Electrophoretic separation and characterisation of gliadin fractions from isolates and nanoparticulate drug delivery systems

Summary Gliadin is a group of polymorphic proteins that are commonly used for the preparation of many pharmaceutical and cosmetic products. The aim of this study was to develop an analytical HPCE procedure for the identification and quantification of gliadin samples. For this purpose, the HPCE buffer composition were optimised to improve the resolution of separations. There-fore, several buffer modifiers were tested for use with 50 mM phosphate buffer in HPCE separations. Twenty percent acetonitrile and 0.05% hydroxypropylmethyl-cellulose provided optimal resolution while maintaining excellent reproducibility. In conclusion, this method can be successfully applied to the elucidation of the different gliadin fractions in protein isolates and nanoparticulate dosage forms.     

高效毛细管电泳用于油田水及甘酪根水热降解物中有机酸分析

采用高效毛细管电泳分离，以间接检测方法对低碳一元及二元羧酸进行分析。结果表明：５种脂肪族一元及二元羧酸６ｍｉｎ内可获得完全分离。本法用于测定油田水及甘酪根降解物中的有机酸，结果与反相高效液相色谱所得结果相吻合。     

Bio-Macromolecular Liquid Chromatography Yesterday and Today: Advancements in the Separation and Study of Nucleic Acids and Their Related Proteins

Abstract

Rapid, repeatable, and resolved chromatographic separations of molecules and analyses of their components have finally arrived in the commercial marketplace. This arrival has been long awaited, particularly with regard to nucleic acids (DNA and RNA). Biotechnological advances of protein and nucleic acid sequencing and synthesis have ushered in not only the instrumentation, but also the requirements of pre- and post-chromatographic column separations. A variety of high performance column supports had to be developed and have resulted in highly resolved separations of nmole quantities, as well as quality resolutions of mmole quantities of macromolecules. In addition, analysis of purified protein and nucleic acid components required development of reason-ably timed, high resolution separations of the more than 20 amino acids in one case, and 60 nucleosides in the other. This review juxtaposes the past with the present, and finally, gives a peek into the future of macromolecule separations.     

Packings and stationary phases for biopolymer separations by HPLC

Summary Packings and stationary phases applied to high resolution separations of proteins, enzymes, and nucleic acids must satisfy a series of distinct criteria that are different from those usually required by HPLC of low molecular weight non-biologically active analytes. These requirements have been met through substantial improvements in classical gel media together with novel developments in silica supports, and have led to a family of products with tailor-made and reproducible properties. Supports consisting of cross-linked organic gels, and inorganic materials (mostly silicas) are now available with graduated particle sizes, pore sizes, porosities and surface areas as well as non-porous beads. A whole range of stationary phases, such as reversed phase, hydrophobic interaction, ion exchanger and affinity packings, were designed for application as chemical sensors for biopolymer recognition in adsorptive chromatography. The phase systems are operated in the gradient mode, giving high resolution and high peak capacities. In addition, aqueous liquid-liquid partitioning systems have been developed for the fractionation of proteins and nucleic acids. Size exclusion media complete the set of HPLC variants enabling a discrimination of proteins according to their size and shape in an isocratic elution mode. Basically, protein purification and isolation is a multistage process where-by the HPLC variants are combined in a logistic sequence, utilizing the different selectivities of the phase systems and thus maximising resolution, speed and throughput.     

Capillary electrophoresis in the evaluation of ischemic injury: simultaneous determination of purine compounds and glutathione

An understanding of tissue energy metabolism and antioxidant status is of major interest in the field of organ preservation for transplantation. Nucleotide and glutathione are indicators of cell damage occurring during ischemia and reperfusion. A high performance capillary electrophoresis (HPCE) method with UV detection (185 nm) for the simultaneous analysis of intracellular free ribonucleotides, nucleosides, bases and glutathione (oxidized and reduced form) in myocardial tissues is described. The method does not involve thiol derivatization. The separations were carried out in an uncoated fused-silica capillary, 60 cm long, 52.5 cm to detector, 75 microm ID, with 20 mM Na-borate buffer, pH 10.00, at 20 kV voltage and reading at 185 nm. Injection was hydrostatic for 12 s and total analysis time was 20 min. The technique enables optimum separation of all the compounds examined and has a resolution similar to that of HPLC analysis, with the advantage of fast simultaneous measurement of cell nucleotide metabolism and redox state, not possible with HPLC.     

