Development of overlapping repeated separation of steviol glycosides with counter current chromatography and a comparison with a conventional repeated separation method

Repeated separation is a valuable method in counter current chromatography, especially on a preparative scale. It can greatly reduce the separation time and the consumption of solvent. In this study, an overlapping repeated separation method was developed. Meanwhile, this method was used to separate steviol glycosides and compared with conventional repeated separation method. The results show that both methods are effective ways for countercurrent chromatography to prepare compounds but the overlapping repeated separation method requires fewer time and solvent than the conventional repeated separation method. So this novel repeated separation method has enormous potential for a preparative separation of target compounds and is very useful for the high‐throughput purification of natural products.     

Development of a relatively cheap and simple automated separation system for a routine separation procedure based on extraction chromatography

A robust analytical method has been developed in our laboratory for the separation of radionuclides by means of extraction chromatography using an automated separation system. The proposed method is both cheap and simple and provides the advantageous, rapid and accurate separation of the element of interest. The automated separation system enables a shorter separation time by maintaining a constant flow rate of solution and by avoiding clogging or bubbling in the chromatographic column. The present separation method was tested with two types of samples (water and urine) using UTEVA-, TRU- and Sr-specific resins for the separation of U, Th, Am, Pu and Sr. The total separation time for one radionuclide ranged from 60 to 100 min with the separation yield ranging from 68 to 98% depending on the elements separated. We used ICP-QMS, multi-low-level counter and alpha spectroscopy to measure the corresponding elements.     

可调移动重叠分离图法用于高效液相色谱多台阶梯度分离条件优化的研究

计算机辅助高效液相色谱（HPLC）分离条件优化可以低成本、快速地得到优化的分离条,因而已较为广泛地用于复杂样品的分离分析。基于移动重叠分离图方法,又发展了一种新型的多台阶梯度分离条件的优化方法可调移动重叠分离图法。该方法通过预测不同流动相条件下各组分的保留时间、峰宽和分离度,绘制出对于样品中各组分的重叠分离区域图。在对当前台阶流动相组成进行优化的同时,考虑其对后面一到两个台阶上流出组分保留的影响,实时地重新绘制对于后面台阶上流出组分的重叠分离区域图。通过观察当前台阶流动相条件对当前台阶和后面台阶上流出组分分离的影响,综合考虑样品中所有组分的分离情况,找到更接近全局最优的分离条件。通过扫描的方法对优化得到的分离条件进行微调,能够进一步提高分离效果。采用文献数据对可调移动重叠分离图法的应用加以说明,在二元流动相体系下,证明了该方法在HPLC方法建立方面的优越性。     

Applicability of dynamic change of pH in the capillary zone electrophoresis of proteins

A method to extend the separation power of CZE is described. The method is based on the separation of sample components at two different pH values during one separation run, and involves dynamic buffering of the pH inside a separation capillary by controlling the flow of H+ ions from the anodic electrode chamber. By changing the anolyte in the chamber, a dynamic pH step is generated, which proceeds rapidly along the capillary and establishes the required new pH value. The use of the method has been demonstrated by the cationic separation of a model mixture of proteins.     

A triple-injection method for microchip electrophoresis

We report here a novel triple injection method for microchip electrophoresis (micro-CE) that results in a higher intensity of DNA peaks. This new method includes a triple-repeated process of a combination of a sample loading voltage and a separation voltage in each interval, namely (loading time) + (separation time) + (loading time) + (separation time) + (loading time), prior to electrophoretic separation. All these injections were electrokinetically controlled by a software. Although the usual sample injection, which included the process of one 60 s electrokinetically application, was limited by the amount of sample, peaks of 40% higher intensity were obtained using the new method within half of the conventional injection time compared to the conventional method. Maximum peak intensity was successfully achieved with integration of the intensities of the triple-repeated peaks by adjusting the application period of the separation voltage. Repetition of the sample loading voltage for an adjusted period with a further adjusted period of separation voltage in each interval may be an effective method for injection of samples that results in peaks with higher intensity.     

Thin-layer chromatography--a useful technique for the separation of enantiomers

Separation of enantiomers has become a well-established technique in many fields of science over the last decade. Unfortunately, even though there are a large number of chiral stationary phases able to perform enantiomeric separation, there is still a great deal of trial and error in developing a method for the separation of enantiomers. Thin-layer chromatography is a very versatile technique, which has brought much advancement in various fields of science. The simplicity of the technique makes it amenable for separation of enantiomers. This paper will present a review of the literature concerning separation of enantiomers. Because of the process of trial and error present in developing a chiral separation method, this paper also presents the mechanism underlying each form of separation. Thus, the methods are presented according to the main mechanism governing the particular separation.     

