Analytical Separation Techniques Research Articles

Recent advances in miniaturization—The role of microchip electrophoresis in clinical analysis

This review is a follow-up from the last review published in 2012 that covers the same topic [22]. Its aim is to cover new innovations and developments since then, involving the role of microchip capillary electrophoresis (MCE) in clinical analysis. MCE has shown great promise in separation science, as it's a state-of-the-art analytical separation technique that offers speed, portability, reduced sample, and reagent requirements, repeatability, can be used online or offline, and can be coupled with many kinds of analytical detectors. It is particularly important in clinical analysis, as it offers online continuous monitoring, can handle small volumes of highly complex biological samples due to its powerful discriminating ability, and it facilitates point-of-care testing. Also microchip separation platforms have been used for sample clean-up, filtration, derivatization, separation and detection, acting as multifunctional platforms ideal for minute sample volumes. In recent times the clinical world has witnessed a need for rapid turnaround times on analysis of very complex samples, and so there has been a shift toward point-of-care testing and other miniaturized analytical techniques as a favorable solution. Research developments in this area have been catalyzed by rapid strides in knowledge in the areas of proteomics, metabolomics, and molecular biology in the quest to understand the physiology of cells, proteins, genes, molecular interactions, and the pathophysiology of disease. Herein, we present selected examples detailing the use of MCE in some of the latest and most exciting clinical applications.

Probing Histidine-Stabilized Gold Nanoclusters Product by High-Performance Liquid Chromatography and Mass Spectrometry

A major hurdle in assessing the biological, chemical and physical properties of current nanoparticles lies in their complex nature in terms of size, shape, and composition. As such, it is vital to develop a high-resolution analytical separation technique to fractionate these nanomaterials. Herein, we demonstrate an unprecedented chromatographic fractionation of gold nanoclusters stabilized with histidine (His-AuNCs) with core diameter smaller than 1 nm. His-AuNCs product has been successfully separated by reverse-phase high-performance liquid chromatography using a binary mixture of methanol and ammonium acetate in water and an optimal solvent elution program. The separated His-AuNCs are online-characterized by UV–vis absorption spectroscopy, and their spectral features are closely related to the number of gold (Au) atom. The absorption band shifts to the lower energy as the number of Au atom increases. The separated His-AuNCs fractions are further collected and anatomized by matrix-assisted laser desorpt...

Evaluation of Several Minor Alkaloid Levels in Tobacco with Separation of R- and S-Nornicotine

Abstract The chiral separation of minor alkaloids from tobacco is of interest because R and S isomers of these compounds have differences in their physiological activity. This difference is also reflected in the physiological properties of tobaccospecificN’-nitrosamines (TSNAs), in particular that of N’-nitrosonornicotine. This compound results mainly from nornicotinenitrosation. The previously reported analytical techniques for the enantiomer separation of minor alkaloids have various shortcomings, such as the need for bidimensional chromatography or poor enantiomer separation. A new method for the analysis of nornicotine, anabasine and anatabine has been developed, based on an original derivatization and a simple gas chromatography/ mass spectrometry (GC/MS) analysis. The method allows separate quantitation of S-nornicotine and R-nornicotine, and the analysis of anabasine and anatabine (without isomer separation). It was found that the proportion of S-nornicotine in the total nornicotine present in tobacco varies, depending on the tobacco type, between 52.6% for a flue-cured tobacco to 91.4% for a Burley. Green tobaccos (freeze dried) showed lower levels of minor alkaloids and S-nornicotine accounted for between 31.6% to 43.8% of the total nornicotine (in the analyzed samples).

A multilevel analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

Diaminofluoresceins are widely used probes for detection and intracellular localization of NO formation in cultured/isolated cells and intact tissues. The fluorinated derivative 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) has gained increasing popularity in recent years because of its improved NO sensitivity, pH stability, and resistance to photobleaching compared to the first-generation compound, DAF-2. Detection of NO production by either reagent relies on conversion of the parent compound into a fluorescent triazole, DAF-FM-T and DAF-2-T, respectively. Although this reaction is specific for NO and/or reactive nitrosating species, it is also affected by the presence of oxidants/antioxidants. Moreover, the reaction with other molecules can lead to the formation of fluorescent products other than the expected triazole. Thus additional controls and structural confirmation of the reaction products are essential. Using human red blood cells as an exemplary cellular system we here describe robust protocols for the analysis of intracellular DAF-FM-T formation using an array of fluorescence-based methods (laser-scanning fluorescence microscopy, flow cytometry, and fluorimetry) and analytical separation techniques (reversed-phase HPLC and LC-MS/MS). When used in combination, these assays afford unequivocal identification of the fluorescent signal as being derived from NO and are applicable to most other cellular systems without or with only minor modifications.

