Comprehensive Multidimensional Separations Research Articles

Liquid chromatography and differential mobility spectrometry—data-independent mass spectrometry for comprehensive multidimensional separations in metabolomics

The benefits of combining drift time ion mobility (DTIMS) with liquid chromatography–high-resolution mass spectrometry (HRMS) have been reported for metabolomics but the use of differential time mobility spectrometry (DMS) is less obvious due to the need for rapid scanning of the DMS cell. Drift DTIMS provides additional precursor ion selectivity and collisional cross-section information but the separation resolution between analytes remains cell- and component-dependent. With DMS, the addition of 2-propanol modifier can improve the selectivity but on cost of analyte MS response. In the present work, we investigate the liquid chromatography–mass spectrometry (LC–MS) analysis of a mix of 50 analytes, representative for urine and plasma metabolites, using scanning DMS with the single modifiers cyclohexane (Ch), toluene (Tol), acetonitrile (ACN), ethanol (EtOH), and 2-propanol (IPA), and a binary modifier mixture (cyclohexane/2-propanol) with emphasis on selectivity and signal sensitivity. 1.5% IPA in the N2 stream was found to suppress the signal of 50% of the analytes which could be partially recovered with the use of IPA to 0.05% as a Ch/IPA mixture. The potential to use the separation voltage/compensation voltage/modifier (SV/CoV/Mod) feature as an additional analyte identifier for qualitative analysis is also presented and applied to a data-independent LCxDMS-SWATH-MS workflow for the analysis of endogenous metabolites and drugs of abuse in human urine samples from traffic control.Graphical

Comprehensive two-dimensional gas chromatography as a boosting technology in food-omic investigations.

This review focuses on the role that comprehensive two-dimensional gas chromatography can play within the investigation workflows of food-omics and related disciplines and subdisciplines, including food metabolomics, nutrimetabolomics, sensomics, and food safety. After a short introductory survey, discussing the intriguing context of system biology and integrationist approaches of investigation, the concepts of analytical dimensions and the key characteristics of comprehensive two-dimensional gas chromatography are introduced. Through a selection of relevant examples, the boosting role of comprehensive two-dimensional gas chromatography within food-omics is described, providing to the reader evidence of how comprehensive multidimensional separations based platforms have introduced new concepts and tools in the analytical measurement of complex biological samples.

Comprehensive Multidimensional Chromatography

As analysts enquire further into the complexity of their samples, powerful separation techniques become an important asset. The ability (or lack thereof) of conventional one-dimensional separations such as liquid and gas chromatography to resolve complicated mixtures was described in detail by Giddings and others in the 1980s. At this point the development of multidimensional separation techniques began to accelerate. These techniques can offer up to an order of magnitude greater peak capacity than conventional chromatographic methods. Today, multidimensional separation methods are used widely throughout many fields in both industry and academia. This review focuses on comprehensive multidimensional techniques in gas and liquid chromatography. Fundamental aspects of the techniques, modulation technology and recent applications in fields that frequently use the technology will be discussed. Multidimensional separation techniques are here to stay and will undoubtedly play increasingly important role in the analysis of complex chemical samples for years to come. Keywords: Comprehensive multidimensional separations, comprehensive two-dimensional gas chromatography, comprehensive two-dimensional liquid chromatography, environmental, flavours and fragrances, instrumentation.

Peak purity assessment in a triple-active fixed-dose combination drug product related substances method using a commercial two-dimensional liquid chromatography system.

Pharmaceutical formulations containing multiple active components challenge the development of analytical methods, especially as the individual active ingredients diverge in their physicochemical properties. Establishing specificity, especially peak purity, is one of the major evaluation criteria when developing a related substances method for drug substances or products. Fixed-dose combination products may not be amenable to common strategies for assessing peak purity, such as performing orthogonal separations, due to the complexity of the separation and/or diversity of the active ingredients. An alternate approach to evaluating peak purity is demonstrated for a triple-active component fixed-dose combination product under development. A commercially available automated two-dimensional liquid chromatography system was used to perform a selective comprehensive multidimensional separation of an active ingredient peak. The first dimension performed the drug product impurity/degradant profiling method; the second dimension assayed these fractions using the drug substance profiling method, which was pseudo-orthogonal to the first dimension. A total of 14 targeted fractions were sampled across the first dimension main peak, with 11 containing detectable analytes and the remaining fractions bracketing the main peak. This degree of sampling allowed profiling of a coeluting degradant present at a 0.2% w/w level throughout the main peak.