Development and validation of an improved method for the analysis of vancomycin by liquid chromatography selectivity of reversed-phase columns towards vancomycin components

The current method prescribed in official monographs for the purity control of vancomycin is inappropriate in that several components are not separated from each other and other components are coeluted with the main component vancomycin B. The method uses an ODS column at pH 3.2. In this study, several changes were introduced in order to improve the separation. The optimization of the separation method at low pH indicated that pH 1.7 was optimum and that the use of dioxane as organic modifier drastically improved the separation. These conditions were used to test a set of more than 40 reversed-phase columns for their selectivity towards vancomycin components. The selection of the most suitable columns was performed by means of principal component analysis. Most of these columns did not allow the separation of didechlorovancomycin from monodechlorovancomycin 1. It was found that neutral to slightly alkaline mobile phases allowed better separation. Further optimization of the separation method and a robustness study were performed by means of experimental design. This optimization indicated that pH 7.7 was optimum and gradient elution was also used to effect complete analysis. The final method uses a Kromasil column and the mobile phase comprises dioxane, water and ammonium formate solution pH 7.7. The separation of monodechlorovancomycin 2 and of some unknown impurities from the main component vancomycin B is described for the first time. The method shows good repeatability, linearity and sensitivity.     

Determination of iodine in biological materials by neutron activation analysis

A method of determination of iodine (total and PBI) in serum, urine and other biological materials has been developed. The method consists in a gamma-spectrometric measurement of¹²⁸I activity after its radiochemical separation. The radiochemical separation procedure includes wet decomposition of the samples in a nitric acid medium followed by a few separation steps, the essential step being the substoichiometric extraction of iodide with a chloroform solution of tetraphenylarsonium chloride. Owing to the application of the substoichiometric separation, a high radiochemical purity of the separated iodine is achieved and the determination of the yield of radiochemical separation is not necessary.     

Characterization of transferrin glycoforms in human serum by CE-UV and CE-ESI-MS

Human transferrin (Tf) is a model glycoprotein for congenital disorders of glycosylation (CDG) diagnosis. In the last few years, new CE-UV methods for intact Tf glycoforms analysis have been developed using nonvolatile BGEs and organic modifiers. However, the use of these BGEs does not allow the coupling of these procedures with electrospray MS (ESI-MS). In this study, a new CE-UV separation method of Tf glycoforms is developed, using a double-layer stable coating and a volatile BGE based on ammonium acetate. The separation method is optimized using standard Tf and their potential is demonstrated applying the method to the analysis of sera Tf from healthy individuals and CDG patients. The CE-UV separation method has been coupled to ESI-MS detection. Main parameters such as sheath liquid composition are optimized in order to obtain a good sensitivity. The CE-ESI-MS method has also been used in serum samples obtaining the separation of the different proteins present in serum and partial separation of Tf glycoforms. Different mass spectra and deconvoluted molecular masses were obtained for each sialoform, allowing unequivocal glycoform identification.     

Elution-extrusion counter-current chromatography separation of five bioactive compounds from Dendrobium chrysototxum Lindl

The elution-extrusion counter-current chromatography (EECCC) method was firstly developed by Berthod in 2003 and has been used in natural products separation in recent years. The advantages of this method have been well documented such as reducing the separation time and solvent consumption. In the EECCC method, the time point of the extrusion step is very important during the whole separation process as it directly affects the resolutions, separation time and solvent consumption. However, how to choose a suitable time point to perform the extrusion step without decreasing the resolution has not been studied yet. In the present study, a strategy for systematically calculating the time point for extrusion was developed in theory and five bioactive compounds from the extract of Dendrobium chrysototxum Lindl. were separated and compared using normal CCC and EECCC method. Our results demonstrated that the accurate time point to perform the extrusion could be calculated and reduced both separation time and solvent consumption without losing separation performance. Using this EECCC method, five bioactive compounds were separated and purified with high purity. The separation time and solvent consumption were decreased from 200 min to 100 min and 5-2.5L during the separation process while the resolutions were still acceptable. Finally, 63 mg, 48 mg, 97 mg, 162 mg and 43 mg of hydroxyl phenanthrenes and bibenzyls with the purity of 98.7%, 98.0%, 98.2%, 99.0% and 98.7%, respectively were isolated from 1.2 g crude extract of D. chrysototxum Lindl. initially purified by column chromatography in one step separation. The purities of compounds were determined by HPLC. Their structures were identified by electrospray ionization-mass spectrometry (ESI-MS) and NMR.     

