Liquid chromatography on unmodified and modified spheron gels I. Nucleosides,bases and related compounds

Summary The ethylene glycol methacrylate gel Spheron and ion exchangers produced by the chemical modification of this gel (such as the cation exchanger Spheron S and anion exchanger Spheron DEAE) are compared with octadecylsilica as column packing materials for reversedphase chromatography of nucleic acid constituents and related compounds. The different separation selectivities of the individual materials can be utilized for the chromatographic separation of these compounds.     

Liquid chromatography on unmodified and modified Spheron gels III. Phenolic compounds

Summary Chromatographic behaviour of phenolic compounds is studied on the ethyleneglycol methacrylate gel Spheron and on ion-exchangers produced by the chemical modification of this gels (cation exchanger Spheron S and anion exchanger Spheron DEAE) as compared to octadecyl silica. The hydrophobic effects obviously predominate in the retention mechanism on Spherons in aqueous methanolic mobile phases, but a selectivity differring from the behaviour on octadecyl silica was found for a number of phenolic compounds This is due to interactions with the functional groups in the unmodified and modified Spheron materials and may be utilized for the separation of phenols by liquid chromatography.     

A critical review of some liquid chromatography systems for the separation of sugars

Summary This contribution to the problems of carbohydrate (sugar) column liquid chromatography, evaluates several currently used systems (reversed phase octadecylated silica gel, amino modified silica gel, cation exchangers on polystyrene and on silica gel basis, polyol derivatised silica gel and anion exchange systems). The elution pattern, analysis time, column efficiency and column life time expectancy are considered to be the important points for this comparison. The application of silica gel-based cation exchanger and of polyol derivatised silica gel is new to this field. The comparison and/or evaluation tries to be critical and to come to a conclusion for the choice of a recommended system.     

Sorption of arsenic,antimony and bismuth on glycolmethacrylate gels with bound thiol groups for direct sampling in electrothermal atomic absorption spectrometry

Batch sorption of arsenic, antimony and bismuth from solutions in 1 M sulphuric acid has distribution coefficients of 10⁴–10⁵. Quantitative sorption on the hydrophilic methacrylate gel containing thiol groups (Spheron Thiol) is possible within 60 min for bismuth or arsenic and 120 min for antimony. Conditions for the electrothermal atomization of arsenic sorbed on Spheron Thiol and injected into the graphite tube as a suspension are optimized. The sensitivities possible are 3.2 ng As ml^-1, 13 ng Sb ml^-1 and 2.8 ng Bi ml^-1; the coefficient of variation for 10 ng of As is 4%. Complete recovery of 40 ng As ml^-1 added to solutions of 5% KCI or 5% MgCl₂ and to river water was obtained.     

Two-dimensional fluorescence difference gel electrophoresis for comparison of affinity and non-affinity based downstream processing of recombinant monoclonal antibody

Although Staphylococcus Protein A (SpA) affinity chromatography is the state of the art capture step for antibody purification, non-affinity methods are more economical. We used two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) to evaluate the purification of a recombinant IgG₁ antibody from cultured cells, with two different processes: (1) SpA capture followed by cation-exchange chromatography (CEX); and (2) CEX capture, followed by anion exchanger, then hydrophobic interaction chromatography. Efficiencies were similar in sodium dodecylsulphate polyacrylamide gel electrophoresis and size-exclusion chromatography; however, 2-D DIGE revealed higher efficiency with SpA than with CEX capture. Thus, 2-D DIGE is a valuable tool for downstream process development.     

Chromatography of inorganic anions on poly(vinyl alcohol) gel columns with acidic eluents

Summary Poly(vinyl alcohol) (PVA) gel shows ionic retention properties for common inorganic anions when an acidic eluent is used. The ionic property of the PVA gel is due to the proton-acceptable nitrogen atoms of the cross-linking agent and the carboxylic residues being comprised in the gel matrix. The extent of the net charge on the gel surface depends on the pH of the eluent. At a pH ranging from 2.3 to 5.3, the PVA gel behaves as a weak anion exchanger with very low ion-exchange capacity. At these conditions four UV-absorbing inorganic anions (bromate, bromide, nitrate, and nitrite) are separated by eluting with aqueous sulfuric acid. Alkyl groups introduced on the gel surface hinder the ionized solute molecules from accessing to the positively charged functional groups on the gel surface. A neutral solute (HNO₂) is retained with non-ionic interactions.     