A Power Function Method for Estimating Base Flow

Analytical base flow separation techniques are often used to determine the base flow contribution to total stream flow. Most analytical methods derive base flow from discharge records alone without using basin-specific variables other than basin area. This paper derives a power function for estimating base flow, the form being aQ(b) + cQ, an analytical method calibrated against an integrated basin variable, specific conductance, relating base flow to total discharge, and is consistent with observed mathematical behavior of dissolved solids in stream flow with varying discharge. Advantages of the method are being uncomplicated, reproducible, and applicable to hydrograph separation in basins with limited specific conductance data. The power function relationship between base flow and discharge holds over a wide range of basin areas. It better replicates base flow determined by mass balance methods than analytical methods such as filters or smoothing routines that are not calibrated to natural tracers or empirical basin and gauge-specific variables. Also, it can be used with discharge during periods without specific conductance values, including separating base flow from quick flow for single events. However, it may overestimate base flow during very high flow events. Application of geochemical mass balance and power function base flow separation methods to stream flow and specific conductance records from multiple gauges in the same basin suggests that analytical base flow separation methods must be calibrated at each gauge. Using average values of coefficients introduces a potentially significant and unknown error in base flow as compared with mass balance methods.

Multiple liquid chromatography separations and nanoESI‐ion trap detection of plasma proteins in search of stroke biomarkers: A pilot study

Stroke is the most common cause of morbidity and death in the Western world, following ischemic heart disease and cancer. Stroke can be of two types, ischemic or hemorrhagic, with ischemic stroke accounting for approximately 85% of the total number of strokes. Well-recognized environmental risk factors for stroke include hypertension, smoking, diabetes mellitus, atrial fibrillation, and atherosclerosis. Computed tomography (CT) scanning is used to diagnose hemorrhagic stroke but is relatively ineffective and may remain normal in patients with mild ischemic strokes. Magnetic Resonance Imaging (MRI) is more sensitive in detecting ischemia than CT, especially in the diagnosis of mild stroke but it is still not 100% sensitive or precise. A simple and low-cost, rapid blood test to confirm a clinical and imaging diagnosis of ischemic stroke would be extremely useful. Based on this, the central idea of this paper is to develop a method that would be applicable to a statistically viable sample set to provide candidate biomarkers for distinguishing stroke types. In search of these candidate biomarkers, different analytical separation techniques have been used to screen for major differences in the proteomes of patients plasma samples with proteomics for identification.

SPECIAL ISSUE: INSTRUMENTATION AND STATIONARY PHASES FOR LIQUID CHROMATOGRAPHY

Analytical separation techniques have proven to be globally needed and will continue to have significant roles in many scientific disciplines. Liquid chromatography (LC) is a powerful separation te...

The composition, origin and fate of complex mixtures in the maltene fractions of hydrothermal petroleum assessed by comprehensive two-dimensional gas chromatography

