Separation Of Complex Mixtures Research Articles

Stage-frit: A straightforward sub-2 μm nano-liquid chromatography column fabrication for proteomic analysis

In this work we demonstrated a facile method for the fabrication of C18 coordination polymer gel in a capillary, called stage-frit, which was efficiently applied to pack sub-2 μm C18 beads into the capillary by a high pressure bomb for the online separation of proteolytic peptides. The back pressure of the column with 10 cm × 75 μm i.d. is regularly lower than 170 bar at a flow rate of 300 nl/min, which could be operated on a common nanoLC system instead of nanoUPLC system due to the good permeability, low back pressure and high mechanical stress of the frit that will totally reduce the cost for the purchase of instrument. The stage-frit allows long-term continuous flow of the solvent and no significant beads loss or pressure instability was observed during the period. The repeatability of retention time for fifteen BSA tryptic peaks was found to be less than 1.08% (RSD) in six time nanoLC-ESI-MS/MS experiments. The average full width at half maximum (FWHM) of peptide peaks is 5.87 s. The sub-2 μm stage-frit nanoLC column showed better sensitivity than the commercial available for large scale proteomic analysis of total tissue proteins from human spleen. The number of identified peptides is approximately 0.4-fold and 0.2-fold higher than that obtained by utilizing commercial columns packed with 3 μm and 1.8 μm C18 materials, respectively. In the field of analytical chemistry, particularly the use of nanoLC systems, stage-frit nanoLC column offers a great potential for the separation of complex mixtures.

Travelling-wave ion mobility time-of-flight mass spectrometry as an alternative strategy for screening of multi-class pesticides in fruits and vegetables

This paper reports a novel approach to screening multi-class pesticides by ion mobility time-of-flight mass spectrometry detection. Nitrogen was selected as mobility gas. After optimization of the different ion mobility parameters, determination of matrix effect on the drift times was conducted using different matrix extracts. The results showed that drift time values are not influenced by the matrix and also are independent of the concentration within the working range for 100 pesticides tested, making drift time a powerful additional identification tool. Based on statistics, 2% variation criteria provides a good fit for all the pesticides targeted, and could be considered as a maximum acceptable criteria associated with the drift time parameter for identification purpose. This 2% value is in agreement with already reported criteria, for instance, for GC or LC retention time in European documents. Finally, the well-known feature of mobility to separate complex mixtures was also tested to obtain purified extracted mass spectra of pesticides present in fruit extract.

Using adsorbents with immobilized metal chelate layers for the gas-chromatographic separation of aromatic hydrocarbons

Adsorbents based of Silipor 200 silica with immobilized layers of nickel acetylacetonate, ethylacetoacetate, and malonate are synthesized. The retention of arenes on the modified phases is studied; that the applicability of these adsorbents to the separation of complex mixtures of aromatic hydrocarbons is demonstrated.

Inlet backflushing device for the improvement of comprehensive two dimensional gas chromatographic separations

Comprehensive two-dimensional gas chromatography (GC×GC) is recognised as a powerful tool for the separation of complex mixtures of volatile and semi-volatile compounds. In the analysis of challenging samples containing highly concentrated, active analytes or those with complicated matrices, it is often the case that less than ideal chromatography is produced. GC×GC chromatograms of such samples typically contain broad, tailing analyte bands. This results in difficulties with quantitation and poor utilisation of the separation space. In this study we investigated the inlet and the modulator as the potential sources of these tailing bands. A simple inlet backflushing device was developed to isolate the inlet from the primary column after the injection, and a similar setup was used to isolate the modulator from the primary column. The device allowed us to divert carrier gas flow back through the inlet at a specified time after the injection, while allowing analytes to pass through the column for separation. Analytes retained within the inlet were prevented from entering the column, and were subsequently removed via the carrier gas split line. The study revealed that the inlet plays a significant role in the development of tailing chromatographic bands, while the modulator simply modulates the already elongated band. Inlet backflushing is a cheap, simple and effective tool that can be used to improve the chromatography of problematic GC×GC analyses of samples consisting of concentrated and active analytes, those derived from natural products and containing complicated matrices.

Targeted Analysis of Protein Phosphorylation by 2D Electrophoresis.

