Influence of pressure on the retention and separation of insulin variants under linear conditions

The effect of pressure on the retention behavior of insulin variants in RPLC on a YMC-ODS C18 column was investigated under linear conditions. The retention factors of these variants increase nearly 2-fold when the average column pressure is increased from 55 to 250 bar while their separation factors remain nearly unchanged. This effect is explained by a change of the partial molar volume of the insulin variants associated with their adsorption that decreases from -99 to -80 mL/mol for mobile-phase concentrations of acetonitrile increasing from 29 to 33% (v/v). This volume change is much larger than the one observed with low molecular weight compounds. For the same pressure variation, the average number Z of acetonitrile molecules displaced from the protein and the stationary phase upon adsorption increases from 22 to 23.3. The pressure-induced relative increase of the term b[S]/[D0]z (which corresponds to the initial slope of the adsorption isotherm) is approximately twice as large for Lispro than for porcine insulin. Because the binding constant of insulin decreases with increasing pressure, this suggests that the number of binding sites on the stationary phase increases even faster. Finally, it was observed that the column efficiency at flow rates higher than 0.6 mL/min increases slightly with increasing pressure. It is suggested that these observations are also valid for other proteins analyzed in RPLC.     

Retention of ionizable compounds on HPLC. 8. Influence of mobile-phase pH change on the chromatographic retention of acids and bases during gradient elution.

The relationships between retention and mobile-phase pH in gradient elution are studied for acids and bases. The apparent pH shift caused by the increasing amount of acetonitrile and methanol has been determined starting from a wide range of pH values. It is shown that good relationships between the retention of ionizable compounds and the pH of the aqueous buffer can be established if the same type of buffer (ammonium acetate in this work) is used for all pH points. Equations are proposed to fit the gradient retention data to the pH of the aqueous buffer. The proposed equation gives an account of the relative variation of the pKa of the compound in the reference to the variation of the pH of the buffer as both parameters change during gradient elution.     

超滤膜分离渗滤液腐植物质的压力影响研究

超滤处理渗滤液膜生物反应器(MBR)出水,考察操作压力(0.3～0.7 MPa)对分离腐植物质(HS)的影响.结果表明,0.6MPa时浓缩液中TOC随体积因数Fv的增加幅度最大,0.4～0.6 MPa时浓缩液中HS浓度均大幅升高,但0.7 MPa时浓缩液HS浓度仅小幅上升,且HS占总TOC的百分比低于进料液;0.3MPa、0.4 MPa时透过液中TOCC随Fv的增加幅度明显高于其他压力条件;各压力下无机盐离子的截留情况比较相似,Fv=5时,Na、K、Mg离子的截留率约为30%,Fe离子约为50%,Ca离子约为15%;重金属离子的变化情况在低压下基本一致,在0.7 MPa时有所不同;0.6 MPa时HS与盐分的分离效果最好,在本实验条件下0.6MPa为最佳操作压力.压力对TOC和HS的浓缩、透过液中TOC的变化以及HS与盐分的分离效果有明显影响,对无机盐离子和重金属离子的变化影响不明显.     

Comprehensive off-line, two-dimensional liquid chromatography. Application to the separation of peptide digests

The separation of the peptide digests of myoglobin and bovine serum albumin was performed with an off-line combination of two commercial, conventional HPLC columns. The first column was packed with a strong ion exchanger and eluted with a KCl gradient. The second column was packed with particles of C18-bonded silica and eluted with an acetonitrile gradient. The conditional peak capacities of the 2D separations achieved exceed 7000 under the experimental conditions investigated. This performance is achieved at the cost of an analysis time of the order of 28 hours. Possible improvements to the separation method described here are discussed.     

Enrichment and identification of cysteine-containing peptides from tryptic digests of performic oxidized proteins by strong cation exchange LC and MALDI-TOF/TOF MS

The extreme complexity of sample and uninformative fragmentation of peptides in MS/MS experiments are two of several real challenges faced by proteomics. In this work, a strategy aimed at tackling these two problems is presented. Briefly, proteins were first oxidized by performic acid to cleave the disulfide bonds and simultaneously convert cysteine residue into its sulfonic form. Then the resultant sulfonic peptides were enriched by SCX chromatography, exploiting the negative solution charge of sulfonic group. The sulfonic peptide could be easily detected by MALDI-MS in negative mode and showed both enhanced fragmentation efficiency and a simplified spectrum in MALDI-MS/MS experiment in positive mode. The strength of the strategy was demonstrated by applying it to bovine serum albumin. Potential use of the strategy in proteomics was also discussed.     