Sedimentary organic matter in hydrothermal systems can be altered by high temperature fluids to generate petroleum. The saturated and aromatic fractions of these hydrothermal oils are compositionally similar to conventional oil with the exception that they often contain higher concentrations of polycyclic aromatic hydrocarbons (PAH) as well as substantial mixtures of coeluting organic compounds that produce dramatically rising signal on the baseline of gas chromatograms termed unresolved complex mixtures (UCMs). Little is known about the compounds that compose UCMs and why or how they form. This is in part due to an inability to discriminate between in situ and migrated components that characterize the petroleum generated in hydrothermal systems. However, UCMs are also a product of the limitations imbedded in analytical separation techniques. With the advent of comprehensive two-dimensional gas chromatography (GC×GC), a revision of what should constitute molecular complexity needs to be considered. We address these problems by comparing the molecular compositions of the maltene fractions of three previously published hydrothermal petroleum samples using time of flight-mass spectrometry (GC×GC–ToF-MS) and 12 hydrothermal petroleum samples in cores from three locales using comprehensive two-dimensional gas chromatography with flame ionization detection (GC×GC–FID). The sediment cores were collected from Middle Valley, located off the axis of the Juan de Fuca Ridge, and the Escanaba Trough, along the Gorda Ridge, both in the NE Pacific Ocean, as well as from the Guaymas Basin in the Gulf of California. We define a UCM in GC×GC data to be a condition in which ⩾25% of the detected peaks within a chromatographic area coelute in either the first or second dimension. In turn, complex (CM) and simple mixtures (SM) are defined as having 5–24% and <5% coelution, respectively. All CM and UCMs were dominated by an array of configurational isomers, which becomes increasingly aromatic with higher molecular weight. We relate this to a multi-molecular complexity metric (MCM) by quantitatively comparing the difference in total peak variance and peak density for a GC×GC chromatogram. MCM values correlate with biomarker thermal maturity ratios for the Escanaba Trough and Guaymas Basin samples indicating that molecular complexity in these hydrothermal environments is in part a function of burial temperatures. Partial Least Squares (PLS) linear regression was applied to the total number of peak retention times as a proxy for the bulk molecular differences between each hydrothermal oil sample. Differences in the sample regressions correlate with the thermal maturity and the degree of PAH alkylation, indicating that this technique can be used to assess the degree of oxidative weathering due to dehydrogenation and hydrocarbon cracking. Subtracted chromatograms were then used to quantitatively track all of the individual molecular changes within the pyrolytic regime at Escanaba Trough. These subtracted chromatograms indicate that high molecular weight PAHs are highly mobile in hydrothermal fluids and may represent a phase partitioning that is occurring at greater depths. This phase condenses just below the seafloor to form an UCM in the near surface sediments. Saturated hydrocarbon biomarkers, such as hopanes, steranes and biphytanes are less mobile and more prone to being cracked and/or aromatized prior to migration toward the ocean floor. Together these techniques suggest that the molecular complexity of hydrothermal petroleum maximizes during the early stages of thermal maturation. The diversity of compounds forming these UCMs then decreases with increasing dehydrogenation, dealkylation and condensation reactions associated with elevated thermal stress and exposure to oxidants within the hydrothermal fluids.

Capillary and microchip electrophoresis: Challenging the common conceptions

Capillary electrophoresis (CE) has long been regarded as a powerful analytical separation technique that is an alternative to more traditional methods such as gel electrophoresis (GE) and liquid chromatography (LC). It is often touted as having a number of advantages over both of these, such as speed, flexibility, portability, sample and reagent requirements and cost, but also a number of disadvantages such as reproducibility and sensitivity. Microchip electrophoresis (ME), the next evolutionary step, miniaturised CE further providing improvements in speed and sample requirements as well as the possibility to perform more complex and highly integrated analyses. CE and ME are seen as a viable alternative to GE, but are often considered to be inferior to LC. This review will consider the strengths and weaknesses of both CE and ME and will challenge the common conceptions held about these.

Simple HPLC method for detection of trace ephedrine and pseudoephedrine in high‐purity methamphetamine

A simple and sensitive HPLC technique was developed for the qualitative determination of ephedrine and pseudoephedrine (ephedrines), used as precursors of clandestine d-methamphetamine hydrochloride of high purity. Good separation of ephedrines from bulk d-methamphetamine was achieved, without any extraction or derivatization procedure on a CAPCELLPACK C18 MGII (250 × 4.6 mm) column. The mobile phase consisted of 50 mM KH2 PO4-acetonitrile (94:6 v/v %) using an isocratic pump system within 20 min for detecting two analytes. One run took about 50 min as it was necessary to wash out overloaded methamphetamine for column conditioning. The analytes were detected by UV absorbance measurement at 210 nm. A sample (20 mg) was simply dissolved in 1 mL of water, and a 50 μL aliquot of the solution was injected into the HPLC. The detection limits for ephedrine and pseudoephedrine in bulk d-methamphetamine were as low as 3 ppm each. This analytical separation technique made it possible to detect ephedrine and/or pseudoephedrine in seven samples of high-purity d-methamphetamine hydrochloride seized in Japan. The presence of trace ephedrines in illicit methamphetamine may strongly indicate a synthetic route via ephedrine in methamphetamine profiling. This method is simple and sensitive, requiring only commonly available equipment, and should be useful for high-purity methamphetamine profiling.

Nanotechnology and chip level systems for pressure driven liquid chromatography and emerging analytical separation techniques: A review

Pressure driven liquid chromatography (LC) is a powerful and versatile separation technique particularly suitable for differentiating species present in extremely small quantities. This paper briefly reviews main historical trends and focuses on more recently developed technological approaches in miniaturization and on-chip integration of LC columns. The review emphasizes enabling technologies as well as main technological challenges specific to pressure driven separations and highlights emerging concepts that could ultimately overcome fundamental limitations of conventional LC columns.

