Separation and Analysis of Nucleic Acids and Their Constituents by Ion-Exclusion and Ion-Exchange Column Chromatography

The recent commercial development of high pressure liquid chromatographic instruments, and the availability of small and uniformly sized resin beads has enhanced the role of column chromatography for the rapid assay of nucleic acid components. A major advancement in the purification and analysis has been due to the recent demonstration that the ion exclusion is more or equally effective than the ion exchange as a separation principle for the resolution of purine and pyrimidine bases, nucleosides, deoxynucleosides and nucleotides^1–3. This article deals with the recent applications of separation principles that have been employed for the separation of nucleic acid constituents. The reader is referred to two review articles of Cohn for the earlier works on this subject^{4, 5}. Separations by chromatography and electrophoresis on paper and thin layers are not described here; several monographs and review articles are available in the literature^6–12.The reader is also referred to a recent methodological study for the base analysis of ribopolynucleotides by chemical tritium labeling which utilizes two dimensional chromatography on thin layers of cellulose as a separation means^{13, 14}.     

Ion chromatographic system with a novel switching suppression device

Ion chromatography (IC) has been a powerful tool for measuring ionic species in environmental samples such as tap, river and drain waters. Suppressor modules (membrane and disposable column types) have been used for reducing the background of a baseline. A new type of suppressor device, which has a suppressor resin and switching valve was developed. Fresh ionic resin is introduced into a groove for each analysis to perform the suppression of the working eluent. The eluent composition for obtaining higher sensitivity and better resolutions among ionic species and carbonate ion was also investigated. Although carbonate buffers are used for ion separation in general, the separation of carbonate ion from other ions was not achieved. A borate eluent resulted in good resolutions and higher sensitivity. A new column was also developed for obtaining higher column efficiency and resolution. The optimization of anion separation using a new IC system (IC-2001) that consists of a new suppressor device, an anion-exchange column (TSKgel SuperIC-Anion, 150x4.6 mm), an autosampler, a conductivity cell and a pump in a compact module is described.     

Chromatographic behaviour of Cs+ in an ion exchange column

The cesium band traveling in an ion exchange column filled with Dowex 50X8 resin has been studied during elution with hydrochloric acid under different conditions. The dependence of the rate of band motion on the separation factor and on the flow rate of the eluent is given. Also the temperature dependence and the acid concentration dependence of the separation factor is shown.     

Separation of lithium isotopes by NDOE bonded Merrifield peptide resin

The novel NDOE (1,12,15-triaza-3,4:9,10-dibenzo-5,8-dioxacycloheptadecane) ion exchange resin was prepared. The ion exchange capacity of NDOE azacrown ion exchanger was 0.2 meq/g dry resin. A study on the separation of lithium isotopes was carried out with NDOE novel azacrown ion exchange resin. The lighter isotope,⁶Li concentrated in the solution phase, while the heavier isotope,⁷Li is enriched in the resin phase. By column chromatography (0.1 cm I.D.×32 cm height) using 2.0M NH₄Cl as an eluent, a separation factor,a=1.0201 was obtained.     

Modified normal-phase ion-pair chromatographic methods for the facile separation and purification of imidazolium-based ionic compounds

Imidazolium- and oligo(imidazolium)-based ionic organic compounds are important in the design of room-temperature ionic liquid materials; however, the chromatographic analysis and separation of such compounds are often difficult. A convenient and inexpensive method for effective thin-layer chromatography (TLC) analysis and column chromatography separation of imidazolium-based ionic compounds is presented. Normal-phase ion-pair TLC is used to effectively analyze homologous mixtures of these ionic compounds. Subsequent separation of the mixtures is performed using ion-pair flash chromatography on normal-phase silica gel, yielding high levels of recovery. This method also results in a complete exchange of the counter anion on the imidazolium compounds to the anion of the ion-pair reagent.     