以meso四-(对磺基苯)卟啉为柱后衍生剂同时测定微量镉、汞、锌的离子色谱法

提出了以TPPS_4为柱后色谱衍生试剂的同时测定微量汞、镉和锌的离子色谱分析法,研究了影响衍生反应速度的因素。在含氯化钠的pH 10.3～12的硼酸钠缓冲介质中,以PAR催化TPPS_4与待测金属离子反应,以硅质键合相阳离子交换剂为固定相、酒石酸—氯化钠为流动相,于10min左右定量分离金属离子并将镉的淋洗顺序调至锌前。用于废水、硅酸盐和大米分析,结果满意。     

A polar-copolymerized method to prepare silica-based anion exchanger for ion chromatography

A novel silica-based strong anion exchanger was developed for ion chromatography by copolymerizing methyltrichlorosilane and 3-chloropropyltrichlorosilane. The method allows the column capacity to be easy control simply by adjusting the ratio of silanes. The unwanted residual silanol groups onto the surface of silica gel could also be greatly reduced by this strategy. The effective column capacity of the column used was measured to be 50.8 μequiv/column (2.03 μequiv/cm). The exchanger was characterized by solid state CP/MAS ¹³C NMR and elemental analysis and its separation performance was evaluated for the separation of common inorganic anions. The results showed that the column had good separation efficiency (e.g. the plate number of nitrite is 80,000/m) and the separation mechanism was observed to be dominantly governed by ion exchange mechanism. The utility of the column was demonstrated for the determination of nitrite and nitrate in saliva sample.     

亲水型高分子包敷大孔硅胶基质的弱阳离子交换填料用于蛋白质分离

报道用亲水型高分子包敷大孔硅在质的羧甲基型（ＣＭ）高效弱阳离子交换色谱分离蛋白质的工作。经ＣＭ柱纯化的溶菌酶比活性提高７倍，活性回收率高于９５％，ＣＭ填料对溶蓖酶的容量达８０ｍｇ／ｇ。     

Manual and automated enrichment procedures for biological samples using lipophilic gels

Aspects of the use of lipophilic gels in manual sample preparation procedures are reviewed. Neutral gels with a controlled hydrophobicity are used for sorbent extraction of non-polar and medium polarity compounds from biological fluids. Acidic amphiphilic compounds can be extracted as ion-pairs with decyltrimethylammonium ions. Solvent or detergent extracts of tissues or faeces can be mixed with hydrophobic gels for transfer of analytes from a solvent to a gel phase, permitting subsequent sample preparation in gel bed systems. Hydrophobic gels, alkyl-bonded silica and polystyrene matrices can be used in series for extraction of compounds with a wide range of polarities. Group fractionations are performed on neutral and ion-exchanging lipophilic gels to yield fractions of neutral, basic and acidic metabolites within selected polarity ranges. Selective isolation of phenolic acids on a strong anion exchanger, of ethynylic steroids on a strong cation exchanger in silver form and of oximes of ketonic steroids on a strong cation exchanger in hydrogen form is possible. A computerized system for automatic sample preparation is also described. It consists of an extraction bed, a cation-exchange column and an anion-exchange column. The pumps and switching valves are arranged so that the columns can operate in series or parallel for isolation of neutral, basic and acidic metabolites of amphiphilic compounds and for regeneration of the column beds. Fractions can be collected, or the effluent from the column beds can be diluted with water to permit sorption on a solid phase. The applicability of the automated method to the analysis of bile acids and metabolites of mono(2-ethylhexyl) phthalate is demonstrated.     

