LC. In a next step, trapping experiments were carried out on a nano-LC system, equipped with a UV-detector, using a commercial reversed-phase column. An in-house built setup, allowing cooling of a segment of the column down to temperatures below T = -20 °C, was used to trap components. Experiments were conducted under isocratic and gradient conditions with methanol as organic solvent. It is demonstrated that, by thermally trapping and elution of components, an enhanced S/N ratio and decreased peak widths can be obtained. At the same time, a significant increase in pressure drop occurs during the cooling process. Limitations and benefits of the technique are further discussed.

Performance evaluation of long monolithic silica capillary columns in gradient liquid chromatography using peptide mixtures.

A systematic study is reported on the performance of long monolithic capillary columns in gradient mode. Using a commercial nano-LC system, reversed-phase peptide separations obtained through UV-detection were conducted. The chromatographic performance, in terms of conditional peak capacity and peak productivity, was investigated for different gradient times (varying between 90 and 1320min) and different column lengths (0.25, 1, 2 and 4m) all originating from a single 4m long column. Peak capacities reaching values up to n=10(3) were measured in case of the 4m long column demonstrating the high potential of these long monoliths for the analysis of complex biological mixtures, amongst others. In addition, it was found that the different column fragments displayed similar flow resistance as well as consistent chromatographic performance in accordance with chromatographic theory indicating that the chromatographic bed of the original 4m long column possessed a structural homogeneity over its entire length.

Design and evaluation of flow distributors for microfabricated pillar array columns.

Five different flow distributors have been compared as a function of the flow rate for their ability to distribute small sample volumes over the entire width of flat rectangular microfabricated pillar array columns. The investigated designs can be divided in two major categories: (1) bifurcating, radially non-interconnecting distributors and (2) radially interconnecting distributors consisting of diamond-shaped pillars, elongated in the direction perpendicular to the flow, providing a high ratio of radial permeability over axial permeability. The quality of the flow distribution was evaluated experimentally by injecting equal volumes of fluorescent tracer into each of the tested designs and calculating the obtained peak variances using the method of moments. Purely bifurcating distributors perform less well than the best possible radially interconnected distributors, because the former inevitably require the use of wide open channels (d > 10 microm), wherein a lot of band broadening can occur. By doubling the aspect ratio of the radially stretched pillars from 5 to 10, the measured peak variance drops to 1/8 of the original value. The best results were obtained with a distributor in which the flow is distributed by a bed of anisotropic pillars with an aspect ratio of 10, but our results indicate that a substantial improvement can still be made by increasing the aspect ratio and adding gradually diverging sidewalls to the inlet.

Use of non-porous pillar array columns for the separation of Pseudomonas pyoverdine siderophores as an example of a real-world biological sample.

We report on the first separation of a complex biomixture in pressure-driven mode using perfectly ordered pillar array columns. The separations were conducted in the reversed-phase mode using a highly aqueous mobile phase, while the outer surface of the non-porous pillars was chemically functionalized with a hydrophobic C8-layer. The samples originated from two different bacterial strains (Pseudomonas aeruginosa PAO1 and Pseudomonas sp. W15Feb38) of fluorescent pseudomonads. These produce fluorescent yellow-green pyoverdines that serve as siderophores to shuttle iron inside the cell. The pyoverdines of both strains were prepared from the supernatant through a crude solid phase extraction without any further purification step. In case of the PAO1 mixture, a separation of 15 components within a column length of 2.5 cm could be observed through the transparent cover glass of the chip. For the W15Feb38 mixture, a separation of eight components could be observed within the same distance. These fast chromatographic separations were compared with those obtained via iso-electrofocusing (IEF), which is the traditionally employed fingerprinting method to characterize pseudomonad strains based on their pyoverdine profiles (siderotyping). With this technique, and despite the injection of a 10,000 times larger sample mass, only nine bands were maximally observed for the PAO1 mixture, whereas maximally six bands were observed in case of the W15Feb38 mixture. The chromatographic pillar array method, yielding a separation in less than 1 min, was also significantly faster than the IEF method, which typically needs 1.5h. The present system can therefore be considered as a potential alternative fingerprinting tool for the fast identification of different strains of fluorescent pseudomonads, including as diagnostic tool for typing strains of the important opportunistic pathogen P. aeruginosa.

Effect of the presence of an ordered micro-pillar array on the formation of silica monoliths.

We report on the synthesis of siloxane-based monoliths in the presence of a two-dimensional, perfectly ordered array of micro-pillars. Both methyltrimethoxysilane- and tetramethoxysilane-based monoliths were considered. The obtained structures were analyzed using scanning-electron microscopy and can be explained from the general theory of surface-directed phase separation in confined spaces. The formed structures are to a large extent nearly exclusively determined by the ratio between the bulk domain size of the monolith on the one hand and the distance between the micro-pillars on the other hand. When this ratio is small, the presence of the pillars has nearly no effect on the morphology of the produced monoliths. However, when the ratio approaches unity and ascends above it, some new types of monolith morphologies are induced, two of which appear to have interesting properties for use as novel chromatographic supports. One of these structures (obtained when the domain size/inter-pillar distance ratio is around unity) is a 3D network of linear interconnections between the pillars, organized such that all skeleton branches are oriented perpendicular to the micro-pillar surface. A second interesting structure is obtained at even higher values of the domain size/inter-pillar distance ratio. In this case, each individual micro-pillar is uniformly coated with a mesoporous shell.