Particle separation in microfluidics using a switching ultrasonic field

We present a new method for separation of micro-sized constituents with positive acoustic contrast factors in a microfluidic channel using ultrasound. The ultrasound field is switched between the first and third resonant modes of the fluid channel, and the suspended constituents are separated onto the side and center pressure nodal lines according to their sizes or acoustic contrast factors. Initial hydrodynamic focusing of the constituents within a region of the channel near to the side nodal line is a crucial step in this separation method. This new method is shown to provide a novel "parallel-stream" separation of two species of particles with good robustness. Prior numerical simulations provide essential information on this operating region and also the voltage cycle to be applied to the ultrasonic actuators for optimal separation. Experiments were conducted using a prototype of the design with polystyrene microspheres of different sizes to demonstrate the efficiency and robustness of the separation process.     

Séparation chromatographique du plomb contenue dans des solutions concentrées de sulfate de zinc

Spectrographic analysis of lead traces in concentrated zinc sulphate solutions is only possible after elimination of zinc and sulphate ions. Two methods of separation on ion exchangers are proposed : the first method consists in a separation of Pb from Zn on an anion exchanger after elimination of sulphate on a cation exchanger; in the second method, the two steps of the separation are realized on the same cation-exchanger column. Both methods are satisfactory, but the second is faster and simpler.The blank of the method is about 1.5 μg Pb (i.e. 0.15 mg Pb/l) for a separation made with 10 ml of electrolyte. The method is therefore suitable for accurate determinations of Pb contents as low as 1 mg/l of concentrated zinc sulpliate electrolyte.     

Rapid analysis of charge variants of monoclonal antibodies with capillary zone electrophoresis in dynamically coated fused-silica capillary

A capillary zone electrophoresis (CZE) method was developed for the rapid analysis of charge heterogeneity of immunoglobulin G (IgG) monoclonal antibodies (mAbs). The separation was carried out in a short, dynamically coated fused-silica capillary. A number of separation parameters were investigated and optimized, including pH, concentration of the separation buffer (ε-amino caproic acid), concentration of the triethylenetetramine (TETA) dynamic coating, the capillary internal diameter and the field strength used for the separation. The effects of between-run flushing of the capillary and the data acquisition rate were also evaluated. Under the optimized conditions, a fast (<5 min), selective and reproducible separation of mAb charge variants was achieved under a very high electric field strength (1000 V/cm). This method also requires only a short conditioning of the capillary, with between-run conditioning completed within 2 min. The method was evaluated for specificity, sensitivity, linearity, accuracy and precision. The same separation conditions were applied to the rapid separation (2-5 min) of charge variants of multiple monoclonal antibodies with pI in the range of 7.0-9.5. Compared with other existing methods for charge variants analysis, this method has several advantages including a short run time, rapid capillary conditioning and simple sample preparation.     

纳米粒子分离方法的研究进展

随着纳米科技的快速发展,纳米粒子的分离已经成为纳米领域的基础性研究课题,同时也是热点与难点问题。该文介绍了几种较为常用的分离纳米粒子的方法,主要包括场流分级法、超速离心法、膜分离法、色谱分离法和磁性分离法,评述了每种方法的优缺点、适用范围、具体应用实例和相关研究进展,并具体讨论了每种分离方法的分离效果、重复性和特异性等。     

Separation method based on affinity reaction between magnetic and nonmagnetic particles for the analysis of particles and biomolecules

A separation method is reported for particle and biochemical analysis based on affinity interactions between particle surfaces under magnetic field. In this method, magnetic particles with immunoglobulin G (IgG) or streptavidin on the surface are flowed through a separation channel to form a deposition matrix for selectively capturing nonmagnetic analytes with protein A or biotin on the surface due to specific antigen (Ag)--antibody (Ab) interactions. This separation method was demonstrated using model reactions of IgG--protein A and streptavidin-biotin on particle surface. The features of this new separation method are (1) the deposited Ag-Ab complex can be examined and further analyzed under the microscope, (2) a kinetic study of complex binding is possible, and (3) the predeposited matrix can be formed selectively and changed easily. The detection limits were about 10(-11) g. The running time was less than 10 min. The selectivities of studied particles were 94% higher than those of label-controlled particles. This method extends the applications of analytical magnetapheresis to nonmagnetic particles. Preliminary study shows that this separation method has a great potential to provide a simple, fast, and selective analysis for particles, blood cells, and immunoassay related applications.     

Electrophoretic separation method for membrane pore‐forming proteins in multilayer lipid membranes

In this paper, we report on a novel electrophoretic separation and analysis method for membrane pore‐forming proteins in multilayer lipid membranes (MLMs) in order to overcome the problems related to current separation and analysis methods of membrane proteins, and to obtain a high‐performance separation method on the basis of specific properties of the lipid membranes. We constructed MLMs, and subsequently characterized membrane pore‐forming protein behavior in MLMs. Through the use of these MLMs, we were able to successfully separate and analyze membrane pore‐forming proteins in MLMs. To the best of our knowledge, this research is the first example of membrane pore‐forming protein separation in lipid membranes. Our method can be expected to be applied for the separation and analysis of other membrane proteins including intrinsic membrane proteins and to result in high‐performance by utilizing the specific properties of lipid membranes.     

