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•A new method for measuring orthogonality in multidimensional separations is introduced.•Our method also diagnoses areas where peaks are clustered in the separation space.•The new ...method comprises of a number of equations which are easily implemented in Microsoft Excel.•We applied the method to 8 computer-generated and 2 experimental multidimensional chromatograms.•The method compared favorably against established methods.
Multi-dimensional chromatographic techniques, such as (comprehensive) two-dimensional liquid chromatography and (comprehensive) two-dimensional gas chromatography, are increasingly popular for the analysis of complex samples, such as protein digests or mineral oils. The reason behind the popularity of these techniques is the superior performance, in terms of peak-production rate (peak capacity per unit time), that multi-dimensional separations offer compared to their one-dimensional counterparts. However, to fully utilize the potential of multi-dimensional chromatography it is essential that the separation mechanisms used in each dimension be independent of each other. In other words, the two separation mechanisms need to be orthogonal. A number of algorithms have been proposed in the literature for measuring chromatographic orthogonality. However, these methods have their limitations, such as reliance on the division of the separation space into bins, need for specialist software or requirement of advanced programming skills. In addition, some of the existing methods for measuring orthogonality include regions of the separation space that do not feature peaks. In this paper we introduce a number of equations which provides information on the spread of the peaks within the separation space in addition to measuring orthogonality, without the need for complex computations or division of the separation space into bins.
Liquid chromatography (LC) is an incredibly successful analytical separation tool. Its versatility is unprecedented because of the many different separation modes and because almost all samples can ...be dissolved in some kind of solvent, ranging from water to organic solvents to strong acids or bases. Conditions can be found to separate almost all pairs of analytes. Here, Pirok et al explore some 160 applications of two-dimensional LC (2D-LC) techniques that have been published in 2016, 2017, or 2018. They also discuss progress in methodology in the same period, obstacles to successful implementation and application of LC X LC techniques and possible remedies.
Online comprehensive two‐dimensional liquid chromatography has become an attractive option for the analysis of complex nonvolatile samples found in various fields (e.g. environmental studies, food, ...life, and polymer sciences). Two‐dimensional liquid chromatography complements the highly popular hyphenated systems that combine liquid chromatography with mass spectrometry. Two‐dimensional liquid chromatography is also applied to the analysis of samples that are not compatible with mass spectrometry (e.g. high‐molecular‐weight polymers), providing important information on the distribution of the sample components along chemical dimensions (molecular weight, charge, lipophilicity, stereochemistry, etc.). Also, in comparison with conventional one‐dimensional liquid chromatography, two‐dimensional liquid chromatography provides a greater separation power (peak capacity). Because of the additional selectivity and higher peak capacity, the combination of two‐dimensional liquid chromatography with mass spectrometry allows for simpler mixtures of compounds to be introduced in the ion source at any given time, improving quantitative analysis by reducing matrix effects. In this review, we summarize the rationale and principles of two‐dimensional liquid chromatography experiments, describe advantages and disadvantages of combining different selectivities and discuss strategies to improve the quality of two‐dimensional liquid chromatography separations.
In this work, a new strategy for the chemometric analysis of two-dimensional liquid chromatography–high-resolution mass spectrometry (LC × LC–HRMS) data is proposed. This approach consists of a ...preliminary compression step along the mass spectrometry (MS) spectral dimension based on the selection of the regions of interest (ROI), followed by a further data compression along the chromatographic dimension by wavelet transforms. In a secondary step, the multivariate curve resolution alternating least squares (MCR-ALS) method is applied to previously compressed data sets obtained in the simultaneous analysis of multiple LC × LC–HRMS chromatographic runs from multiple samples. The feasibility of the proposed approach is demonstrated by its application to a large experimental data set obtained in the untargeted LC × LC–HRMS study of the effects of different environmental conditions (watering and harvesting time) on the metabolism of multiple rice samples. An untargeted chromatographic setup coupling two different liquid chromatography (LC) columns hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) together with an HRMS detector was developed and applied to analyze the metabolites extracted from rice samples at the different experimental conditions. In the case of the metabolomics study taken as example in this work, a total number of 154 metabolites from 15 different families were properly resolved after the application of MCR-ALS. A total of 139 of these metabolites could be identified by their HRMS spectra. Statistical analysis of their concentration changes showed that both watering and harvest time experimental factors had significant effects on rice metabolism. The biochemical insight of the effects of watering and harvesting experimental factors on the changes in concentration of these detected metabolites in the investigated rice samples is attempted.
The non-enzymatic glycation of proteins and their advanced glycation end products (AGEs) are associated with protein transformations such as in the development of diseases and biopharmaceutical ...storage. The characterization of heavily glycated proteins at the intact level is of high interest as it allows to describe co-occurring protein modifications. However, the high heterogeneity of glycated protein makes this process challenging, and novel methods are required to accomplish this.