Perspectives on analyses of nucleic acid constituents: the basis of genomics

The recent mapping of the human genome was a tremendous achievement made possible to a large degree by the development of analytical methods for sequencing purine and pyrimidine bases in nucleic acids. In the last 3 decades, the number of analyses of nucleic acids and their constituents by HPLC and capillary electrophoresis (CE) has exploded. These techniques have been used not only for genomics, but also for the determination of free nucleotides, nucleosides and their bases in body fluids and tissues. Although a large number of HPLC and CE papers have been published on nucleic acid constituent applications, relatively little has been written on the mechanisms of the separations. However, to optimize analytical conditions knowledgeably and rapidly, it is important to know why and how these separations occur and the factors that affect them. The HPLC methods for the analysis of nucleic acid constituents and the information available on some of the mechanisms of separation of nucleotides, nucleosides and their bases, as well as the analysis of these compounds by CE and the factors that affect these separations are discussed.     

Rapid, high-resolution high-performance liquid chromatographic analysis of antibiotics

A HPLC column devised for high separation speed combined with highly practical operating features has been found useful for separating antibiotics. Important characteristics involve compromises in packing particle size, column configuration and support-stationary phase combinations. We determined that these columns are useful for rapid, high-resolution separations with unmodified state-of-the-art HPLC equipment without the extra-column band-broadening effects typical of so-called “fast” HPLC columns. The proposed columns feature efficient sterically-protected monofunctional silane stationary phases that provide good separation reproducibility and high column stability. The combination of these unique bonded silanes and a highly purified, less-acidic silica support give superior peak shapes for antibiotic compounds. The proposed column configuration can halve separation times and double peak heights without loss in resolution, compared to widely used analytical columns. Increased mobile phase flow-rates permit even faster separations of antibiotics with only modest loss in resolution and peak heights for trace analyses in biological systems.     

High efficiency HPLC with long packed columns

Coupling several 25 cm × 2 mm ID analytical columns together is a simple and easy way to achieve high resolution HPLC (20,000–50,000 plates). How to do this with commercially available instruments, injectors and detectors is described in the paper. An attractive feature of the long narrow column approach is that the same HPLC set-up can be used for both fast-low and slow-high efficiency separations. High efficiency is essential for really complex natural mixtures, e.g. the hop and beer bitter acids. Some examples are shown.     

Specific applications of capillary electrochromatography to biopolymers, including proteins, nucleic acids, peptide mapping, antibodies, and so forth

