Development of capillary column packed with thiol-modified gold-coated polystyrene particles and its selectivity for aromatic compounds

Three types of thiol compounds (n-octadecanethiol, thiophenol, and 2-phenylethanethiol) were used to modify the gold-coated polystyrene particles (dp. 5microm) to prepare a stationary phase for capillary liquid chromatography through the formation of self-assembled monolayer. The column with n-octadecanethiol-modified gold-coated polystyrene particles (C18-Au) demonstrated the higher affinity to phenanthrene and anthracene than small aromatics compared to the ODS column. In addition, the shape selectivity between phenanthrene and anthracene in the C18-Au column was much higher than that in the ODS column (separation factors: 1.82 and 1.14, respectively). The relationship between the retention factor and acetonitrile content in the mobile phase revealed that the retention behaviors in the C18-Au column was more sensitive on the acetonitrile content than those in the ODS column. Relatively higher affinity for phenanthrene and anthracene was commonly observed in all the three thiol-modified Au columns than that for the conventional ODS column, whereas separations of benzene and nitro- and chlorobenzenes were quite different among the three thiol-modified Au and ODS columns.     

In flow activation of diol-silica with cyanogen bromide and triethylamine for preparing high-performance affinity chromatographic columns

A new coupling strategy using pre-packed diol-silica supports to obtain affinity columns for high-performance affinity chromatography (HPAC) is described. These columns were prepared by "in flow" activation in which solutions containing anhydrous solutions of CNBr and triethylamine are separately pumped to a mixer and then onto a pre-packed diol-silica column. Recycling the amino ligand to be coupled several times over the activated silica diol columns results in ligand immobilization. DNA (the Op 1 lac operator), 6-aminohexyl-Cibacron and a peptide (melittin) were all successfully "in flow" coupled to freshly activated columns. Methods for CNBr activation of pre-packed diol-silica column were developed for one, two or three pump HPLC systems. The supports were successfully used for the HPAC purification of a Lac repressor-beta-galactosidase fusion protein, alcohol dehydrogenase, and calmodulin. Columns prepared by in flow activation/coupling procedures were shown to be stable for at least 14 months. Also, in flow activated silica columns could be stored in anhydrous acetone for at least 3 months prior to coupling. Our experiments with these affinity ligand columns (DNA-silica, aminohexyl-Cibacron F3GA-silica, and melittin-silica), suggests that this is a very successful coupling protocol for producing a variety of HPAC columns.     

High-performance catalytic chromatography. The adapter approach

A sequence-specific DNA that binds EcoRI endonuclease was immobilized on glycidioloxypropyl-silica and Sepharose by cyanogen bromide (CNBr)-activated coupling. Elution of bound enzyme by conventional affinity strategies (increase of salt concentration) or by catalysis-induced elution (adding a Mg2+ cofactor required for catalysis) was compared. Greater yield and fold-purification was obtained with catalysis-induced elution for both DNA-silica and DNA-Sepharose columns, and silica gives higher performance than Sepharose. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed primarily a single band for EcoRI endonuclease for catalysis-induced elution from DNA-silica columns. Since catalysis-induced elution decreases the lifetime of DNA affinity columns, an alternative approach for preparing re-usable DNA columns was also developed. In this approach, a single stranded adapter DNA sequence is first coupled to silica or Sepharose and then annealed with another DNA sequence that contains a complementary, single stranded tail and the duplex binding site for EcoRI endonuclease. After use, replacing the hydrolyzed DNA regenerates the column. For this adapter approach, Sepharose gives better purity than silica and comparable yields and catalytic based elution gave the highest purity and yield, regardless of support. Substrate DNA with either a tail (for annealing to the column) at one end or both ends were compared and the former gave higher purity. Finally, enzyme binding to the substrate in solution ("trapping") or on a pre-bound substrate column was compared and trapping gave higher yield and similar purity to the alternative. Thus, trapping with a single tailed substrate oligonucleotide on a Sepharose adapter column and using catalytic elution gave the highest performance.     

金属螯合亲和色谱中固定金属与蛋白质的作用

在不同PHNaCl的磷酸缓冲体系，比较了牛血清蛋白（BSA）、核糖核酸酶（RNase)、细色素C（Cyt-C)和溶菌酶（Lys)在IDA裸柱和一些金属螯合柱上的保留特性，考察了固定金属对蛋白质保留行为的影响，指出蛋白质在强结合IDA－Cu柱上的保留主要受固定金属和蛋白质间配位作用支配，在弱亲和的IDA－Ni,IDA-Co和IDA－Zn柱上的保留主要受静电作用控制，配位作用为辅，讨论了金属螯合亲和色谱中影响蛋白质和金属配位的主要因素，金属离子的电荷和半径，配位原子对中心离子外层d轨道的影响，以及蛋白质表面配位的组氨酸数目，离解常数和取向，影响金属螯合配体和蛋白质静电作用的主要因素为溶液的PH和蛋白质的等电点pI.     