Comprehensive multidimensional separations of peptides using nano-liquid chromatography coupled with micro free flow electrophoresis.

The throughput of existing liquid phase two-dimensional separations is generally limited by the peak capacity lost due to under sampling by the second dimension separation as peaks elute off the first dimension separation. In the current manuscript, a first dimension nanoliquid chromatography (nLC) separation is coupled directly with a second dimension micro free flow electrophoresis (μFFE) separation. Since μFFE performs continuous separations, no complicated injection or modulation is necessary to couple the two techniques. Analyte peaks are further separated in μFFE as they elute off the nLC column. A side-on interface was designed to minimize dead volume in the nLC × μFFE interface, eliminating this as a source of band broadening. A Chromeo P503 labeled tryptic digest of BSA was used as a complex mixture to assess peak capacity. 2D nLC × μFFE peak capacities as high as 2,352 could be obtained in a 10 min separation window when determined according to the product of the first and second dimension peak capacities. After considering the orthogonality of the two separation modes and the fraction of separation space occupied by peaks, the usable peak capacity generated was determined to be 776. The 105 peaks/min generated using 2D nLC × μFFE was nearly double the previously reported maximum peak capacity production rate achieved using online LC × LC.

Limits of Detection and Quantification in Comprehensive Multidimensional Separations. 1. A Theoretical Look

Comprehensive multidimensional separations (e.g., GC×GC, LC×LC, etc.) are increasingly popular tools for the analysis of complex samples, due to their many advantages, such as vastly increased peak capacity, and improvements in sensitivity. The most well-established of these techniques, GC×GC, has revolutionized analytical separations in fields as diverse as petroleum, environmental research, food and flavors, and metabolic profiling. Using multidimensional approaches, analytes can be quantified at levels substantially lower than those possible by one-dimensional techniques. However, it has also been shown that the modulation process introduces a new source of error to the measurement. In this work, we present the results of a study into the limits of quantification and detection (LOQ and LOD) in comprehensive multidimensional separations using GC×GC and the more popular "two-step" integration algorithm as an example. Simulation of chromatographic data permits precise control of relevant parameters of peak geometry and modulation phase. Results are expressed in terms of the dimensionless parameter of signal-to-noise ratio of the base peak (S/N(BP)) making them transportable to any result where quantification is performed using a two-step algorithm. Based on these results, the LOD is found to depend upon the modulation ratio used for the experiment and vary between a S/N(BP) of 10-17, while the LOQ depends on both the modulation ratio and the phase of the modulation for the peak and ranges from a S/N(BP) of 10 to 50, depending on the circumstances.