Chiral separation of β‐blockers by MEEKC using neutral microemulsion: Analysis of separation mechanism and further elucidation of resolution equation

Based on the investigation of the effect of microemulsion charge on the chiral separation, a new chiral separation method with MEEKC employing neutral microemulsion was established. The method used a microemulsion containing 3.0% (w/v) neutral surfactant Tween 20 and 0.8% (w/v, 30 mM) dibutyl l ‐tartrate in 40 mM sodium tetraborate buffer to separate the enantiomers of β‐blockers. The effect of major parameters on the chiral separation was investigated. The applied voltage had little effect on the resolution, but the chiral separation could be improved by suppressing the EOF. Nine racemic β‐blockers obtained relatively good enantioseparation after appropriate concentrations of tetradecyl trimethyl ammonium bromide were added into the microemulsion to suppress the EOF. These results were explained based on the analysis of the separation mechanism of the method and deduced separation equations. The resolution equation of the method was further elucidated. It was found that the fourth term in the resolution equation, an additional term compared to the conventional resolution equation for column chromatography, represents the ratio of the relative movement distance between the analyte and microemulsion droplets relative to the effective capillary length. It can be regarded as a correction for the effective capillary length. These findings are significant for the development of the theory of MEEKC and the development of new chiral MEEKC method.     

Pressure-driven sample injection with quantitative liquid dispensing for on-chip electrophoresis.

A novel pressure-driven sample injection method was developed as an alternative to electrokinetic injection, and electrophoretic separation was carried out on a microfabricated device employing this method. This method enables a defined volume of liquid dispensing, followed by instantaneous injection driven by pneumatic pressure, greatly simplifying the injection procedure. A particular microstructure, called a "metering chamber", has been designed for the quantitative dispensing of an ultra-low volume of sample liquid; a "hydrophobic passive valve" equipped with an air vent channel is employed for injecting a dispensed sample into the separation channel. The reproducibility of dispensing was 3.3% (n = 15), expressed by the variation of dispensed volumes. The electrophoretic separation of DNA fragments was performed using this injection method, varying the injection volumes from 0.45 to 4.0 nL, and the separation efficiencies were compared. This precise injection method, easily variable in injection volumes, is highly suitable for quantitative as well as qualitative electrophoretic analyses.     

Microchip-based plasma separation from whole blood via axial migration of blood cells

Highly efficient cell-free plasma separation from 200 μL of human whole blood was realized via axial migration of blood cells and cross-flow filtration in a microchip. Although various analyses of small volumes of blood have been reported, a large volume of blood is necessary for obtaining blood cells and plasma for the conventional plasma separation technique of centrifugation. A highly efficient plasma separation method using small volumes of blood without hemolysis is an important issue. We developed a plasma separation method based on a microchip with a filter, which utilizes the axial migration of blood cells observed in blood vessels. Clogging and hemolysis on the filter can be prevented by the axial migration of the blood cells. Using this method, 65% of the plasma from 200 μL of whole blood was successfully separated without hemolysis. When the plasma separation microchip interfaced with a micro-ELISA system was applied to C-reactive protein (CRP) analysis, the CRP concentration obtained by the microchip showed good correlation with that obtained by conventional centrifugation. Total analysis time, including plasma separation, was achieved in only 25 min.     

Characterization of pharmaceutical drugs by a modified nonaqueous capillary electrophoresis--mass spectrometry method

A simple method for the separation and characterization of a group of nine basic compounds, comprising seven tricyclic antidepressant and two bronchodilator drugs, by nonaqueous capillary electrophoresis (NACE) employing ultraviolet and mass spectrometry detection is described. After optimization of the electrophoresis separation conditions, including the compositions of the electrolyte and the organic solvent, a reliable separation of all nine basic analytes was achieved in 80 mM ammonium formate dissolved in a methanol-acetonitrite (80:20 v/v) mixture, having an apparent pH of 8.7. The volatile nonaqueous electrolyte system used with a normal electroosmotic flow polarity also provided an optimal separation condition for the characterization of the analytes by mass spectrometry. When results were compared with reversed-phase gradient and isocratic high-performance liquid chromatography (HPLC) methods, the NACE method provided greater efficiency, achieving baseline resolution for all nine basic compounds in less than 30 min. The NACE method is suitable for use as a routine procedure for the rapid separation and characterization of basic compounds and is a viable alternative to HPLC for the separation of a wide range of pharmaceutical drugs.