Identification of the factors that influence the reproducibility of chromatographic retention data

Principal component analysis was used to identify the parameters that influence the column-to-column and batch-to-batch reproducibility of retention times and retention factors measured on Symmetry C18, Kromasil C18, Luna C18 (2) and Vydac RP C18, all reversed-phase silica columns. We devised a procedure that allows the determination of the differences in column volume and packing density between two columns, provided that these columns are packed with identical stationary phases (i.e., phases that originate from the same batch). Principal component analysis of the retention times confirmed that the column-to-column variations of the column volume and the total porosity of the bed are the factors that influence the reproducibility of the retention times, the column volume being the major factor. For the fluctuations of the retention factors, the column phase ratios (or the bed porosities) and some specific, secondary retention mechanisms are responsible. All the C18 columns investigated proved to behave in a very similar fashion. Two principal components were always sufficient to characterize the variations of either the retention times or the retention factors.     

Photochemical modifications of lac repressor--II. Tryptophan photochemistry as a probe in studying the allosteric behaviour of the protein

Irradiation of lac repressor under aerobic conditions in the near UV region (295-400 nm) decreases the Trp fluorescence of the protein. A total loss of fluorescence corresponds to the destruction of all tryptophanyl residues. Irradiation with light of wavelength between 250 and 400 nm quenches fluorescence completely when only half of the Trp residues ae destroyed. An internal photodynamic effect, in which N-formylkynurenine, a principal photoproduct of Trp, sensitizes further the destruction of the other Trp residues, accounts for our results. Experiments performed in the presence of sodium azide suggest that singlet oxygen is not involved in the destruction of Trp, but may be responsible for histidine degradation. Irradiating the repressor complexed with non-operator E. coli DNA has the same effect on Trp residues as irradiating repressor alone. On the contrary, when repressor is complexed to lac operator, both tryptophanyl residues seem to be destroyed simultaneously. This indicates that binding of specific operator DNA at the DNA site induces changes in the environment of the tryptophanyl residues (mainly tor Trp 220) which cannot further transfer in excitation energy to the photoproduct of the other Trp. A prolonged irradiation destroys the complex, leading to the same result observed for non-specific complex or for repressor alone. These results are discussed in terms of the proximity of Trp from the inducer binding site and the allosteric behaviour of the repressor.     

反相高效液相色谱法同时分析三聚氰酸和三聚氰胺

建立了同时分析三聚氰酸和三聚氰胺的反相高效液相色谱法．发现三聚氰酸和三聚氰胺在C8柱上的保留比在C18柱上明显要强,这一结果表明三聚氰酸和三聚氰胺在反相固定相中的保留并非以通常的疏水分配作用为主导．方法的紫外检测下限为0．034～0．31mg／L,标准曲线线性范围为0．5-100mg／L．方法不经特殊的样品前处理即可用于奶粉和游泳池水等样品中三聚氰酸和三聚氰胺的同时分析．     

Effect of extra-column volume on practical chromatographic parameters of sub-2-μm particle-packed columns in ultra-high pressure liquid chromatography

Effects of extra-column volume on apparent separation parameters were studied in ultra-high pressure liquid chromatography with columns and inlet connection tubings of various internal diameters (id) using 50-mm long columns packed with 1.8-μm particles under isocratic conditions. The results showed that apparent retention factors were on average 5, 11, 18, and 41% lower than those corrected with extra-column volumes for 4.6-, 3.0-, 2.1-, and 1.0-mm id columns, respectively, when the extra-column volume (11.3 μL) was kept constant. Also, apparent pressures were 31, 16, 12, and 10% higher than those corrected with pressures from extra-column volumes for 4.6-, 3.0-, 2.1-, and 1.0-mm id columns at the respective optimum flow rate for a typical ultra-high pressure liquid chromatography system. The loss in apparent efficiency increased dramatically from 4.6- to 3.0- to 2.1- to 1.0-mm id columns, less significantly as retention factors increased. The column efficiency was significantly improved as the inlet tubing id was decreased for a given column. The results suggest that maximum ratio of extra-column volume to column void volume should be approximately 1:10 for column porosity more than 0.6 and a retention factor more than 5, where 80% or higher of theoretically predicted efficiency could be achieved.     

Elution behavior for a large sample size of uranyl ions on reversed-phase columns using alpha-hydroxyisobutyric acid as an eluent

Large sample sizes of uranyl ions are eluted on a strenedivinylbenzene copolymer phase and an octadecyl phase column, respectively, using alpha-hydroxyisobutyric acid (alpha-HiBA) as an eluent. Chromatograms are obtained from variations of the uranyl sample amounts, eluent concentrations, concentrations of the sample matrix, and the pH of the sample solution for both columns, respectively. Column capacities are estimated from the loading factors measured from the retention times of the peaks. Bandwidths of the peaks and apparent column efficiencies are measured as a function of the loading factor and calculated using the equations derived from the assumptions of a Langmuir isotherm for a single solute. Comparison between the experiment and the calculation reveals that the former showed a broader bandwidth and worse column efficiency than the latter for both columns. The two columns are compared with regards to the retention time, peak shape, column capacity, column efficiency, etc. The PRP-1 column shows a rectangular-, triangle-type peak shape, longer retention time, lower column capacity, and better column efficiency, and the LC-18 column shows a distorted Gaussian curve, shorter retention time, higher column capacity, and worse column efficiency. Column capacity, peak shape, and retention time are dependent on the eluent concentration rather than the alpha-HiBA concentration in the sample solutions.     