Tandem hollow-fiber flow field-flow fractionation

Reinjection of one ore more collected fractions of eluted samples is recognized as a useful procedure in analytical separation techniques, among which field-flow fractionation (FFF), to improve the actual separation of complex samples. Hollow-fiber flow FFF (HF5) is a micro-channel subset of flow FFF (F4), which has recently reached a performance comparable to that of standard, flat-channel F4. To further improve HF5 of complex protein samples, we present a new device and method for in-line, reinjection HF5 that we call tandem HF5 (HF5/HF5). HF5 is ideally suited for tandem operation because (1) small channel volume and low operation flow rates allow reducing dilution and volume of the collected fractions, and (2) the relaxation/focusing step that takes place between the 1st and 2nd run (refocusing) allows reestablishing the volume and concentration of the sample plug before the 2nd elution. HF5/HF5 proves particularly effective in the case of oligomeric proteins since it allows collecting and reinjecting the bands that correspond to each separated oligomeric form. This provides information on the dynamic equilibria between the different oligomers. For HF5/HF5 operations, a modified, prototype HF5 instrumentation is presented which includes a “trap” constituted of a four-port, two-way valve positioned downstream the UV detector and a collection loop. The effect of refocusing conditions on HF5/HF5 performance is investigated by varying refocusing time. With a complex protein samples such as blood serum, HF5/HF5 can improve detectability of the low abundance components since overloading effects due to high-abundance components are reduced. This is shown for serum lipoproteins: while after the 1st run high density lipoproteins (HDLs) are not separated from high-abundance serum proteins, after the 2nd run it is shown possible to separate the HDL subclasses.

25 Years Full of Chemical Discovery

25 Years Full of Chemical Discovery

Book Review: Ion-Pair Chromatography and Related Techniques

Book Review: Ion-Pair Chromatography and Related Techniques

Emerging Analytical Separation Techniques with High Throughput Potential for Pharmaceutical Analysis, Part I: Stationary Phase and Instrumental Developments in LC

In recent years, a trend of change has been observed within pharmaceutical industry. As modern drug discovery has reached a remarkable level of complexity and drugs need to be discovered, developed and produced against strict timelines and within cost- and regulatory constraints, industry seeks "lean" solutions to increase productivity. Among them, increasing the sample throughput of the ever-growing number of necessary (routine) analyses has become a popular target to cut precious time. For the last thirty years, High-Performance Liquid Chromatography (HPLC) has been the leading technology when it comes to various analyses in pharmaceutical industry; however, its necessity of serial analyses taking typically 10-45 min has been a sample throughput-limiting barrier. Lately, the fundamentals of HPLC have been exploited to raise new technologies that can speed up analyses to ground breaking limits, without compromising separation efficiency. This paper reviews some promising technologies, i.e. totally porous sub-2microm particles accompanied by pressures up to 1000 bar (Ultra-Performance Liquid Chromatography or UPLC), fused-core particle technology, monolithic supports and High Temperature Liquid Chromatography (HTLC), having the potential to take LC to the next level in pharmaceutical industry. As each analytical method has its own demands, the advances of the above technologies are discussed for different applications in pharmaceutical analysis where high-throughput analysis can be meaningful, i.e. in a drug discovery and development setting, and in quality operations. Both chemical and biological pharmaceuticals are considered. We discuss the perspectives of these technologies and their realizations up to now in high-throughput pharmaceutical analysis.

Emerging Analytical Separation Techniques with High Throughput Potential for Pharmaceutical Analysis, Part II: Novel Chromatographic Modes

In this review paper, the high-throughput potential of some novel chromatographic modes is surveyed. The modes are Hydrophilic Interaction Liquid Chromatography (HILIC), Supercritical Fluid Chromatography (SFC), and Polar Organic Solvent Chromatography (POSC). Their high throughput potential will be discussed in three domains, i.e. drug discovery, bio-analysis in clinical drug development, and quality control (QC) testing, and is illustrated with some examples.