Präparative Trennung und quantitative Bestimmung von catena-Phosphaten(V) mit Flüssigkeits-Chromatographie

Separation on a Preparative Scale and Quantitative Aanalysis of Oligophosphates by Means of Liquid Chromatography New methods for the chromatographic analysis and isolation of oligophosphates have been developed. The qualitative separation has been optimized for thin layer chromatography. Quantitative analysis and isolation of the various oligophosphate species has been achieved using liquid chromatography. For ion exchange and molecular sieve-gel columns packing material, column parameters, and eluent were optimized. The on line detection system included a differential refractometer coupled to a strip chart recorder. The applicability of the technique was demonstrated by the quantitative analysis of oligophosphate mixtures. Good reproducibility and accuracy was found. It has been ascertained that the stability of polyphosphates is sufficient to allow efficient separations.     

转Bt基因植物表达产物Cry1Ab蛋白的制备纯化方法研究

以转Bt基因水稻为试材,研究其表达产物Cry1Ab蛋白的提取、分离及纯化的方法。实验结果表明,DEAE-纤维素填料对Bt蛋白有较好捕获效果。根据生物信息学方法预测了目标蛋白和主要共存蛋白的等电点和疏水性差异。合理地选择了阴离子交换色谱与疏水作用色谱组合方法。提取液经DEAE-Sephadex A-50柱层析及Phenyl-Sepharose Fast Flow疏水层析分离后,目标蛋白得到了显著的纯化。考察了疏水层析中用不同洗脱液洗脱Cry1Ab蛋白对活性回收率和纯度的影响,结果表明：以0．25mol／L KSCN作洗脱液对活性影响最小,HIC一步纯化倍数可达8倍,总纯化倍数达100倍。     

An overview of sequential injection chromatography

New generation of sequential injection analysis (SIA) called sequential injection chromatography (SIC) has already been consolidated as a good alternative of high performance liquid chromatography (HPLC) for fast analysis of simple samples. Benefits of flow methods are automation, miniaturization and low sample and mobile phase consumption. Implementation of short monolithic chromatographic column into SIA opens new area—on-line chromatographic separation of multi-compound sample in low-pressure flow system, with the advantage of flow programming and possibility of sample manipulation. In the presented review the potential of SIC and its comparison with HPLC for determination of pharmaceutical mixtures is discussed and outlines past and recent trends focused on separation with SIC.     

柱色谱分离蔗糖酰化产物中蔗糖-6-乙酸酯的研究

通过蔗糖C-6位羟基的选择性乙酰化反应,合成的蔗糖-6-乙酸酯是进一步制备卤代蔗糖衍生物的重要中间体[1]。三氯蔗糖是目前被广泛关注的一种新型卤代蔗糖衍生物[2],它是以天然的蔗糖为原料,经半化学合成而得到的强力甜味剂,具有高甜度、低热值、无毒和抗龋齿等特点。在众多的三氯蔗糖的合成方法中[3-5],必须首先要对活泼的蔗糖C-6位羟基进行保护,即蔗糖的选择性酰化反应。蔗糖的乙酰化产物中除蔗糖-6-乙酸酯外,还有未转化的原料蔗糖和部分反应副产物。因此,为了减轻后续工艺合成的难度,降低制备成本,使最终产物三氯蔗糖容易提纯,必须对酰化…     

Fractionation of nitrogen stable isotopes by ion exchange chromatography

The ion exchange chromatography displacement technique was used for the enrichment of ¹⁵N. The runs were conducted in laboratory and bench scales using two systems of columns filled with Wofatit KPS ion exchange resin (medium porosity type). Ammonia NH₄ ⁺/NH₃ aq. was chosen as the isotopic exchange system. The ammonium bands formed in the columns were eluted by means of sodium hydroxide solution. Hydrodynamic patterns in the column beds was evaluated in terms of dimensionless Reynolds number. The results show that separation process can be performed in the turbulent or laminar flow regime.     