Experimental investigation of the band broadening arising from short-range interchannel heterogeneities in chromatographic beds under the condition of identical external porosity.

The interesting possibilities offered by micropillar array columns to investigate the relation between bed heterogeneity and band broadening in chromatographic columns are illustrated and investigated. The perfect control over the microscopic bed structure offered by the photolithographic fabrication technique could be used to produce a heterogeneous bed displaying a short-range interchannel heterogeneity and having exactly the same particle size and external porosity as the perfectly ordered bed that was used as the reference system. According to this approach, any observed difference in band broadening could be directly owed to the effect of the bed heterogeneity. Knowing that the only difference between the investigated beds was their degree of heterogeneity, the obtained plate height measurements provided a unique test to distinguish among the abilities to recognize short-range interchannel heterogeneity of the different existing plate height models. Interpreting the data with the classical van Deemter and Knox models, it was found that the increased bed heterogeneity leads not only to an increase in the A-term constant but also to a clear increase in the C-term constant.

Experimental investigation of the band broadening originating from the top and bottom walls in micromachined nonporous pillar array columns.

We report on the experimental investigation of the effect of the top and bottom wall plates in micromachined nonporous pillar array columns. It has been found that their presence yields an additional c-term type of band broadening that can make up a significant fraction of the total band broadening (at least if considering nonporous pillars and a nonretained tracer). Their presence also induces a clear (downward) shift of the optimal velocity. These observations are, however in excellent quantitative agreement with the theoretical expectations obtained from a computational fluid dynamics study. The presently obtained experimental results, hence, demonstrate that the employed high aspect ratio Bosch etching process can be used to fabricate micromachined pillar arrays that are sufficiently refined to achieve the theoretical performance limit.

Optimum kinetic performance of open-tubular separations in microfluidic devices.

A survey is made of the different factors contributing to the kinetic performance of open-tubular separation channels. Being representative for most of the channels used in microfluidic devices, the main focus is on channels with a rectangular format. Kinetic plots of t(0)/N(2 )versus N are established to allow for a visual selection of the ideal channel format and dimensions. These plots for example show that in the pressure-driven mode a channel with a flat-rectangular crosssection (top and bottom wall covered by a retentive layer) can always yield slightly faster (some 15%) separations than a cylindrical capillary, provided the channel depth is optimized. If the channel depth is fixed, the optimal w/d-ratio depends on the required plate number. In electrically driven flows, the situation is reversed and rectangular channels with a small width are to be preferred, and the cylindrical capillary format becomes the best format.

Pressure-driven reverse-phase liquid chromatography separations in ordered nonporous pillar array columns.

Building upon the micromachined column idea proposed by the group of Regnier in 1998, we report on the first high-resolution reversed-phase separations in micromachined pillar array columns under pressure-driven LC conditions. A three component mixture could be separated in 3 s using arrays of nonporous silicon pillars with a diameter of approximately 4.3 microm and an external porosity of 55%. Under slightly retained component conditions (retention factor k' = 0.65-1.2), plate heights of about H = 4 microm were obtained at a mobile phase velocity around u = 0.5 mm/s. In reduced terms, such plate heights are as low as hmin = 1. Also, since the flow resistance of the column is much smaller than in a packed column (mainly because of the higher external porosity of the pillar array), the separation impedance of the array was as small as E = 150, i.e., of the same order as the best currently existing monolithic columns. At pH = 3, yielding very low retention factors (k' = 0.13 and 0.23), plate heights as low as H = 2 microm were realized, yielding a separation of the three component mixture with an efficiency of N = 4000-5000 plates over a column length of 1 cm. At higher retention factors, significantly larger plate heights were obtained. More experimental work is needed to investigate this more in depth. The study is completed with a discussion of the performance limits of the pillar array column concept in the frame of the current state-of-the-art in microfabrication precision.

An automated injection system for sub-micron sized channels used in shear-driven-chromatography.

This paper describes a method to automatically and reproducibly inject sharply delimited sample plugs in the shallow (i.e., sub-micron) channels typically used in shear driven chromatography. The formation of asymmetric plugs, which typically occurs during loading of the sample in wide channels, is circumvented by etching a slit in the middle of the channel that is connected to a micro-well and a vacuum system with syringes for the supply of both the analyte and the mobile phase. The design of the injection slit was supported by a series of CFD simulations to optimize its shape and that of the corresponding injection well. The system was intensively tested experimentally and showed good reproducibility, both for the width and the area of the injected peaks (relative standard deviations are max. 4 and 6%, respectively). The concentration of the injected plug was found to be approximately 80% of the original sample concentration. It was also observed that with the current setup the lower limit of the peak width was about 120 microm. This is a consequence of the fact that the peak width originating from the convection filling step becomes negligible to the contribution of diffusion during the filling and flushing time. Being fully automated and perfectly closed, the presently proposed injection system also paves the way to integrate other functionalities in shear driven chromatography, i.e. gradient elution and parallelization.