Methodological Aspects about Determination of Phenolic Compounds and In Vitro Evaluation of Antioxidant Capacity in the Honey: A Review

The antioxidant activity of honey varies greatly depending on the honeys floral source. There is little knowledge about the profiles of antioxidant substances in honey from different floral sources. The variation in these profiles might be responsible for the widely varying ability of honey to protect against oxidative reactions. Honey is rich in polyphenol compounds, which act as natural antioxidants, and are becoming increasingly popular because of their potential role in contributing to human health. These compounds can also be used as indicators in studies about the floral and geographical origin of the honey. Therefore, we overviewed the current analytical methods for measuring polyphenols and antioxidant capacity in honey. The analytical procedure to determine individual phenolic compounds involves their extraction from the sample, analytical separation and quantification. The analytical separation techniques widely employed are gas chromatography, high-pressure liquid chromatography and capillary electrophoresis. The techniques to evaluate the antioxidant capacity are based on colorimetric assays such as DPPH, FRAP, TEAC (ABTS) and microplate fluorescence reader like ORAC assay. Keywords: Phenolic compounds, Honey, Antioxidant capacity, TEAC, FRAP, DPPH, ORAC

Advances in analytical separations

This special issue of Analytical and Bioanalytical Chemistry contains a selection of 12 manuscripts originating from lectures and posters presented at the 27th International Symposium on Chromatography, held in Munster, Germany, from September 21 to 25, 2008. Both the conference and the articles impressively demonstrate that analytical separations are far from only being mature routine techniques with limited potential for innovation. Yes, these techniques are established tools in industry and academia owing to their high separation power and excellent performance/cost ratio. However, they also are one of the most important driving forces for innovation inmany established and emerging areas of science. The topics covered in these contributions range from the development of stationary phases to the development and application of complex hyphenated techniques. As probably the best example, liquid chromatography/mass spectrometry has strongly pushed liquid chromatography to new heights, and its application areas and significance in industry are still growing rapidly. It is therefore not surprising that it also plays a major role in this special issue. Two manuscripts on electrodriven analytical separation techniques complement the chromatography manuscripts for the analysis of ionic compounds. We wish the readers interesting and rewarding reading of the articles in “Advances in Analytical Separation.” Uwe Karst (right) has held the Chair of Analytical Chemistry at the University of Munster since 2005. His research interests include hyphenated techniques and speciation analysis.

Systematic NMR Analysis of Stable Isotope Labeled Metabolite Mixtures in Plant and Animal Systems: Coarse Grained Views of Metabolic Pathways

BackgroundMetabolic phenotyping has become an important ‘bird's-eye-view’ technology which can be applied to higher organisms, such as model plant and animal systems in the post-genomics and proteomics era. Although genotyping technology has expanded greatly over the past decade, metabolic phenotyping has languished due to the difficulty of ‘top-down’ chemical analyses. Here, we describe a systematic NMR methodology for stable isotope-labeling and analysis of metabolite mixtures in plant and animal systems.Methodology/Principal FindingsThe analysis method includes a stable isotope labeling technique for use in living organisms; a systematic method for simultaneously identifying a large number of metabolites by using a newly developed HSQC-based metabolite chemical shift database combined with heteronuclear multidimensional NMR spectroscopy; Principal Components Analysis; and a visualization method using a coarse-grained overview of the metabolic system. The database contains more than 1000 1H and 13C chemical shifts corresponding to 142 metabolites measured under identical physicochemical conditions. Using the stable isotope labeling technique in Arabidopsis T87 cultured cells and Bombyx mori, we systematically detected >450 HSQC peaks in each 13C-HSQC spectrum derived from model plant, Arabidopsis T87 cultured cells and the invertebrate animal model Bombyx mori. Furthermore, for the first time, efficient 13C labeling has allowed reliable signal assignment using analytical separation techniques such as 3D HCCH-COSY spectra in higher organism extracts.Conclusions/SignificanceOverall physiological changes could be detected and categorized in relation to a critical developmental phase change in B. mori by coarse-grained representations in which the organization of metabolic pathways related to a specific developmental phase was visualized on the basis of constituent changes of 56 identified metabolites. Based on the observed intensities of 13C atoms of given metabolites on development-dependent changes in the 56 identified 13C-HSQC signals, we have determined the changes in metabolic networks that are associated with energy and nitrogen metabolism.

Influence of Acetyl and Amide Groups on Peptides RP-LC Retention Behavior

Reversed phase liquid chromatography (RP-LC) is still one of the most efficient analytical techniques for separation of complex biologically active analytes, including proteins and peptides. However, the separation of those analytes is not easy to perform in practice. Therefore, the appropriate optimization of the separation conditions is necessary. The aim of the study was to explore the influence of acetyl and amide groups in peptides on their retention in gradient elution reversed phase LC. Moreover, the investigation regards the influence of temperature and gradient time for such peptides. Those results were suggested in view of their usefulness in predictions of peptides RP-LC retention on the basis of quantitative structure retention relationships for proteomic research.