Comprehensive multidimensional separations

Multidimensionality in the instrumental separation sciences represents improved capabilities for discovery of molecular species. The literature is replete with reports where new separation tools have allowed new compounds—or previously unreported compounds—to be detected and identified in all manner of samples. Thus multidimensional separation (MDS) methods normally involve sampling narrow zones of eluate from a first-dimension column, to be re-separated on a second dimension normally of somewhat different mechanism, to resolve compounds that are poorly resolved or completely unresolved in the first stage. This is the aim of all chromatographic methods—to resolve compounds. Therefore the MDS method succeeds by providing greater capacity for the total analysis. However, the case where multidimensionality involves just one separation stage as one of the dimensions of the analytical system also provides improved analytical capability. This is most readily recognised in the form of gas chromatography– mass spectrometry (GC–MS) and liquid chromatography (LC)–MS methodologies. In this case the two dimensions are fully orthogonal—both dimensions provide independent mechanisms of analysis. The extent to which multi-(two-)dimensio nal separations are orthogonal is a matter of some discussion, and this is especially the case for MDGC. Here the only possible mechanism is based on phase selectivity and temperature, and the independence of the mechanisms is still a useful research subject. The advent of ‘powerful’MS solutions might be apparently sufficient for many sample analyses, and we have been served well by these methods. But the overriding question should be whether an improved quality of analysis can be achieved by an MDS approach. Can MS provide an unambiguous molecular assignment where an insufficient separation is obtained? Clearly, a single MS dimension was found to be lacking in certain cases, otherwise there would have been little motivation to develop MS/MS approaches which have found many unique applications, both for specificity of molecular structural information, but especially where component overlap compromises a single MS stage. We can ask the same question for the separation stage. Given the acknowledged success of MDGC (but still the lack of widespread adoption in most GC laboratories), where does the technology of comprehensive 2D gas chromatography (GC×GC) and its liquid chromatography counterpart sit? Given that GC×GC has now about a 20-year history since it was first proposed by Professor John Phillips, have comprehensive multidimensional separations (CMS) captured the hearts and minds of the chromatographer? If not, can we rationalize the reason why? Whilst MDGC provides a relatively simple interpretation of a chromatographic result, CMS completely overturns the chromatography experiment—the way it is conducted, the conditions and technology of modulation, the need to select appropriate columns and dimensions—and optimisation, the need for appropriate detection technologies, and then there is the matter of data handling and interpretation. Literally, every aspect of both GC and LC are challenged by CMS technology. This requires development of methods and, more importantly, systems that are dedicated to CMS. But if chromatographers were not up to challenges, then perhaps we would still be using packed columns (or TLC)! The way forward to considering MDS was illuminated by Giddings, and his insights in a series of landmark papers. However, these were not supported by parallel experimental developments—they were simply impossible given the technology of the time. So his were very much ‘thought experiments’. Phillips made the first experimental developments that Published in the special issue Comprehensive Multidimensional Separations with Guest Editors James Harynuk and Philip Marriott.

Selective comprehensive multidimensional separation for resolution enhancement in high performance liquid chromatography. Part II: Applications

In this second paper of a two-part series, we demonstrate the utility of an approach to enhancing the resolution of select portions of conventional 1D-LC separations, which we refer to as selective comprehensive two-dimensional HPLC (sLC×LC), in three quite different example applications. In the first paper of the series we described the principles of this approach, which breaks the long-standing link in online multi-dimensional chromatography between the timescales of sampling the first dimension (1D) separation and the separation of fractions of 1D effluent in the second dimension. In the first example, the power of the sLC×LC approach to significantly reduce the analysis time and method development effort is demonstrated by selectively enhancing the resolution of critical pairs of peaks that are unresolved by a one-dimensional separation (1D-LC) alone. Transfer and subsequent 2D separations of multiple fractions of a particular 1D peak produces a two-dimensional chromatogram that reveals the coordinates of the peaks in the 2D separation space. The added time dimension of sLC×LC chromatograms also facilitates the application of sophisticated chemometric curve resolution algorithms to further resolve peaks that are otherwise chromatographically unresolved. This is demonstrated in this work by the targeted analysis of phenytoin in urban wastewater effluent using UV diode array detection. Quantitation by both standard addition and external calibration methods yielded results that were not statistically different from 2D-LC/MS/MS analysis of the same samples. Next, we demonstrate the utility of sLC×LC for reducing ion suppression due to matrix effects in electrospray ionization mass spectrometry through the analysis of cocaine in urban wastewater effluent. Finally, we explore the flexibility of the approach in its application to two select regions of a single 1D separation of triclosan and cocaine. The diversity of these applications demonstrates the power and versatility of the sLC×LC approach, which will benefit tremendously from further optimization and advances in valve technology that specifically address the needs of this new technique.

Selective comprehensive multi-dimensional separation for resolution enhancement in high performance liquid chromatography. Part I: Principles and instrumentation

