Cyclic electrophoretic and chromatographic separation methods

A review is given of the application of cyclic analytical methods in capillary electroseparation (CE) and liquid chromatography (LC) systems. Cyclic methods have been used since the early sixties in chromatographic systems to overcome pressure limitations to resolution. From the early nineties on they have also been applied in capillary electroseparation systems to overcome voltage limitations. Some basic theory is given, outlining the temporal development of resolution in cyclic CE and LC systems and calculating the maximal resolution that can be obtained as a function of the operational parameters of pressure and electrical field. Simple equations are given for the temporal change in the peak capacity and the loss of peaks from the systems as it occurs in some cyclic systems. Finally, a circular open tubular chromatographic system is proposed using integrated pumping and continuous detection. The performance of such a system is discussed using magnetohydrodynamic and alternating current electroosmotic pumping as examples of integrated pumps and Shah Convolution Fourier transform detection as an example of a continuous detection method.     

Achiral and chiral analysis of duloxetine by chromatographic and electrophoretic methods,a review on the separation methodologies

Duloxetine (DLX) is a widely used antidepressant drug belonging to the class of selective serotonin and norepinephrine reuptake inhibitors (SNRIs); its efficacy has been demonstrated in the treatment of not only major depressive disorders but also diabetic neuropathic pain, generalized anxiety disorder, fibromyalgia or stress urinary incontinence. It is a chiral substance and is used in therapy in the form of the enantiopure S‐DLX, which is twice as active as R‐DLX. Several methods have been published for the achiral and chiral determination of DLX in pharmaceuticals, biological materials and environmental samples, the majority using liquid chromatography and capillary electrophoresis coupled with different detection techniques (UV detection, fluorescence, mass spectrometry). The aim of the current review is to provide a systematic survey of the analytical techniques used for the determination of DLX from different matrices.     

Advances in sample preparation in electromigration, chromatographic and mass spectrometric separation methods

The quality of sample preparation is a key factor in determining the success of analysis. While analysis of pharmaceutically important compounds in biological matrixes has driven forward the development of sample clean-up procedures in last 20 years, today's chemists face an additional challenge: sample preparation and analysis of complex biochemical samples for characterization of genotypic or phenotypic information contained in DNA and proteins. This review focuses on various sample pretreatment methods designed to meet the requirements for the analysis of biopolymers and small drugs in complex matrices. We discuss the advances in development of solid-phase extraction (SPE) sorbents, on-line SPE, membrane-based sample preparation, and sample clean-up of biopolymers prior to their analysis by mass spectrometry.     

Capillary electrophoretic analysis of flavonoids.

Combining the effects of electrophoresis and electroendosmosis, flavonoids were separated in less than ten minutes in a fused silica capillary tube with a borate buffer adjusted to pH 10. An increase in the concentration of borate from 0.1 to 0.2 M resulted in longer migration times due to a decrease in electroosmotic flow, but also in improved selectivity and higher resolution of flavonoids. The calibration curve of rutin showed a detection limit of 0.02 mg/mL and linearity over its pharmaceutical concentration range. Using an internal standard of known concentration, the content of rutin in a methanolic extract of Sambuci flos could be determined with a coefficient of variation as small as 3.8% by the molar ratio-peak area ratio method.     

树状大分子接枝型聚合物色谱分离材料研究进展

随着色谱固定相制备技术和材料科学领域的不断发展,目前已经有大量修饰方法和新型材料被用于固相萃取、高效液相色谱以及离子色谱聚合物固定相填料的功能化修饰。其中聚酰胺-胺(PAMAM)树状大分子由于其独特的结构和性质,在色谱分离材料结构完善和性能提升中也发挥了重要的作用。该文主要综述了PAMAM树状大分子在以聚合物为基质的色谱分离材料修饰中的应用,并对其今后的发展进行了展望。     

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

A model system for selective solubilization and fast separation of proteins from the rat liver membrane fraction and purified rat liver plasma membranes for their further proteomic analysis is presented. For selective solubilization, high-pH solutions and a concentrated urea solution, combined with different detergents, are used. After extraction, proteins are separated by anion-exchange chromatography or a combination of anion- and cation-exchange chromatography with convective interaction monolithic supports. This separation method enables fast and effective prefractionation of membrane proteins based on their hydrophobicity and charge prior to one-dimensional (1-D) and 2-D electrophoresis and mass spectrometry. By use of this sample preparation method, the less-abundant proteins can be detected and identified.     