Separation of lithium isotopes by N4S2 azacrown ion exchanger

The novel N₄S₂ azacrown ion exchange resin was prepared. The ion exchange capacity of N₄S₂ ion exchanger was 0.34 meq/g dry resin. A study on the separation of lithium isotopes was carried out with N₄S₂ azacrown ion exchange resin. The lighter isotope,⁶Li is concentrated in the resin phase, while the heavier isotope,⁷Li is enriched in the solution phase. With column chromatography [0.1 cm (I.D.)×32 cm (height)] using 2.0M NH₄Cl as an eluent, separation factor, a=1.034 was obtained.     

Opportunities and challenges of high-pressure ion exchange chromatography for nuclide separation and enrichment

With the rapid development of the nuclear industry, more-stringent requirements are proposed for high-level radioactive waste liquid treatment and the enrichment of isotope products. High-pressure ion exchange chromatography has been widely accepted for the fine separation of elements and nuclides due to its advantages, such as high efficiency, environmental friendliness, ease of operation, and feasibility for large-scale industrial applications. Here, we summarized the evolution of high-pressure ion exchange chromatography and the relevant research progress in ion exchange equilibrium and related separation technology. The prospects for application of high-pressure ion exchange chromatography to rare earth elements, actinide elements and isotope separation were discussed. High-pressure ion exchange chromatography represents a promising strategy for the extraction of rare earth elements and actinide elements from high-level radioactive waste liquid, as well as being an effective method for the automated production of high purity isotope products with great environmental benefits.     

Quantification of lactobionic acid and sorbitol from enzymatic reaction of fructose and lactose by high-performance liquid chromatography

Experimental conditions for complete separation and quantification of mixtures containing lactobionic acid, sorbitol, lactose and fructose are discussed for the first time. These mixtures appear in the enzymatic bioconversion of fructose and lactose catalyzed by glucose-fructose oxidoreductase (GFOR) and glucono-delta-lactonase (GL) enzymes of Zymomonas mobilis cells. The high-performance liquid chromatography (HPLC) separation was carried out in a strong cation ion exchange resin (hydrogen form) based on a copolymer of styrene divinylbenzene (PS-DVB). A stationary phase of beta-cyclodextrin was also evaluated. An efficient separation was obtained with PS-DVB column eluted with sulfuric acid 0.450 mM solutions (pH 3.0-3.2) at 75 degrees C. The formation of lactones was observed, which is associated with the dissolution of lactobionic acid crystals; however, by dissolving the lactobionic acid crystals on alkaline calcium hydroxyde solution in equimolar ratio a single lactobionic acid chromatographic peak without lactobionolactone is obtained.     

用离子交换树脂脱除氨基酸与盐混合液中的盐

在用蛋白质酸性水解备氨基酸时,因中和残留的酸会使水解液中带入大量的盐。在进一步用离子交换色谱法分离混合氨基酸时,首先需脱掉其中的盐。本文用苯乙烯系强酸性阳离子交换树脂的盐型柱,根据氨基酸与苯乙烯系强酸性阳离子交换树脂之间既存在离子间的静电作用,又存在疏水作用,且二者之间存在协同作用,而盐在盐型苯乙烯系强酸性阳离子交换树脂柱上不保留的原理,用水作为洗脱剂,使盐和氨基酸（配制的盐和氨基酸混合液及含盐的毛发水解液）得到分离,本方法脱除氨基酸中的盐简单易行,用水作为洗脱剂即廉价由不造成污染,盐型树脂不用再生即可用于下次运行,研究了各种条件对分离性能的影响。     

Study of ion chromatography with ion-exchange fibers as the stationary phase

The application of ion-exchange fibers as the stationary phase in ion chromatography for the separation of inorganic anions has been studied. Results indicate that a separator column packed with VS-2 anion-exchange fibers and a suppressor column packed with VS-1 cation-exchange fibers have a similar separation efficiency to small-particle resin columns, but that the column pressure drop (ΔP) in fiber columns in only one-tenth of that in resin columns, at the same flow-rate. This allows the separation to be performed efficiently at a higher flow-rate and with lower presure, as shown for the separation of an anion mixture.     