An approach to enhancing the resolution of select portions of conventional one-dimensional high performance liquid chromatography (HPLC) separations was developed, which we refer to as selective comprehensive two-dimensional HPLC (sLC×LC). In this first of a series of two papers we describe the principles of this approach, which breaks the long-standing link in on-line multi-dimensional chromatography between the timescales of sampling the first dimension (1D) separation and the separation of fractions of 1D effluent in the second dimension. This allows rapid, high-efficiency separations to be used in the first dimension, while still adequately sampling 1D peaks. Transfer, transient storage, and subsequent second dimension (2D) separations of multiple fractions of a particular 1D peak produces a two-dimensional chromatogram that reveals the coordinates of the peak in both dimensions of the chromatographic space. Using existing valve technology we find that the approach is repeatable (%RSD of peak area <1.5%), even at very short first dimension sampling times – as low as 1s. We have also systematically studied the critical influence of the volume and composition of fractions transferred from the first to the second dimension of the sLC×LC system with reversed-phase columns in both dimensions, and the second dimension operated isocratically. We find that dilution of the transferred fraction, so that it contains 10–20% less organic solvent than the 2D eluent, generally mitigates the devastating effects of large transfer volumes on 2D performance in this type of system. Several example applications of the sLC×LC approach are described in the second part of this two-part series. We anticipate that future advances in the valve technology used here will significantly widen the scope of possible applications of the sLC×LC approach.

Chemometrics in comprehensive multidimensional separations

Chemometric methods have critical importance for the discovery of the information/knowledge buried or concealed in high-dimensional datasets acquired from comprehensive multidimensional separations (CMDS), and for interpretation of experiments or chemical processes. In this work, employment of new developments in chemometrics making full use of the data to maximize the potential of CMDS to resolve mathematically a variety of practical problems is reviewed whilst providing the authors' point of view. During the past several years, chemometrics has been successfully applied to many areas of concern to CMDS investigation, including experimental parameter optimization, data quality improvement, identification and quantification of target chemical components, pattern recognition technique for clustering and classification, multivariate model establishment to correlate chromatographic properties and molecular descriptors. On the basis of the high-dimensionality characteristics of CMDS, some special aspects such as evaluation of orthogonality and image processing have also been included in this review. It is expected that an overview of the diverse ways in which chemometrics can aid CMDS investigations will prove valuable to interested users in this area through a comprehensive survey of previous research contributions. Chemometrics lends itself well to the powerful separation capability of CMDS.

Theoretical description of a new analytical technique: Comprehensive online multidimensional fast Fourier transform separations

Comprehensive multidimensional separations are today dominated by systems that are fundamentally limited to highly asymmetrical online separations sacrificing separation space, or to lengthy, time consuming offline separations. With the exception of pulse-modulated methods, separations have thus been limited to two dimensions. It is proposed that some of the limitations and shortcomings of these methods may be ameliorated or overcome by employing multi-dimensional detection whereby each analyte is effectively labelled in the frequency domain by a series of pulsed-injections, and a symmetrical, comprehensive online analysis performed with the resulting signal processed by sequential Fourier analysis. A semi-empirical computer model of this system was developed and its feasibility positively demonstrated in simulations of high-efficiency separations in two dimensions. Separations of higher dimensionality were shown to be possible but involved signal-processing challenges beyond the present work. By eliminating wrap-around effects and enabling the separation of physically unseparated peaks, the technique facilitates significant improvements in peak capacity per unit of analysis time as well as greatly improved signal to noise ratios. Because these comprehensive online multidimensional Fourier transform separations depend heavily upon the practical lifetime of imposed injection pulses, it is envisaged that this method will leverage emerging high-efficiency micro- and nanoscale separations technologies.

Comprehensive multidimensional separation methods by hyphenation of single-photon ionization time-of-flight mass spectrometry (SPI-TOF-MS) with GC and GC×GC

One- and comprehensive two-dimensional gas chromatography were hyphenated with soft photoionization mass spectrometry. The characteristics of these two- and three-dimensional comprehensive separation techniques are discussed in detail. Using the innovative electron beam pumped excimer light source (EBEL) for single-photon ionization (SPI), organic molecules with ionization energies (E ( i )) of below 9.8 eV can be detected by a time-of-flight mass spectrometer (TOF-MS). SPI with 126 nm vacuum ultraviolet (VUV) photons enables the universal and soft ionization of organic molecules. SPI-TOF-MS hyphenated to one-dimensional gas chromatography results in a comprehensive two-dimensional separation method (GCxMS). To demonstrate this, diesel fuel was analyzed, and the resulting GCxMS chromatograms are discussed in depth. A three-dimensional separation method was also realized by combining comprehensive two-dimensional gas chromatography (GCxGC) with SPI-MS. In the resulting separation space, constituents originating from mineral oil diesel blended with biodiesel were dispersed along the two GC separation axes, while the molecular mass axis served as a third separation dimension.