Two-dimensional (2D) gel electrophoresis combines isoelectric focusing in the first and SDS polyacrylamide gel electrophoresis in the second dimension to separate complex mixtures of proteins with unequalled resolution and sensitivity. It is well suited for the analysis of posttranslational protein modifications as most of them affect the isoelectric point and, therefore, the focusing behavior of the protein in the first dimension. It is particularly useful for low-abundance proteins, as it provides a first indication of PTMs, before establishing methods for protein isolation. For targeted proteomics of more abundant proteins, 2D electrophoresis itself may be the method of choice for the isolation of posttranslationally modified isoforms of the protein of interest for mass spectrometric analyses. Protein phosphorylation can be detected by use of phospho-specific stains or antibodies, or by comparing spot patterns of a protein sample before and after phosphatase treatment. Here we describe a simple method, combining 2D gel electrophoresis and western blot analysis with dephosphorylation by λ-phosphatase to analyze the phosphorylation status of oxophytodienoic acid reductase 3 in protein extracts from different organs of tomato and Arabidopsis plants.

Role of UHPLC in evaluating as-synthesised ligand-protected gold nanoparticles products

This paper reports an efficient and fast UHPLC method to separate complex mixtures of gold nanoparticles synthesised in various conditions.

Implementing stationary-phase optimized selectivity in supercritical fluid chromatography.

The performance of stationary-phase optimized selectivity liquid chromatography (SOS-LC) for improved separation of complex mixtures has been demonstrated before. A dedicated kit containing column segments of different lengths and packed with different stationary phases is commercially available together with algorithms capable of predicting and ranking isocratic and gradient separations over vast amounts of possible column combinations. Implementation in chromatographic separations involving compressible fluids, as is the case in supercritical fluid chromatography, had thus far not been attempted. The challenge of this approach is the dependency of solute retention with the mobile-phase density, complicating linear extrapolation of retention over longer or shorter columns segments, as is the case in conventional SOS-LC. In this study, the possibilities of performing stationary-phase optimized selectivity supercritical fluid chromatography (SOS-SFC) are demonstrated with typical low density mobile phases (94% CO2). The procedure is optimized with the commercially available column kit and with the classical isocratic SOS-LC algorithm. SOS-SFC appears possible without any density correction, although optimal correspondence between prediction and experiment is obtained when isopycnic conditions are maintained. As also the influence of the segment order appears significantly less relevant than expected, the use of the approach in SFC appears as promising as is the case in HPLC. Next to the classical use of SOS for faster baseline separation of all solutes in a mixture, the benefits of the approach for predicting as wide as possible separation windows around to-be-purified solutes in semipreparative SFC are illustrated, leading to significant production rate improvements in (semi)preparative SFC.

Multidimensional capillary electrophoresis.

Multidimensional separation where two or more orthogonal displacement mechanisms are combined is a promising approach to increase peak capacity in CE. The combinations allow dramatic improvement of analytical performance since the total peak capacity is given by a product of the peak capacities of all methods. The initial reports were concentrated on the construction of effective connections between capillaries for 2D analysis. Today, 2D and 3D CE systems are now able to separate real complex biological or environmental mixtures with good repeatability, improved resolution with minimal loss of sample. This review will present the developments in the field of multidimensional CE during the last 15 years. The endeavors in this specific field were on the development of interfaces, interface-free techniques including integrated separations, microdevices, and on-line sample concentration techniques to improve detection sensitivity.

Retention time prediction for dereplication of natural products (CxHyOz) in LC–MS metabolite profiling

The detection and early identification of natural products (NPs) for dereplication purposes require efficient, high-resolution methods for the profiling of crude natural extracts. This task is difficult because of the high number of NPs in these complex biological matrices and because of their very high chemical diversity. Metabolite profiling using ultra-high pressure liquid chromatography coupled to high-resolution mass spectrometry (UHPLC–HR-MS) is very efficient for the separation of complex mixtures and provides molecular formula information as a first step in dereplication. This structural information alone or even combined with chemotaxonomic information is often not sufficient for unambiguous metabolite identification. In this study, a representative set of 260 NPs containing C, H, and O atoms only was analysed in generic UHPLC–HR-MS profiling conditions. Two easy to use quantitative structure retention relationship (QSRR) models were built based on the measured retention time and on eight simple physicochemical parameters calculated from the structures. First, an original approach using several partial least square (PLS) regressions according to the phytochemical classes provided satisfactory results with an easy calculation. Secondly, a unique artificial neural network (ANN) model provided similar results on the whole set of NPs but required dedicated software. The retention prediction methods described in this study were found to improve the level of confidence of the identification of given analytes among putative isomeric structures. Its applicability was verified for the dereplication of NPs in model plant extracts.