In this study, we investigated two novel LC-HRMS methods to study glycated reference proteins at the intact protein level: low-flow hydrophilic-interaction liquid chromatography (HILIC) and native size-exclusion chromatography (SEC). Model proteins were exposed to conditions that favored extensive glycation and the formation of AGEs. After glycation, complicated MS spectra were observed, along with a sharply reduced signal response, possibly due to protein denaturation and the formation of aggregates. When using HILIC-MS, the glycated forms of the proteins could be resolved based on the number of reducing monosaccharides. Moreover, some positional glycated isomers were separated. The SEC-MS method under non-denaturing conditions provided insights into glycated aggregates but offered only a limited separation of glycated species based on molar mass. Overall, more than 25 different types of species were observed in both methods, differing in molar mass by 14–162 Da. 19 of these species have not been previously reported.
The proposed strategies show great potential to characterize highly glycated intact proteins from native and denaturing perspectives and provide new opportunities for fast clinical diagnoses and investigating glycation-related diseases.
The profile of glycated intact proteins analyzed by LC-MS. Display omitted
•Two methods are applied to the study of the process of glycation: HILIC-HRMS (denaturing) and SEC-HRMS (non-denaturing).•The separation of glycoconjugates (HILIC) and aggregates (SEC) provides insights into the glycation process.•The methods allow monitoring of co-occurring glycations and advanced glycation end products (AGEs).•Dynamic changes in the monosaccharide-conjugated proteins and AGEs are observed during protein glycation.
Bottom-up proteomics provides often small amounts of highly complex samples that cannot be analysed by direct mass spectrometry (MS). To gain a better insight in the sample composition, liquid ...chromatography (LC) and (comprehensive) two-dimensional liquid chromatography (2D-LC or LC × LC) can be coupled to the MS. Low-flow separations are attractive for HRMS analysis, but they tend to be lengthy. In this work, a low-flow, online, actively modulated LC × LC system, based on hydrophilic-interaction liquid chromatography (HILIC) in the first dimension and reversed-phase liquid chromatography (RPLC) in the second dimension, was developed to separate complex mixtures of peptides. Miniaturization permitted the analysis of small sample amounts (1–5 μg) and direct coupling with micro-ESI MS (1 μL min−1). All components were focused and automatically transferred from HILIC to RPLC using stationary-phase-assisted active modulation (C18 traps) to deal with solvent-incompatibility or dilution issues. Optimization of the setup was performed for the HILIC columns and the RPLC columns to provide a more efficient separation and higher identification rates than obtained using one-dimensional (1D) LC. A 60% increase in peak capacity was obtained with the 2D setup compared to a 1D-RPLC separation and a 17–34% increase in the number of proteins identified was achieved for the samples analysed (2D-yeast-8280 peptides and 2D-kidney tissue-8843 peptides), without increasing the analysis time (2 h).
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•Increased LC × LC peak capacity versus 1D-LC without an increase in analysis time.•Complete sample transfer between HILIC and RPLC dimensions.•Sample focusing on trap column to overcome dilution and solvent incompatibility.•Increased protein identification rate.•Application of HILIC × RPLC to kidney tissue and IMR-90 cell line.
Asymmetrical flow field-flow fractionation (AF4) has attracted considerable attention as a size-based separation technique, due to its mild separation conditions, broad working range (from ...approximately 103 to 109 Da molecular mass or from 1 nm to 1 μm particle diameter), and versatility. AF4 is primarily being used to measure particle size, polydispersity, and physical stability of various systems, such as (bio)-macromolecules and nanoparticles. In comparison with size-exclusion chromatography (packed column), AF4 (open channel) allows separation while preserving labile structures. Monitoring of interactions between different compounds and in very complex matrices is possible. Preservation of the structure and correlation of structural characteristics with activity and functionality can bolster the development of new therapeutic strategies for diseases and new materials with improved properties. In this review, a detailed overview is presented of developments in AF4 for interaction studies between various systems, such as protein-protein, polymer-polymer, nanoparticle-drug, and nanoparticle-protein. The prospects and obstacles for AF4, and other less-commonly used types of FFF, for studying interactions within complex and fragile systems are covered. Coupling AF4 to a variety of detection systems can greatly contribute to the understanding of the interaction/association processes and provide information on the interaction kinetics. This review is intended to provide comprehensive documentation on the types of information (structural, morphological, chemical) on molecular interactions that can be retrieved by AF4.
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•Applications of field-flow fractionation (FFF) to study molecular interactions between various types of analytes are reviewed.•Fragile structures and weakly-associated complexes can be preserved during FFF separation.•FFF can be coupled to various detectors to provide structural, morphological and chemical information.•Asymmetrical flow field-flow fractionation (AF4) can be used to study the formation of protein corona.•For certain classes of analytes, interactions with the currently available membranes are unavoidable.
•Modular device with a rotating mechanism for spatial multi-dimensional LC.•Prototypes fabricated through 3D-printing (Digital Light Processing).•Adequate flow confinement in first dimension ...demonstrated experimentally.•Successful transfer from first to second dimension.