Systematic optimization of micellar electrokinetic chromatographic separation of flavonoids

Summary A simple and rapid systematic optimization scheme was described for the micellar electrokinetic chromatographic separation of a group of flavonoids. The scheme employed an interpretative optimization approach to predict the optimum conditions for the separation of a group of flavonoids by micellar electrokinetic chromatography. By performing a set of nine pre-planned experiments conducted over the maximum working range for the system, global optimum separation conditions could be determined. To validate the optimization procedure, additional experiments were performed using the optimum experimental conditions derived from the optimization scheme. The results showed that satisfactory separation of all the peaks could be obtained. In addition, the application of the method in micropreparative micellar electrokinetic chromatography of the flavonoids was demonstrated.     

Analytical separation and detection methods for flavonoids

Flavonoids receive considerable attention in the literature, specifically because of their biological and physiological importance. This review focuses on separation and detection methods for flavonoids and their application to plants, food, drinks and biological fluids. The topics that will be discussed are sample treatment, column liquid chromatography (LC), but also methods such as gas chromatography (GC), capillary electrophoresis (CE) and thin-layer chromatography (TLC), various detection methods and structural characterization. Because of the increasing interest in structure elucidation of flavonoids, special attention will be devoted to the use of tandem-mass spectrometric (MS/MS) techniques for the characterization of several important sub-classes, and to the potential of combined diode-array UV (DAD UV), tandem-MS and nuclear magnetic resonance (NMR) detection for unambiguous identification. Emphasis will be on recent developments and trends.     

Liquid-liquid and solid-phase extractions of phenols from virgin olive oil and their separation by chromatographic and electrophoretic methods

The high oxidative stability of virgin olive oil is related to its high monounsaturated/polyunsaturated ratio and to the presence of antioxidant compounds, such as tocopherols and phenols. In this paper, the isolation of phenolic compounds from virgin olive oil, by different methods, was tested and discussed. Particularly liquid-liquid and solid-phase extraction methods were compared, assaying, for the latter, three stationary phases (C8, C18 and Diol) and several elution mixtures. Quantification of phenolic and o-diphenolic substances in the extracts was performed by the traditional Folin-Ciocalteau method and the sodium molybdate reaction, respectively. Furthermore, the quantification of phenolic compounds in the extracts and in a standard mixture was carried out both with diode array and mass spectrometric detection and capillary zone electrophoresis.     

Combining immunoassays with chromatographic and electrophoretic separation techniques — a review

The combination of immunoassays with separation techniques such as chromatography and electrophoresis can provide both selectivity and sensitivity that is competitive with any method currently available for molecular analysis. Immunoassays can be carried out on-line and off-line, pre and post separation. The on-line post separation mode is the most promising for routine analysis because of the high throughput that can be achieved but also provides the greatest challenge with regard to compatibility of the interfaced systems. This paper reviews the various approaches that have been researched from a practical immunochemical point of view with emphasis on the special problems incurred with matrix compatibility for on-line post separation systems.     

Analysis of cyanobacterial pigments and proteins by electrophoretic and chromatographic methods

Cyanobacteria are a diverse and ubiquitous group of prokaryotes with several unifying features. Amongst these is the macromolecular structure known as the phycobilisome, which is composed of water-soluble phycobiliproteins covalently bound by linker peptides or proteins in a configuration designed to optimize energy transfer to the photosynthetic reaction center of the organism. Phycobiliproteins are highly fluorescent by virtue of their covalently bound, linear tetrapyrrole chromophores known as bilins. Analysis of these prosthetic pigments, along with other non-water soluble pigments, such as the chlorophylls and carotenoids, can provide insight into microbial diversity. The effects of environmental growth conditions and stresses can also be probed by measuring pigment and protein concentrations. This review will focus, therefore, on applications of various chromatographic and electrophoretic methods for the analysis of cyanobacterial pigment and protein constituents. Although the greatest emphasis will be placed on the measurement of bilins and phycobiliproteins, this review will also consider other pigments and proteins important to cyanobacterial growth and survival, such as chlorophyll a, carotenoids, ectoenzymes, linker and membrane proteins, and extracellular proteins.     