Separation of biopolymers is an obvious application of capillary electrochromatography (CEC) technology, since speed and resolution should increase significantly over high-performance liquid chromatography (HPLC). All too often, HPLC chromatograms of polymers show poorly resolved envelopes of overlapping peaks from oligomers. The practical limitation of column length and pressure drop has hindered development of high resolution separations of many polymers in HPLC. However, this generally applies only to packed beds of small particles, and not to continuous (or monolithic) beds, as introduced by Hjerten et al. [S. Hjerten, Ind. Eng. Chem. Res. 38 (1999) 1205; S. Hjerten, C. Ericson, Y.-M. Li, R. Zhang, Biomed. Chromatogr. 12 (1998) 120; C. Ericson, S. Hjerten, Anal. Chem. 71 (1999) 1621; J.-L. Liao, N. Chen, C. Ericson, S. Hjerten, Anal. Chem. 68 (1996) 3468; S. Hjerten, A. Vegvari, T. Srichaiyo, H.-X. Zhang, C. Ericson, D. Eaker, J. Capillary. Elec. 5 (1998) 13; C. Ericson, J.-L. Liao, K. Nakazato, S. Hjerten, J. Chromatogr. A 767 (1997) 33; S. Hjerten, D. Eaker, K. Elenbring, C. Ericson, K. Kubo, J.-L. Liao, C.-M. Zeng, P.-A. Lidstrom, C. Lindh, A. Palm, T. Srichiayo, L. Valtcheva, R. Zhang, Jpn. J. Electroph. 39 (1995) 1]. Throughout this review we will refer to such packings as monolithic or continuous beds, but they are identical type packings, formed by the in situ polymerization in the capillary or column. CEC capillaries can be much longer, and contain smaller particles than is practical for HPLC. This improves resolution significantly. CEC is able to capitalize on existing mobile phase technology developed over 30 years to improve separations. The requirement that the mobile phase simultaneously promote the separation and mobile phase mobility needs to be considered. In RPLC, this dual role is not much of a problem. It may be much more important in other modes, particularly ion-exchange (IEC). As the field develops, it is becoming clear that CEC is not just a simple extension of HPLC. Instruments, column technology and operating optima are clearly different than HPLC. CEC will develop into its own unique field. Open tubular HPLC is almost precluded by the high pressures required for forcing liquids through 10 microm or smaller capillaries. Electroosmotic pumping (EOF) avoids the pressure constraints and provides better flow profiles. Compared to HPCE, the ability to interact with the stationary phase may enable separations that would be difficult with electrophoresis alone. Since the mobile phase can be less complex than micellar electrokinetic chromatography (MEKC), CEC also avoids the problem of high background signals from the micelle forming compounds. Thus CEC-MS (mass spectrometry) is expected to be even more powerful than HPCE-MS. The fortuitous, simultaneous development of matrix assisted laser desorption-time of flight MS (MALDI-TOF-MS) technology will enable extension of the mass range to above 100 000 Da. Lack of familiarity is the perhaps the largest liability of CEC compared to other techniques. This paper critically compares the state-of-the-art of CEC with HPLC and HPCE, with a particular emphasis on separation of biopolymers. The goal is to help the reader overcome the fear of the unknown, in this case, CEC.     

High-performance capillary electrophoresis using open tubes and gels

Summary This paper overviews several aspects of high performance capillary electrophoresis (HPCE), a promising new method of analytical and micropreparative separation of biochemically important samples. The basic migration equations of electrophoresis are first presented and the benefit of high fields for rapid analysis and high performance emphasized. Since power is generated with high voltages, Joule heating results and this heat must be dissipated. The use of capillary columns is shown to be important in efficient heat removal and in minimizing the temperature differences within the column. The various factors influencing band broadening are next described, and it is shown how plate counts close to 10⁶ can be achieved. Various results from our laboratory on open tube and gel columns are then presented to illustrate the potential of this method. Chiral resolution of dansylated amino acids using a chiral metal chelate micelle in open tube HPCE is shown. With the gel columns, the baseline separation of a 2-chain variant from methionine growth hormone (met-hGH) under non-denaturing conditions at fields close to 1000 V/cm is presented. Finally, the micropreparative purification of a 20-mer oligonucleotide using the gel column is described.     

Separation of DNA restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields

This paper presents results on the separation of DNA restriction fragments by high performance capillary electrophoresis (HPCE). Capillaries containing polyacrylamide with low amounts of crosslinking agent (i.e. 0.5% C) were first studied. The greater molecular accessibility offered with columns of low crosslinking, relative to higher crosslinked gels (e.g. 5% C), permitted high efficiency separations of double stranded DNA fragments up to 12,000 base pairs in length. Capillaries containing no crosslinking agent, i.e. linear polyacrylamide, were then examined. Ferguson plots (i.e. log mobility vs. %T) were used to assess the size selectivity of linear polyacrylamide capillaries. In another study, it was determined that the relative migration of DNA species was a strong function of applied electric field and molecular size. Lower fields yielded better resolution than higher fields for DNA molecules larger than about 1000 base pairs, albeit at the expense of longer separation time. Based on these results, we have examined pulsed field HPCE and have demonstrated the use of this approach to enhance separation.     