Spatial comprehensive two-dimensional liquid chromatography (xLC×xLC) may be an efficient approach to achieve high peak capacities in relatively short analysis times, thanks to parallel second-dimension separations 1,2. A key issue to reach the potential of xLC×xLC is to achieve adequate flow control and confinement of the analytes to the desired regions, i.e. confinement in the first-dimension direction and subsequently homogeneous flow in the second dimension. To achieve these goals we propose the TWIST concept (TWo-dimensional Insertable Separation Tool), a modular device that includes an internal first-dimension (1D) part that is cylindrical and rotatable. This internal part features a series of through-holes, each of which is perpendicular to the direction of the 1D flow. The internal part is inserted in the cylindrical casing of the external part. The internal diameter of the casing is marginally larger than the external diameter of the internal part. The external part also comprises a flow distributor and second-dimension (2D) channels. During the 1D injection and development, the channel is placed in a position where the through-holes are facing the wall of the external part, such that the liquid remains confined within the 1D channel. Thereafter, to realize the transfer to the second dimension (2D injection), the 1D channel is rotated, so that the holes of the internal part are aligned with the holes on the external part, allowing a transversal flow of the 2D mobile phase from the distributor through the 1D channel and eventually into the 2D area.
•Absence of stationary-phase material in 1D channel deteriorates analyte transfer.•Porous materials in both 1D and 2D lead to reduced band broadening.•Thin or no spacers in the 2D are preferred in ...terms of band broadening.•Fabrication imperfections of 3D-printed devices increase A-term dispersion.•Pressure resistance is affected by the thickness of the encasing of devices.
Conventional one-dimensional column-based liquid chromatographic (LC) systems do not offer sufficient separation power for the analysis of complex mixtures. Column-based comprehensive two-dimensional liquid chromatography offers a higher separation power, yet suffers from instrumental complexity and long analysis times. Spatial two-dimensional liquid chromatography can be considered as an alternative to column-based approaches. The peak capacity of the system is ideally the product of the peak capacities of the two dimensions, yet the analysis time remains relatively short due to parallel second-dimension separations. Aspects affecting the separation efficiency of this type of systems include flow distribution to homogeneously distribute the mobile phase for the second-dimension (2D) separation, flow confinement during the first-dimension (1D) separation, and band-broadening effects during analyte transfer from the 1D separation channel to the 2D separation area.
In this study, the synergy between computational fluid dynamics (CFD) simulations and rapid prototyping was exploited to address band broadening during the 2D development and analyte transfer from 1D to 2D. Microfluidic devices for spatial two-dimensional liquid chromatography were designed, simulated, 3D-printed and tested. The effects of presence and thickness of spacers in the 2D separation area were addressed and leaving these out proved to be the most efficient solution regarding band broadening reduction. The presence of a stationary-phase material in the 1D channel had a great effect on the analyte transfer from the 1D to the 2D and the resulting band broadening. Finally, pressure limit of the fabricated devices and printability are discussed.
•Drug isomers can confidently be differentiated by GC–VUV.•Small differences in VUV spectra are sufficient to distinguish drug isomers.•GC–VUV provides orthogonal selectivity to GC–MS.•Traditional ...GC–MS data also provides information for isomer differentiation.•ROC curves and LR values can be retrieved from match score distributions.
Currently, forensic drug experts are facing chemical identification challenges with the increasing number of new isomeric forms of psychoactive substances occurring in case samples. Very similar mass spectra for these substances could easily result in misidentification using the regular GC–MS screening methods in combination with colorimetric testing in forensic laboratories. Building on recent work from other groups, this study demonstrates that GC–VUV is a powerful technique for drug isomer differentiation, showing reproducible and discriminating spectra for aromatic ring-isomers. MS and VUV show complementary selectivity as VUV spectra are ring-position specific whereas MS spectra are characteristic for the amine moieties of the molecule. VUV spectra are very reproducible showing less than 0.1‰ deviation in library match scores and therefore small spectral differences suffice to confidently distinguish isomers. In comparison, MS match scores gave over 10‰ deviation and showed significant overlap in match score ranges for several isomers. This poses a risk for false positive identifications when assigning compounds based on retention time and GC–MS mass spectrum. A strategy was developed, based on Kernel Density Estimations of match scores, to construct Receiver Operating Characteristic (ROC) curves and estimate likelihood ratios (LR values) with respect to the chemical differentiation of drug related isomers. This approach, and the added value of GC–VUV is demonstrated with the chemical analysis of several samples from drug case work from the Amsterdam area involving both compounds listed in Dutch drug legislation (3,4-MDMA; 3,4-MDA; 4-MMC; 4-MEC and 4-FA) as well as their unlisted and thus uncontrolled isomers (2,3-MDMA; 2,3-MDA; 2- and 3-MMC; 2- and 3-MEC and 2- and 3-FA).
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