Evaluation of the impact of peak description on the quantitative capabilities of comprehensive two-dimensional liquid chromatography

Comprehensive, two-dimensional liquid chromatography (LC×LC) is a powerful technique for the separation of complex mixtures. Most studies using LC×LC are focused on qualitative efforts, such as increasing peak capacity. The present study examined the use of LC×LC–UV/vis for the separation and quantitation of polycyclic aromatic hydrocarbons (PAHs). More specifically, this study evaluated the impact of different peak integration approaches on the quantitative performance of the LC×LC method. For well-resolved three-dimensional peaks, parameters such as baseline definition, peak base shape, and peak width determination did not have a significant impact on accuracy and precision. For less-resolved peaks, a dropped baseline and the summation of all slices in the peak improved the accuracy and precision of the integration methods. The computational approaches to three-dimensional peak integration are provided, including fully descriptive, select slice, and summed heights integration methods, each with its own strengths and weaknesses. Overall, the integration methods presented quantify each of the PAHs within acceptable precision and accuracy ranges and have comparable performance to that of single dimension liquid chromatography.

Towards point of care testing for C. difficile infection by volatile profiling, using the combination of a short multi-capillary gas chromatography column with metal oxide sensor detection

Rapid volatile profiling of stool sample headspace was achieved using a combination of short multi-capillary chromatography column (SMCC), highly sensitive heated metal oxide semiconductor sensor and artificial neural network software. For direct analysis of biological samples this prototype offers alternatives to conventional gas chromatography (GC) detectors and electronic nose technology. The performance was compared to an identical instrument incorporating a long single capillary column (LSCC). The ability of the prototypes to separate complex mixtures was assessed using gas standards and homogenized in house ‘standard’ stool samples, with both capable of detecting more than 24 peaks per sample. The elution time was considerably faster with the SMCC resulting in a run time of 10 min compared to 30 min for the LSCC. The diagnostic potential of the prototypes was assessed using 50 C. difficile positive and 50 negative samples. The prototypes demonstrated similar capability of discriminating between positive and negative samples with sensitivity and specificity of 85% and 80% respectively. C. difficile is an important cause of hospital acquired diarrhoea, with significant morbidity and mortality around the world. A device capable of rapidly diagnosing the disease at the point of care would reduce cases, deaths and financial burden.

The power of ion mobility-mass spectrometry for structural characterization and the study of conformational dynamics

Mass spectrometry is a vital tool for molecular characterization, and the allied technique of ion mobility is enhancing many areas of (bio)chemical analysis. Strong synergy arises between these two techniques because of their ability to ascertain complementary information about gas-phase ions. Ion mobility separates ions (from small molecules up to megadalton protein complexes) based on their differential mobility through a buffer gas. Ion mobility-mass spectrometry (IM-MS) can thus act as a tool to separate complex mixtures, to resolve ions that may be indistinguishable by mass spectrometry alone, or to determine structural information (for example rotationally averaged cross-sectional area), complementary to more traditional structural approaches. Finally, IM-MS can be used to gain insights into the conformational dynamics of a system, offering a unique means of characterizing flexibility and folding mechanisms. This Review critically describes how IM-MS has been used to enhance various areas of chemical and biophysical analysis.