A practical interface for microfluidics and nanoelectrospray mass spectrometry

We report a new method for fabricating nanospray ionization tips for MS, formed from glass substrates and the inert polymer, parylene-C. Using a single photolithography step, the emitters are formed contiguously with microchannels, such that no dead volumes are observed. In addition, because the devices are very thin (approximately 0.3 mm) and the tips are formed at rectangular corners, the Taylor cone volumes are small, which makes the method attractive for future integration with microfluidic separations. Device performance was demonstrated by evaluating diverse analytes, ranging from synthetic polymers, to peptides, to nucleic acids. For all analytes, performance was similar to that of conventional emitters (pulled-glass capillaries and the Agilent HPLC Chip) with the advantage of rapid, batch fabrication of identical devices.     

Mechanistic Principles in Chiral Separations Using Liquid Chromatography and Capillary Electrophoresis

Due to the importance of chiral separations of drugs, pharmaceuticals, agrochemicals and xenobiotics by high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), it is important to have the knowledge of the enantiomeric recognition mechanisms so that scientists may design and module the new chiral selectors for rapid, inexpensive and reproducible chiral separations; specially at preparative scale. The mechanisms of the chiral separation by HPLC and CE using polysaccharides, cyclodextrins, macrocyclic glycopeptide antibiotics, Pirkle type, ligand exchangers, crown ethers and other several types of chiral selectors have been discussed. Various complex formation and different types of interactions responsible for chiral resolution have been presented in detail.     

Determination of thiopental in human serum and plasma by high-performance capillary electrophoresis-micellar electrokinetic chromatography.

The quantitation of thiopental in human serum and plasma was investigated using high-performance capillary electrophoresis (HPCE) in a micellar configuration and the results were compared with reversed-phase high-performance liquid chromatography (HPLC). Thiopental and an internal standard (carbamazepine for HPCE and thiamylal for HPLC) were extracted from serum or plasma using pentane and a phosphate buffer (pH 6.4). HPCE analysis took place in a phosphate-borate buffer with 50 mM sodium dodecyl sulphate using an automated instrument and HPLC was performed with a C8 column and a mobile phase of phosphate buffer-acetonitrile (65:35, v/v). HPCE and HPLC data from 66 patient samples compared well based on linear regression analysis. However, estimates obtained with the inclusion of the internal standard were lower than those based on the sample peak only. This example allows the elucidation of the advantages of using HPCE as an assay methodology for the therapeutic monitoring of thiopental and other drugs.     

(R)‐N,N,N‐Trimethyl‐2‐Aminobutanol‐Bis(Trifluoromethane‐Sulfon)Imidate Chiral Ionic Liquid Used as Chiral Selector in HPCE,HPLC, and CGC

Abstract

In this work, the (R)‐N,N,N‐trimethyl‐2‐aminobutanol‐bis(trifluoromethanesulfon)imidate chiral ionic liquid was first used in chromatography and exhibited a excellent chiral recognition ability in high performance capillary electrophoresis (HPCE), high performance liquid chromatography (HPLC), and capillary gas chromatography (CGC), which also showed for the first time that chrial ionic liquid was an effective chiral selector in HPCE and HPLC. The compounds that have been separated using this chiral ionic liquid at least included alcohol, amine, acid, and amino acid, et al. enantiomers. As the chiral ionic liquid can be easily synthesized from relatively inexpensive materials, it should have a great potentiality for chiral separation in chromatographic science.     