葫芦脲在色谱固定相中的研究进展

葫芦脲是超分子化学中继冠醚、环糊精、杯芳烃之后发展起来的又一类新型高度对称的桶状大环主体分子,是一种由多个甘脲单元组成的大环穴状配体,被誉为"第四代超分子化合物"。由于其具有特殊的分离选择性和稳定性,在超分子化学和色谱的交叉领域备受关注。本文从葫芦脲的结构特征出发,侧重概述了葫芦脲同系物及其衍生物在色谱固定相中的研究现状和进展。     

Trace analysis of surfactants using chromatographic and electrophoretic techniques

Because of the widespread use, increased application of new formulations and immense impact on organisms and ecology surfactants are still in the focus of analytical chemistry. The development of methods with higher selectivity and lower detection limits is important to meet the requirements of greater responsibility for health of people and environment. Efficient separation methods, like HPLC, GC and CE, in combination with sensitive detection, like MS, are to be preferred over collective techniques which can suffer from interfering components. A review on trace analysis of ionic and neutral surfactants including sample preparation steps is presented, considering especially those methods which provide information about homologous and isomeric distribution of surfactant mixtures. Examples for the determination of linear alkylbenzene sulfonates in river water by HPLC and CE are discussed to show the capability of these methods for environmental analyses. As future trends increased applications of LC/MS (very high sensitivity) and also of CE (robustness and possibility for rapid method development) can be predicted.     

Trace analysis of surfactants using chromatographic and electrophoretic techniques

Because of the widespread use, increased application of new formulations and immense impact on organisms and ecology surfactants are still in the focus of analytical chemistry. The development of methods with higher selectivity and lower detection limits is important to meet the requirements of greater responsibility for health of people and environment. Efficient separation methods, like HPLC, GC and CE, in combination with sensitive detection, like MS, are to be preferred over collective techniques which can suffer from interfering components. A review on trace analysis of ionic and neutral surfactants including sample preparation steps is presented, considering especially those methods which provide information about homologous and isomeric distribution of surfactant mixtures. Examples for the determination of linear alkylbenzene sulfonates in river water by HPLC and CE are discussed to show the capability of these methods for environmental analyses. As future trends increased applications of LC/MS (very high sensitivity) and also of CE (robustness and possibility for rapid method development) can be predicted. Received: 30 July 1998 / Revised: 28 October 1998 / Accetped: 1 November 1998     

Recent innovations in protein separation on microchips by electrophoretic methods

Microchips for analytical purposes have attracted great attention over the last 20 years. In the present review, we focus on the most recent development of microchips for electrophoretic separation of proteins. This review starts with a short recalling about the microchips covering the basic microchip layout for CE and the commercial chips and microchip platforms. A short paragraph is dedicated to the surface treatment of microchips, which is of paramount importance in protein analysis. One section is dedicated to on-line sample pretreatment in microchips and summarizes different strategies to pre-concentrate or to purify proteins from complex matrixes. Most of the common modes used for CE of proteins have already been adapted to the chip format, while multidimensional approaches are still in progress. The different routes to achieve detection in microchip are also presented with a special attention to derivatization or labeling of proteins. Finally, several recent applications are mentioned. They highlight the great potential of electrophoretic separations of proteins in numerous fields such as biological, pharmaceutical or agricultural and food analysis. A bibliography with 151 references is provided covering papers published from 2000 to the early 2007.     

Intact protein separation by chromatographic and/or electrophoretic techniques for top-down proteomics

Mass spectrometry used in combination with a wide variety of separation methods is the principal methodology for proteomics. In bottom-up approach, proteins are cleaved with a specific proteolytic enzyme, followed by peptide separation and MS identification. In top-down approach intact proteins are introduced into the mass spectrometer. The ions generated by electrospray ionization are then subjected to gas-phase separation, fragmentation, fragment separation, and automated interpretation of mass spectrometric and chromatographic data yielding both the molecular weight of the intact protein and the protein fragmentation pattern. This approach requires high accuracy mass measurement analysers capable of separating the multi-charged isotopic cluster of proteins, such as hybrid ion trap-Fourier transform instruments (LTQ-FTICR, LTQ-Orbitrap). Front-end separation technologies tailored for proteins are of primary importance to implement top-down proteomics. This review intends to provide the state of art of protein chromatographic and electrophoretic separation methods suitable for MS coupling, and to illustrate both monodimensional and multidimensional approaches used for LC-MS top-down proteomics. In addition, some recent progresses in protein chromatography that may provide an alternative to those currently employed are also discussed.     