Separation of complex sugar mixtures on a hydrolytically stable bidentate urea-type stationary phase for hydrophilic interaction near ultra high performance liquid chromatography

A stationary phase bearing both bridged bis-ureido and free amino groups (USP-HILIC-NH2 -2.5SP) for high-speed hydrophilic interaction liquid chromatography separations was prepared using a one-pot two-step procedure starting from 2.5 μm totally porous silica particles. Highly polar compounds, such as polyols, hydroxybenzoic acids, and sugars, were successfully analyzed in shorter times and with higher peak efficiency, when compared to results obtained with a bidentate urea-type column packed with 5 μm particles. Increased sugarophilicity and better peak shape were attested for the USP-HILIC-NH2 -2.5SP column (100 × 3.2 mm id) when compared with two commercially available UHPLC columns, namely an acquity BEH amide packed with totally porous 1.7 μm microparticles and a HILIC Kinetex column packed with core-shell 2.6 μm particles. Finally, the new column was employed in the separation of complex mixture of sugars (mono-, di-, and oligosaccharides) and in the analysis of beer samples. The resulting chromatograms showed good selectivity and overall resolution, while the catalyzing effect of the free amino moieties resulted in excellent peak shapes and in the absence of split peaks due to sugar anomerization phenomena.

Metal–organic frameworks as stationary phases for mixed-mode separation applications

Polymorphic metal-organic framework (MOF) materials offer a platform for small-scale separation of complex mixtures of polycyclic aromatic hydrocarbons (PAHs) and polar compounds. Retention factors show dependence on both analyte dimensions and polarity, suggesting mixed-mode separation, allowing complete resolution of some analytes from multi-component mixtures.

Metabolite fingerprinting by capillary electrophoresis-mass spectrometry.

Changes in metabolite concentrations in response to specific diseases, treatments, diets, or other factors can be used to understand the complex mechanisms that control and regulate the human body and potentially detect the onset of disease prior to the observation of symptoms in a patient. Different analytical and chemometric platforms are necessary to detect as many metabolites as possible in different biological fluids. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) is a particularly attractive, although still not common, approach for metabolomics for the detection of mainly polar and ionic metabolites. Among its main features, CE provides the capability to separate complex mixtures with high resolution and minimum sample treatment. However, the routine, automated use of CE-MS is not without challenges. In this chapter we describe a well-tested method for fingerprinting serum and urine using CE-TOF-MS. We describe below a sensitive and quite robust method for metabolomics with CE-MS including sample treatment, separation conditions, instrumental setup, and identification of 76 metabolites in the profile. Useful advice for daily practice is also included for every step of the procedure.

A “plug-and-use” approach towards facile fabrication of capillary columns for high performance nanoflow liquid chromatography

Capillary columns used for nanoflow liquid chromatography play an important role in modern proteomics. High quality columns are needed to provide high peak capacity and highly reproducible separations. This is extremely important when multiple separations were compared in parallel in searching for potential biomarkers. Herein, we introduce a “plug-and-use” fritting technology for fabrication of high quality and highly reproducible capillary columns. Due to the identical length, good permeability, and stability of the prefabricated frits adopted, the capillary columns presented excellent performance consistency in terms of retention time, peak width as well as peak capacity at a column-to-column level (relative standard deviations, RSDs, at 0.4–0.9%, 2.1–3.6%, and 2.7%, respectively, n=6) for separations of complex mixtures of protein digest. For capillary columns packed with 5μm particles, high separation efficiency was demonstrated by the minimum plate height of 11μm, approaching the theoretical performance limit of such material. For separations of protein digests, the columns demonstrated excellent peak capacities of 110 and 300 for 20 and 360min gradients, respectively. The simple fabrication, good performance as well as consistent quality of such columns provide a reliable tool for high throughput separations requiring the use of multiple high performance capillary columns in parallel.

GC models for separation optimization in pressure-tuneable tandem capillary columns operated isothermally. Part 1: Theoretical aspects

GC column selectivity can be continuously adapted to suit analytical needs using a flow-tuneable tandem system. Its application for the separation of complex mixtures requires a deep understanding of the theory in this area. Although a number of researchers have developed specific models, a general and exhaustive theory is still missing. In this paper, we have made an implementation of pre-existing models on tandem-column assemblies operated isothermally. In particular, we have investigated the effect of column length and diameter, phase thickness, and oven temperature on chromatographic parameters, such as capacity factor, selectivity, and intrinsic resolution. A new approach for the correct choice of the working temperature has been proposed.