Ion-exchange separation of proteins by polyallylamine-grafted cellulose gel

A cellulose-based anion exchanger bearing water-soluble polycation was tested for separation of proteins. The exchanger was obtained by partial oxidation of cellulose gel by aq. NaIO4 followed by Schiff base formation with polyallylamine (PAA, molecular mass 5000). The retention behavior of proteins for three grades of PAA-cellulose gels, with amino group contents of 0.35, 0.59 and 0.96 mmol/g cellulose, was examined at several pH values and compared with that for conventional DEAE-cellulose gel with amino group content of 1.07 mmol/g cellulose. The retention of proteins by PAA-cellulose gels was remarkably greater than that for the DEAE-cellulose gel. Pairs of proteins having close isoelectric points and molecular masses (human and bovine serum albumins; beta-lactoglobulin A and B) could be separated by the PAA-cellulose gel columns. Such efficiency can be ascribed to high local density of grafted polyallylamine, in contrast to the random and sparse charge distribution in DEAE-cellulose.     

Design and evaluation of an on-line microcolumn pre-concentration technique for graphite furnace atomic absorption spectrometry

Summary A simple method is described for trace element pre-concentration on a microcolumn of inner diameter 1 mm and 25 mm length. The column packed with methacrylate gel with bound 8-quinolinol groups (Spheron Oxin) is incorporated into the tip of the sampling arm of the autosampler. The sample solution, water, eluent and air streams are switched with an eight-port valve. The flow-rate at pre-concentration is about 0.14 ml/min. After washing out the residues of the sample matrix from the column, the collected elements are eluted with 1 mol/l HNO₃ directly into the graphite tube of the atomizer. The reproducibility of the method was found to be better than 5%. The method was used for the determination of Pb in NaCl.

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Entwurf und Abschätzung einer on-line Mikrosäulen-Vorkonzentrierungstechnik für die Graphitofen-Atomabsorptionsspektrometrie

     

Determination of molybdenum(VI) in natural water and rock by ion-exchange absorptiometry combined with flow analysis

Ion-exchange absorptiometry combined with flow analysis has been applied to the determination of trace amounts of molybdenum(VI) in natural water and rock. By using a pretreatment column packed with Sephadex G-25 gel, molybdenum(VI) in a sample solution can be sorbed selectively on the gel at pH 3.5. The molybdenum(VI) in the column was desorbed with EDTA as the molybdenum(VI)-EDTA complex, and the solution was introduced into a Tiron solution stream. The yellow complex formed between molybdenum(VI) and Tiron in the flow system was then concentrated on a QAE-Sephadex A-25 anion exchanger packed in a flow-through silica micro-cell. The attenuation of incident light by the molybdenum(VI)-Tiron complex on the anion exchanger in the cell was continuously recorded with a spectrophotometer at 410 nm. The complex on the anion exchanger was easily desorbed with sodium nitrate, so the flow-through cell could be used repeatedly. The minimum amount that could be detected corresponded to 15 ng of molybdenum(VI). Molybdenum(VI) in three or four sample solutions could be determined within 1 h.     

离子色谱法分析金属离子的研究进展

综述了离子色谱法(IC)分析金属离子的研究进展,对目前应用于分析金属离子的阳离子交换IC、阴离子交换IC和螯合离子色谱进行了评述。阳离子交换IC是IC分析金属离子的主要形式,固定相为强酸(磺酸)型阳离子交换剂和弱酸(羧酸)型阳离子交换剂,结合适当的检测方法,阳离子交换IC可以测定碱金属、碱土金属、过渡金属、稀土离子、铵离子及低相对分子质量的有机胺类分子等。阴离子交换IC可以分析碱土金属、过渡金属、稀土离子等,对金属离子的分析具有更好的选择性,并可以实现金属离子和无机阴离子的同时测定。螯合离子色谱可以对复杂基体中的痕量金属离子进行测定。引用文献125篇。     

静态涂覆液态离子交换剂柱的离子色谱——十六烷基吡啶季铵盐涂覆柱的性能和应用

Small和Gjerde发展了离子色谱和单柱离子色谱以后,在检测系统和高效固定相方面也有令人注目的进展。例如:近年来利用烷基键合相或中性聚合物和离子对试剂相结合的离子色谱技术,使固定相材料     

Is Multidimensional High Performance Liquid Chromatography (HPLC) an Alternative in Protein Analysis to 2D Gel Electrophoresis?