Determining sulfur-containing amino acids by capillary electrophoresis: a fast novel method for total homocyst(e)ine human plasma

A high-performance capillary electrophoresis (HPCE) method based on laser-induced fluorescence detection is presented here. It enables the determination of sulfur-containing amino acids within 15 min. Fluorescence of sulfur-containing amino acids in plasma is linear over a range of 50-150 micromol/L for L-methionine, 5-100 micromol/L for L-homocysteine, and 50-200 micromol/L for L-cysteine. For homocysteine, we were able to detect 1 fmol injected, equivalent to a plasma concentration of 10 nmol/L. A similar sensitivity is present for cysteine, an even lower one being found for methionine. The intra- and interassay relative standard deviations are < 1%. High-performance liquid chromatography (HPLC) methods are commonly employed for quantifying blood concentrations of sulfur-containing amino acids. A comparative analysis of HPCE and HPLC quantitation of homocysteine has been carried out in 61 blood samples. Plasma concentrations measured by HPCE were in good agreement with those obtained employing an HPLC-based method, a satisfactory correlation being observed between the concentrations obtained by the two methods (r= 0.9972). Thus, the HPCE-based procedure presented here for the measurement of sulfur-containing amino acids in plasma is a simple, fast, accurate, and very sensitive method, suitable for routine determinations in clinical studies.     

毛细管电色谱手性分离进展

 :比较全面地评述了毛细管电色谱(CEC)在手性拆分领域中的应用和发展,包括CEC的不同操作模式、手性试剂和手性固定相。92篇。     

Direct determination of amino acids and carbohydrates by high-performance capillary electrophoresis with refractometric detection

This is an initial report to propose a novel approach in high-performance capillary electrophoresis (HPCE) for the direct detection of compounds without natural absorbance in the UV and visible spectral range, such as amino acids and carbohydrates. A refractometry detector with the 2 nl cell (Applied Systems, Minsk, Belarus) was employed to identify amino acids and carbohydrates without derivatization. The first results are provided on separation of seven free amino acids in the phosphate running buffer and three free carbohydrates in the borate-sodium dodecyl sulfate running buffer and detection by refractometer. Fused capillaries of 50 or 75 microm internal diameter and separation voltage (10-23 kV) were applied. Detection limits ranged typically from 10 to 100 fmol and the response was linear over two orders of magnitude for most of the amino acids and carbohydrates. The HPCE system demonstrated good long-term stability and reproducibility with a relative standard deviation, less than 5% for the migration time (n=10).     

超滤、高效液相色谱和毛细管电泳的多维组合用于胎脑垂体组织培养液的分离分析

采用超滤、高效液相色谱和毛细管电泳的多维组合分离分析了胎脑垂体组织培养液的成分，比较了各种工具的不同组合产生的分离效果，考察了获取最多信息量或达到最佳分离的条件，获得了满意结果。     

Operational parameters of voltammetric high-performance liquid chromatographic detectors with copper electrodes and application to a determination of some fodder biofactors

Two versions of an amperometric detector with a copper working electrode have been constructed and tested for high-performance liquid chromatography (HPLC). The performance of the detectors was studied using selected amino acids. The dependence of the detector response on the mobile phase flow-rate was studied in the range common in both macro- and microcolumn HPLC (5 microliter/min to 1.0 ml/min). It has been found that the detection sensitivity generally increases with decreasing flow-rate, i.e., the detector response is governed by the rate of the complexation reaction between the cupric ions and the solutes. This fact makes amperometric detection with a copper electrode especially useful for microcolumn separations. For all 20 amino acids studied, calibration curve parameters and detection limits have been determined; the latter vary from 0.4 to 18 ng in the injected volume. The amino acids can also be sensitively detected in a medium of 0.1-1.0 M ammonia, which is promising for the use of strong anion exchangers in amino acid separations. Choline can also be detected at a copper electrode, with a detection limit of 40 ng. An HPLC method with amperometric detection at a copper electrode has been developed for the determination of lysine, methionine and choline in fodder biofactors.