Measurement of mobile-phase volume in reversed-phase liquid chromatography and evaluation of the composition of liquid layer formed by solvation of packing materials

The mobile-phase volumes (Vm) in reversed-phase liquid chromatography (RPLC) with alkyl-bonded silica, defined as the difference between the total volume of eluent in the column (V0) and the volume of the eluent solvent layer formed by solvation of the bonded phase (VL), are determined by the method derived from the eluent electrolyte effect on the retention of ionic analytes. The validity of the Vm values obtained is evaluated by comparing them with the retention volumes of various organic compounds and inorganic ions, which have been suggested as unretained markers, and those obtained from a linear dependence of the logarithmic retention factor on the carbon numbers of homologous series. From the results obtained, it has been concluded that the solvated liquid phase on a column packing material should be assigned to a part of the stationary phase and the method developed for determination of the Vm value based on the ion partition model gives the most reasonable value as the mobile-phase volume in RPLC. The volume and the solvent composition of the solvated liquid phase on C1, C8, and C18 bonded silica are estimated, and the effects of organic modifiers and the physicochemical structures of the packing materials on these values are discussed.     

Influence of argon pressure on the composition of Co-early transition metal films fabricated by r.f. magnetron sputtering in the composite target mode

The effects of argon pressure and the solute element on compositional changes have been studied in binary Co-early transition metal (ETM) thin films fabricated by r.f. magnetron sputtering using the composite target mode. The solute concentration of the deposited film increases linearly with the area fraction of solute element of target and with the logarithm of argon pressure in the range 0.5–10 m torr. The compositional changes are discussed by considering not only the sputtering yield but also the argon pressure, which is related to the collision effect, and the difference in atomic weights between Co and ETM elements.     

Theory and application of the two-mode gradient elution in liquid chromatography involving simultaneous changes in temperature and mobile-phase composition

The theory of the dual-mode gradient elution in liquid chromatography involving any type of simultaneous changes in column temperature and mobile-phase composition is developed following Drake's approach. The theory was tested in the retention prediction of six alkylbenzenes in aqueous eluting systems modified by acetonitrile. Significant delay phenomena, i.e., a lag between the programmed gradient temperature and the temperature in the oven, and a lag between the oven temperature and the effective temperature the analyte feels in the column, were detected. When these phenomena were taken into account, the retention prediction obtained for all solutes under all dual-mode gradient conditions was excellent. The average percentage error between experimental and predicted retention times is below to 2%.     

Separation of closely related large peptides by micellar electrokinetic chromatography with organic modifiers.

Large peptides with similar electrophoretic mobilities were separated by micellar electrokinetic chromatography (MEKC) with organic modifiers. [Leu13]motilin and [Met13]motilin differ by only one neutral amino acid residue. Because the electrophoretic mobilities of these peptides are almost identical, these peptides were not separated by capillary zone electrophoresis (CZE). Such large peptides have not been separated by conventional MEKC either, because they interacted strongly with the micelle. However, they were completely separated by MEKC when an organic solvent was added to the micellar solution. Some insulins, larger peptides than motilin, from different origins, which have very similar electrophoretic mobilities, were also successfully separated by the same technique. The size of peptides which were separated without organic modifiers was examined.     

乳状液膜法分离钨钼的研究——酸性体系

研究了以三烷基氧化膦（ＴＲＰＯ）为载体、ＮａＯＨ为反萃试剂的液膜体系在酸性介质中进行的钨钼分离．其特点是利用钨、钼与Ｈ２Ｏ２生成的过氧化合物的迁移性能的显著差异来分离钨钼．考察了影响钨钼分离的诸因素．经二级间歇液膜处理，Ｍｏ／ＷＯ３（重量比）从０．５％降到０．００１％以下，达到要求（最高级钨酸产品要求钼含量不大于０．００８％）．本文提供了一种适用于从盐酸分解钨酸钙精矿制取钨酸时分离钨钼的新方法．     

Capillary electrochromatography for separation of peptides driven with electrophoretic mobility on monolithic column.

A mode of capillary electrochromatography for separation of ionic compounds driven by electrophoretic mobility on a neutrally hydrophobic monolithic column was developed. The monolithic column was prepared from the in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate to form a C12 hydrophobic stationary phase. It was found that EOF in this hydrophobic monolithic column was very poor, even the pH value of mobile phase at 8.0. The peptides at acidic buffer were separated on the basis of their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase; therefore, different separation selectivity can be obtained in CEC from that in capillary zone electrophoresis (CZE). Separation of peptides has been realized with high column efficiency (up to 150,000 plates/meter) and good reproducibility (migration time with RSD <0.5%), and all of the peptides, including some basic peptides, showed good peak symmetry. Effects of the mobile phase compositions on the retention of peptides at low pH have been investigated in a hydrophobic capillary monolithic column. The significant difference in selectivity of peptides in CZE and CEC has been observed. Some peptide isomers that cannot be separated by CZE have been successfully separated on the capillary monolithic column in this mode with the same buffer used.     