Comprehensive two-dimensional liquid chromatography

Having nearly exhausted the possibilities for generating peak capacity through improvements in column technology, chromatographers are increasingly looking to alternative ways of maximising chromatographic separation. In recent years there has been increasing activity in the field of comprehensive multidimensional separations to meet analysis demands. Comprehensive two-dimensional liquid chromatography (LCxLC) approaches offer high peak capacity which leads to significantly improved analytical performance over single-column liquid chromatography. There are several closely related avenues available for achieving an LCxLC separation and this review pays special attention to the different valve-based interfaces that have been used to comprehensively couple the first and second dimension columns in LCxLC systems. A brief discussion of column choices for selected applications and the conditions employed is also presented.

Modulation Ratio in Comprehensive Two-dimensional Gas Chromatography

Comprehensive two-dimensional chromatography employs a serially coupled two-column arrangement where effluent from the first column is collected or sampled and then introduced to the second column according to a chosen modulation period. This is effected by use of a modulator at or near the column junction. One of the considerations in applying the technique is the period of the modulator, which determines the sampling duration of the first column effluent. Here, we propose that the sampling rate can be most effectively described by a new term, called the modulation ratio (MR). This is defined as the ratio of 4 times the first column peak standard deviation (4sigma) divided by the modulation period (PM) or 1.6985 times the half-height width of the peak (wh): MR = 4sigma/PM = wb/PM = (wh x 1.6985)/PM. The 4sigma value is more commonly recognized as the peak base width (wb). The use of 4sigma as the numerator is preferred to simply sigma because when the PM value used for an experiment is equal to sigma, then the MR value is calculated to be 4, implying that the primary peak will be modulated approximately 4 times as is normally recommended for a comprehensive multidimensional separation. The less well-defined term of modulation number (NM) has been previously used and proposed as the number of modulations per peak and, therefore, is intended to convey the manner in which the primary column peak is sampled; this is a subjective and not well-characterized value. The use of MR should provide users with a meaningful and strictly defined value when reporting experimental conditions. The utility of MR is demonstrated through a mathematical model of the modulation process for both Gaussian and tailing peaks, supported by an experimental study of the modulation ratio. It is shown that for the analysis of trace compounds where precise quantitative measurements are being made, the experiment should be conducted with an MR of at least 3. Conversely, for semiquantitative methods or the analysis of major components, an MR of approximately 1.5 should suffice.

Multidimensional LC-LC and LC-CE for high-resolution separations of biological molecules.

In multidimensional separations, two or more independent separation methods are coupled in an effort to resolve complex mixtures. The displacement mechanisms of each method should be orthogonal, such that little correlation exists between the retention of compounds in each dimension. When multiple orthogonal separation methods are coupled such that all sample components are subjected to complete analysis on all dimensions, the method is considered "comprehensive". The primary advantage of comprehensive multidimensional separations over their one-dimensional counterparts is the potential for dramatically enhanced resolution. High resolving power can be achieved because the peak capacity of a comprehensive multidimensional separation is roughly equal to the product of the individual peak capacities of each dimension. In this review, the theory and instrumentation of two-dimensional liquid chromatography (LC-LC) and liquid chromatography-capillary electrophoresis (LC-CE) separations are discussed. Some applications of these techniques to the separation of biological molecules are highlighted. Future directions for the development of multidimensional separations are also considered.

Comprehensive two-dimensional separations using microcolumns

The late Professor John B. Phillips has been the leading pioneer in developing the fundamental theory, instrumentation, and appreciation of comprehensive multidimensional separations. The comprehensive approach to high resolution separations easily surpasses any other approaches to achieving extremely high peak capacity. The practical implementation of comprehensive multidimensional techniques places the highest demands on performance of the separate dimensions in terms of speed, sensitivity, and reproducibility. In this paper, the history, requirements, instrumentation, and applications of comprehensive two-dimensional column separations are reviewed. Combinations of GC×GC, SFC×GC, SFE×GC, LC×LC, LC×CE, and ITP×CE are described, since these are the major comprehensive multidimensional techniques reported to date. © 2000 John Wiley & Sons, Inc. J Micro Sep 12: 241–254, 2000