The interactive modes of High Performance Liquid Chromatography (HPLC) of proteins provide a platform for the construction of a multidimensional HPLC system coupled to mass spectrometry. We present a system composed of both anion and cation exchanger columns, in the first dimension, and n‐octadecyl bonded 1.5 μm nonporous silica columns in the second dimension. Both columns are operated under gradient conditions. A system suitability test with standard proteins showed that the total analysis can be performed within about 20 minutes. The fractions taken from the ion exchanger column are directly analyzed within one minute on the reversed phase column at a high flow rate. Two reversed phase columns are applied and operated alternatively: while the first column performs the separation within one minute, the analytes leaving the first dimension are enriched in an on‐column focusing mode on top of the second column. The sample clean‐up and enrichment is performed on a novel type of restricted access cation exchanger column with internal sulfonic acid groups and external diol groups. The columns exhibit a molecular weight exclusion limit for globular proteins of about 15 kDa. Our next studies will be directed towards the analysis of proteins and peptides from extracts of fibroblasts.     

Chromatographic Properties of HPLC Sorbents Modified with Zinc Octa-4,5-Carboxyphthalocyanate

The adsorption of the zinc octa-4,5-carboxyphthalocyanate modifier on known HPLC sorbents of different types (Silasorb-C₁₈, Diasorb-130-C₄, Diasorb-130-C₁₆, Spheron C 100 (LC), Spheron C 1000, and Hema S 1000 QL) was studied. The optimum conditions were determined for the modification of the sorbents in the static and dynamic modes. The mechanism of the retention of modifiers and adsorbates was considered, and the amount of the modifier on the surface of the adsorbents was calculated. The effect of the modifier on the retention of adsorbates was demonstrated. Mobile phases were selected for the selective and efficient separation of model mixtures of phenols.     

Chromatography of inorganic ions o sulfoethyl cellulose layer in hydrochloric acid and in hydrochloric acid-ammonium thiocyanate media

Summary Chromatographic behavior of 51 inorganic ions has been systematically studied on layers of sulfoethyl (SE) cellulose, a strongly acidic exchanger, in hydrochloric acid and in acid-ammonium thiocyanate media. The sorption of most of the ions on he SE-cellulose decreases with increasing concentration of the acid and the thiocyanate. The characteristic retention of some metal ions of SE-cellulose layer can be recognized over a low concentration of the acid or the salt. Feasibilities for separations of analytical interest are also presented in both systems.     

Expanded Bed Adsorption Chromatography for Recovery of Phycocyanins from the Microalga Spirulina Platensis

We carried out the purification of C-phycocyanin and allophycocyanin from Spirulina platensis taking advantage of the adsorption properties of the expanded beds. Initially, phycobiliproteins were released from the microalga cells by osmotic shock. Next, phycocyanins were recovered by applying the centrifuged cell suspension directly to the anion exchanger Streamline-DEAE using expanded bed columns, equilibrated with 50 mM sodium phosphate buffer, pH 7.0. After adsorption, washing was carried out in the expanded-bed mode. Having removed unbound proteins and cellular debris, the bed was allowed to sediment and phycocyanins rich solution was eluted with a downward flow of 500 mM sodium phosphate buffer, pH 7.0. Finally, we utilized conventional gel filtration and ion exchange chromatography methods for separation and purification of C-phycocyanin and allophycocyanin. The purification steps were monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the purity of recovered phycocyanins was confirmed by absorption and emission spectroscopy. The main advantage of this new method is the high yield achieved in the steps of product extraction and adsorption by expanded bed adsorption, so reducing both processing times and costs.     

Advances in capillary electrophoresis: the challenges to liquid chromatography and conventional electrophoresis

In the 1980s, capillary electrophoresis (CE) developed rapidly into a first-class analytical separation technique. Its advances in instrumentation and method development will not only enhance or complement existing mature separation techniques such as liquid chromatography and conventional slab gel electrophoresis, but will also severely challenge these separation methods. A brief overview of the most striking achievements of CE in the 1980s is given. which illustrates the challenges to liquid chromatography and conventional slab gel electrophoresis, and some detailed discussions are presented to highlight the advantages of CE. New developments in CE that can be expected for the 1990s include especially column technology, separation chemistry and instrumentation, which will serve further to diversify and improve the applicability of this technique in areas which are poorly addressed by other separation methods. This paper considers and speculates on the technological advancements that can be expected to emerge for CE in the 1990s.