Influence of operating parameters on the retention of chromatographic particles by thermal field-flow fractionation

We have investigated the retention behavior of chromatographic particles in thermal field-flow fractionation (FFF). Retention time is found to increase with increasing temperature drop across the channel thickness, as expected for species exhibiting a thermophoretic mobility. Experiments have been performed with a vertically oriented channel rather than by using the classical horizontal configuration as this leads to much more reproducible retention data. In acetonitrile, silica-based particles are more retained than octadecyl-bonded silica particles, which confirms our previous finding, by means of a different method, that the thermophoretic mobility of the latter is smaller than that of the former. Whatever the type of particles and the nature of the carrier liquid, the relative retention time is observed to decrease with increasing carrier flow rate. This indicates that a hydrodynamic lift force acts on particles to move them away from the accumulation wall, as is usually observed in all FFF experiments with micrometer-sized particles. However, upward and downward flow directions in the vertical channel lead to similar retention data, indicating that inertial lift forces have a minor influence on retention. In addition, the relative retention time steadily decreases with increasing sample concentration, suggesting that the hydrodynamic lift force increases significantly with sample concentration. Accordingly, we speculate that a new transport phenomenon, called shear-induced hydrodynamic diffusion, not previously accounted for in the modeling of retention in FFF, is controlling the migration of the particles in the FFF channel. Implications of the influence of this phenomenon in other FFF experiments are discussed.     

乳状液膜法分离钨钼的研究弱碱性体系

进行了以氯化甲基三烷基铵（Ｎ２６３）为载体的乳状液膜在弱碱性条件下分离钨、钼的研究．利用钨、钼形成同多酸能力的差异，通过载体选择性迁移钨来分离钨、钼．考察了影响钨、钼分离的各种因素，得到了分离钨、钼的最佳操作条件．按某厂生产仲钨酸铵的料液中含钼８．９×１０－４ｍｏｌ／ｄｍ３计算，经一级间歇式分离即可降至１．６×１０－４ｍｏｌ／ｄｍ３达到要求（含钼低于２．１×１０－４ｍｏｌ／ｄｍ３）     

Influence of column temperature on the electrophoretic behavior of myoglobin and alpha-lactalbumin in high-performance capillary electrophoresis

The influence of column temperature on the electrophoretic behavior of myoglobin and alpha-lactalbumin in high-performance capillary electrophoresis (HPCE) is presented. The major effect of temperature is to shorten the analysis time by decreasing the viscosity, but specific temperature effects on the protein migration behavior were also observed. Myoglobin, under high field (350 V/cm), was essentially temperature stable from 20 to 45 degrees C, but at constant current, a second form of myoglobin could be detected at both 214 and 410 nm. The initial form appeared to correspond to the Fe3+ and the second to the Fe2+ oxidation state of the heme iron. The rate of conversion from Fe3+ to the reduced Fe2+ in myoglobin, under given electrophoretic conditions, followed first-order kinetics with a rate constant at 30 degrees C of 304 s-1. A second protein, alpha-lactalbumin type III, demonstrated a conformational transition that resulted in asymmetric peaks and sigmodial mobility plots versus temperature in the transition region.     

Influence of the gas composition on the synthesis of boron-doped carbon nanotubes by ECR-CVD

Boron-doped carbon nanotubes (CNTs) were synthesized on porous silicon by electron cyclotron resonance chemical vapor deposition (ECR-CVD). CH₄, B₂H₆ and H₂ were used as the source gases and Fe₃O₄ nanoparticles as the catalyst. The effect of gas composition on the boron-doped CNTs growth characteristic was investigated. Scanning electron microscopy , transmission electron microscopy and X-ray photoelectron spectroscopy were used to evaluate the structure and composition. The results show that adding B₂H₆ influences the growth characteristic of CNTs greatly. CNTs tend to unaligned growth with boron. The increase of the B₂H₆ ratio leads to changes in the structure of CNTs from straight tubular to bamboo-like and makes the outer diameters bigger, the tube walls thicker, cause a rough surface and a decrease in the growth rate of the CNTs. An increase in the B/C ratio within the samples was detected as the B₂H₆ ratio in the precursor gas was increased.