Study of the selectivity of reversed-phase columns for the separation of polycarboxylic acids and polyphenol compounds

A systematic investigation was undertaken into the relative separation performance of five reversed-phase chromatography columns including some commercially new hybrid packed columns for a series of polycarboxylic acids and polyphenol compounds. Information theory (IT) and factor analysis (FA), together with a basic evaluation of retention information (band shape, retention factor and elution order) were used to compare four columns to a conventional C18 column. The results revealed very little difference in retention behaviour between the Phenomenex Aqua C18 column, the Waters XTerra RP C18 column, and the conventional Phenomenex Luna C18 column. However, there were notable differences in the retention processes between the Phenomenex Synergi polar-RP column, which is an ether-linked phenyl base with polar endcapping, and the Luna C18 column. The most significant differences were observed between the Luna C18 column and a Phenomenex Luna Cyano column. However, the limited degree of retention of the polycarboxylic acids and polyphenol compounds on the Luna Cyano column permits only limited use for the separation of these types of compounds. Overall, the Phenomenex Synergi polar-RP column exhibited the best performance for the separation of the test solutes compared to that of the conventional C18 column, with IT yielding an Informational Similarity of 0.99 and FA a moderate correlation coefficient of 0.70. The Phenomenex Synergi polar-RP column gave the best peak shape and offered substantial selectivity differences thereby providing a good alternative over the conventional C18 column for separating polycarboxylic acids and polyphenols.     

Characterization of stationary phases based on monosubstituted benzene retention indices using correspondence factor analysis and linear solvation energy relationships in RPLC

In reversed-phase liquid chromatography (RPLC), the comparison of experimental results obtained from different columns is a complex problem. A correspondence factor analysis (CFA) and a linear solvation energy relationship (LSER) were applied on retention data to characterize second-order intermolecular interactions responsible for retention on a set of RPLC columns. Seven octadecyl-C₁₈ columns with different packing materials are obtained from different manufacturers and one octyl-C₈ column. The retention data were determined under isocratic conditions using a methanol–water (65:35, v/v) mobile phase. The chromatographic retention indices based on alkan-2-ones and alkyl aryl ketones retention index scales are calculated using a multiparametric least-squares regressions iterative method. The CFA and LSER results permitted to highlight that the retention indices were appropriate for studying the second-order retention mechanisms on the eight chromatographic systems investigated and exhibited the best reproducibility. Although many earlier studies have reported the use of chemometric methods to characterize chemical factors affecting retention in RPLC using retention factors as retention parameters, this is the first study based on retention indices.     

Application of a flow-tunable, serially coupled gas chromatographic capillary column system for the analysis of complex mixtures

Summary A capillary column gas chromatographic system employing two serially coupled fused-silica columns and a simple coupling element is described. The system is operated in flow-tunable mode (flow-tunable tandem system). The very fact of continuous tuning over a large polarity (selectivity) range, ultimately determined by the two constituent columns, offers several possibilities in the analysis of complex mixtures. In this paper two applications are discussed in detail: optimization of peak separation and peak identification. For these applications it is feasible to use, retention data collected from experiments on the tandem system, and empirical formulas. A relatively simple theoretical mathematical model valid for the flow-tunable tandem system, however, furnishes an easy way of calculating retention data on the system from data collected from the individual single columns, thus, creating a new possibility for optimization and peak identification. Optimization and peak identification processes using the empirical and theoretical models are both demonstrated by analysis of solvent mixtures. Dedicated to the memory of Dr. Tibor Tóth Presented at Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Retention in coupled capillary column supercritical fluid chromatography with lipids as model compounds

Summary We have investigated to which extent retention data, acquired on single capillary columns, can be used for predicting retention factors in a coupled column system. For this purpose we utilized a model mixture of 18 lipid components with widely different vapor pressures and polarities. The sample was chromatographed on two columns, SB-biphenyl-30 (70% methyl-30% biphenylpolysiloxane) and SB-cyanopropyl-50 (50% methyl-50% cyanopropylsiloxane). Experimental retention factors, acquired in coupled column systems with two columns connected in different order, were thus compared with values calculated from runs on each single column. The agreement between calculated and experimental values generally was better than 5% without any pressure drop correction.To study the possibility of predicting retention behavior in a wide pressure range from a limited number of experiments, we also investigated the relation between solute retention and mobile phase density. We found that all data could be fitted to second order equations, which gives the possibility to optimize the resolution with respect to pressure from a limited number of runs at different pressures.