Separation of carbohydrates using hydrophilic interaction liquid chromatography

A strategy was developed to rapidly evaluate chromatographic properties of hydrophilic interaction chromatography (HILIC) columns for separating carbohydrates. Seven HILIC columns (Silica, Diol, TSK Amide-80, XAmide, Click Maltose, Click β-CD, and Click TE-Cys columns) were evaluated by using three monosaccharide and seven disaccharides as probes. The influence of column temperature on the peak shape and tautomerization of carbohydrates, as well as column selectivity were investigated. The influence of surface charge property on the retention was also studied by using glucose, glucuronic acid, and glucosamine, which indicated that buffer salt concentration and pH value in mobile phase was necessary to control the ionic interactions between ionic carbohydrates and HILIC columns. According to evaluation results, the XAmide column was selected as an example to establish experimental schemes for separation of complex mixtures of oligosaccharide.

Conceptual Design of Distillation-Based Hybrid Separation Processes

Hybrid separation processes combine different separation principles and constitute a promising design option for the separation of complex mixtures. Particularly, the integration of distillation with other unit operations can significantly improve the separation of close-boiling or azeotropic mixtures. Although the design of single-unit operations is well understood and supported by computational methods, the optimal design of flowsheets of hybrid separation processes is still a challenging task. The large number of operational and design degrees of freedom requires a systematic and optimization-based design approach. To this end, a structured approach, the so-called process synthesis framework, is proposed. This article reviews available computational methods for the conceptual design of distillation-based hybrid processes for the separation of liquid mixtures. Open problems are identified that must be addressed to finally establish a structured process synthesis framework for such processes.

Monolithic columns in liquid phase separations

The separation of chemical entities in a complex sample mixture is a common problem in chemical/biochemical analysis. Since the vast majority of sample mixtures exist in solution or are brought into solution, liquid phase separations have become a preferred choice for resolving sample mixtures. Within this context and in its broader sense, liquid chromatography (LC) continues to be an indispensable technique in most analytical/bioanalytical laboratories. This is the result of numerous theoretical and technological advances, which have had a remarkable impact on chemical analysis—one recent example is the field of proteomics, which relies heavily on LC to separate complex mixtures of peptides from proteolytic digests. Indeed, it is difficult to imagine an analytical laboratory without state-of-the-art LC equipment. Column technology is one of the most investigated research topics in chromatographic techniques, and LC is not an exception. This is not surprising since it is within the column that the chromatographic processes occur, making the column the “heart” of the separation system. The development of new separation media is essential in achieving a high degree of selectivity, high efficiency, and faster separations to deal with the increasing demands imposed by the complexity of samples in chemical analysis. In recent years, we have seen a remarkable evolution in column technology for LC. Although columns packed with silica microparticulates have predominated in LC and ancillary techniques, monolithic columns have emerged as a viable alternative for liquid phase separations. A monolithic column can be defined as a continuous solid matrix that is porous in nature, containing interconnected flow paths, and can take various formats (e.g., rod-like, flat bed, tubular); the retentive phase is at the surface of this material. The porous, rod-like structures have been the most popular. These porous structures are generally classified into polymeric or silica-based monoliths and can be fabricated by in situ polymerization of organic monomeric precursors in porogenic solvents, for polymeric monoliths, or by sol–gel chemistry in the case of silica monoliths. The main attraction of monolithic columns compared with particle-packed columns is their high axial permeability. This provides low back pressures with excellent flow characteristics, fast mass transfer, and fast separations, without sacrificing separation efficiency. After about two decades of developmental progress since the initial seminal work of Hjerten (1989), Svec (early 1990s), and Tanaka (mid 1990s), monolithic columns have found applicability in different separation modes: reversed-phase chromatography, ion-exchange chromatography, hydrophilic interaction chromatography, size-exclusion chromatography, molecular imprints, and affinity chromatography, to mention a few, and a variety of such modes are commercially available. Monoliths have been used in various formats beyond LC, including solid-phase extraction, sample preconcentration, and 96-well disc arrays for the separation of many compounds in, for example, the pharmaceutical, environmental, microfluidics, and molecular bioprocessing fields. Today, developmental work and applications of monolithic columns in liquid phase separations continue. A quick search in the Web of Knowledge (Thomson Published in the topical collection Monolithic Columns in Liquid Phase Separations with guest editor Luis A. Colon.