Overview of chromatographic and electrophoretic methods for the determination of branched-chain amino acids

The significance of branched-chain amino acids in diseases was clearly shown over the years. This review aims to describe the available techniques for their analytical determination. The article provides examples of the use of various analytical methods. The methods are divided into two categories: derivatization and non-derivatization approaches. Separation is achieved through different chromatography or capillary electrophoresis techniques and can be combined with different detectors such as flame ionization, ultraviolet, fluorescence, and mass spectrometry. It compares the application of various derivatization reagents or detection as such for different detectors.     

Chromatographic, capillary electrophoretic and capillary electrochromatographic techniques in the analysis of flavonoids

An overview is presented of chromatographic methods currently in use to determine flavonoids, including free aglycones, their corresponding glycosides, one by one, and, in the presence of each other. As a basis of selection, the following approaches can be distinguished: critical evaluation of the preliminary steps (extraction/isolation and hydrolysis) as well as the separation, identification and quantitation of constituents both on the basic research level and/or subsequently to various work up procedures. Chromatographic techniques were discussed after extraction/isolation of various flavonoids from several natural matrices. Papers were classified and compared from analytical point of view, primarily on the chromatographic, secondly on the detection techniques applied.     

Enzymatic release of oligosaccharides from glycoproteins for chromatographic and electrophoretic analysis

For most separations-based analyses of glycoprotein oligosaccharides, the first step is release of the oligosaccharides from the polypeptide. Historically, O-linked and N-linked oligosaccharides have been released from glycoproteins using chemical means, such as alkaline degradation (β-elimination) or hydrazinolysis. In the last two decades, a growing repertoire of enzymes, including endoglycosidases and glycoamidases, able to release glycoprotein oligosaccharides under mild conditions, have become available. This review traces the discovery characterization and use of these glycoprotein oligosaccharide releasing enzymes. Emphasis is placed on providing information of practical value for the researcher wishing to incorporate enzymatic oligosaccharide release into their study of glycoprotein oligosaccharide structure and function.     

Comparative validation of amisulpride determination in pharmaceuticals by several chromatographic, electrophoretic and spectrophotometric methods

Depleted uranium (DU) is a by-product of the uranium enrichment process for nuclear fuel. According to the Commission Decision 2002/657/EC, a confirmatory method for the quantification of DU in freeze-dried fish was developed by isotope ratio dynamic reaction cell inductively coupled plasma-mass spectrometry (IR-DRC-ICP-MS). A preliminary study was performed to determine the following parameters: instrumental detection limit (IDL), isotopic ratio measurement limit (IRML), percentage of DU (P(DU)) in presence of natural uranium (NU) and limit of quantification (LoQ(DU)). The analyses were carried out by means of IR-DRC-ICP-MS. Ammonia was the reaction gas used for the dynamic reaction cell. In addition, a sector field inductively coupled plasma mass spectrometer (SF-ICP-MS) was employed to calculate the within-laboratory reproducibility. For the confirmatory method the following parameters were determined: (a) trueness; (b) precision; (c) critical concentrations alpha and beta (CC(alpha), CC(beta)); (d) specificity; (e) stability. Trueness was assessed by using the recovery tests. The recovery and within-laboratory reproducibility were determined by fortifying the blank digested solution of dogfish tissue: six aliquots were fortified at 1, 1.5 and 2 times the LOQ(DU) with 25.0, 37.5 and 50.0 ng L(-1) or 4.16, 6.24, 8.32 microg kg(-1) with a recovery of -8.2, +9.5 and +9.6%, respectively and a within-laboratory reproducibility (three analytical run) of 15.5, 8.0 and 11.0%, respectively. The results for the decision limit and the detection capability were: CC(alpha) = 11.69 ng L(-1) and CC(beta) = 19.8 ng L(-1). The digested solutions resulted to be stable during testing time (60 days) and the method can be considered highly specific as well.