Quantitative Analysts of 5-Hydroxytryptamtne in Biological Material by High Perfornance Liquid Chromatography and Electrochmical Detection

Abstract

A quantitative method for the analysis of 5-hydroxytryptamine in biological material is described. The method is based on high performance liquid chromatography (HPLC) with electrochemical detection. A simple purification on a weakly acidic ion exchange resin prior to the analysis gives quite clean samples and permits concentration of diluted samples. The chromatographic separation is performed on a reverse phase column with organic modifier added to an aqueous eluent. With this analytical system 25 pg of 5-hydroxytryptamine can be detected.     

Carbon nanotubes‐A resin for electrochemically modulated liquid chromatography

Electrochemically modulated liquid chromatography is a special form of ion exchange chromatography in which the separation process is controlled by applying an electric potential to the stationary phase. This form of chromatography has so far only been applied in research studies. The present study shows that multiwalled carbon nanotubes are an effective resin material for an electrochemically modulated chromatography process. The experiments are carried out in a newly designed column that enables the packing of nanomaterials. We investigate the influence of the applied potential on the retention and elution of maleic acid, determine the dynamic binding capacity, and calculate the utilization degree of the electrical charge in the adsorption process. Moreover, the stability of the resin and the membrane over more than 200 working hours are presented. In addition to the stability, their sturdiness and inexpensive price are important qualities that make multiwalled carbon nanotubes interesting for application as the stationary phase in an electrochemically driven process. The investigated chromatography technique represents a promising separation process for future applications as a preparative step in biotechnology as well as other life science fields.     

Procedures for the enhancement of selectivity in liquid phase chemiluminescence detection

An overview of liquid phase chemiluminescence (CL) processes is presented and the potential for CL detection in liquid chromatography (LC) is discussed, with particular reference to the luminol and peroxyoxalate reactions. Post column ion displacement from a solid phase reagent [a cation exchange resin in the copper(II) form] followed by catalysis of the luminol reaction is used for the quantification of mixtures of weak acids after separation by ion-exclusion chromatography. Polyaromatic hydrocarbons (PAHs) released during the combustion of biomass fuels are separated by reversed-phase chromatography and quantified by their sensitizing effect on the peroxyoxalate reaction. This procedure is also used for the determination of carboxylic acids in non-aqueous media after selective pre-column derivatization with a fluorescent label (9-anthracenemethanol).     

Multiple-injection technique for isolating a target protein from multicomponent mixtures

An integrated chromatographic process comprising ion exchange (IEC) and hydrophobic interaction chromatography (HIC) for isolating a target protein form multicomponent mixtures has been analyzed. The model mixture contained immunoglobulin G that was the key product of the separation process, cytochrome C and ovalbumin. The adsorption characteristics and the mass transport kinetics of the model proteins have been determined along with their dependencies on the operating variables such as pH, temperature and the salt concentration for IEC as well as HIC media. Limitations of the process efficiency resulting from kinetic effects, solubility constraints and the necessity of the mobile phase exchange between chromatographic steps have been discussed. To improve the performance of the integrated process the multiple-injection technique has been suggested. This technique consisted in loading feed mixtures dissolved in a good solvent onto the column by several small-volume injections under conditions of strong protein adsorption. It allowed diminishing interactions between the sample-solvent and protein and elimination of undesired effects such as band splitting and band broadening. For the process design and optimization a dynamic model has been used accounting for thermodynamics and kinetics of the process. The optimization results indicated superiority of the multiple-injection technique over standard isocratic injections in terms of the process yield and productivity.     

多维高效液相色谱分离模式组合

简述了多维高效液相色谱法的特点及发展简况,重点对分子排阻色谱／反相色谱、离子交换色谱／反相色谱、正相色谱／反相色谱、分子排阻色谱／离子交换色谱、液固色谱／反相色谱、亲合色谱／反相色谱、非手性柱／手性柱等的联用模式及实际应用进行了概括和总结。