高压离子交换排代法富集锂同位素(Ⅰ)——精馏型平衡塔板理论在富集同位素中的应用

本文根据精馏型平衡塔板理论推导出富集系数很小和有限带长时的同位素稳定谱带理论方程组,并对某些具体情况进行了分析。     

High enrichment of 15N by chromatographic chemical process

Nitrogen isotope enrichment experiments were conducted to obtain highly enriched (15)N by ion-exchange process. (15)NH(4)Cl ((15)N=80%) as feeding materials were used to perform the chromatographic operation with two different flow rates and column diameters. Both separation coefficient (epsilon) and height equivalent to a theoretical plate (HETP) have same values in two run experiments. The value of HETP was more enlarged when high enrichment of (15)N was obtained in comparison with that of low enrichment. 99.756% (15)N and 13.63 g (15)N whose percentage was over 99.0% were successfully achieved by 25 m chromatographic migration with the flow rate and column diameter at 50 cm(3)/mL, 3.0 cm, respectively. High flow rate and large column diameter have advantages to the enrichment of (15)N by ion exchange process.     

高压离子交换排代法富集锂同位素(Ⅱ)——HETP计算和富集条件

本文根据“精馏平衡塔板型”富集同位素理论提出了富集同位素时计算HETP的方法,寻找出富集和实验参数之间的关系,并进行了讨论;表明Glueckanf塔板理论和Snyder公式适用于简单排代机制的轻同位素富集过程。     

Band broadening inside the chromatographic column: the interest of a liquid stationary phase

Band broadening inside chromatographic columns was studied by Giddings 40 years ago. This theory is revisited pointing out that the band width depends only on the band position, x, inside the column and the height equivalent to a theoretical plate, H, and not on the solute affinity for the stationary phase. The band standard deviation, sigma, inside the column is simply sigma = square root [xH]. This property can be used in countercurrent chromatography (CCC), a chromatographic technique that works with a liquid stationary phase. Two possibilities are presented: 1-extrusion of the liquid stationary phase called elution-extrusion method, and 2-slow motion of the stationary phase in the same direction as the mobile phase, called cocurrent CCC method. A mixture of five steroids, prednisone, prednisolone acetate, testosterone, estrone and cholesterol, with partition coefficient varying from 0.1 to 40, is used with a 53 mL CCC column to show the method capabilities. The elution-extrusion method is discontinuous; however, it allows saving dramatic amounts of solvent and time. Cholesterol could be fully resolved in 2h and 120 mL instead of 7 h and 1.2 L using the classical elution way. The cocurrent CCC method is continuous and was able to resolve cholesterol at baseline in 40 min using 110 mL. Detection is difficult due to the fact that two immiscible liquid phases enter the detector.     

Effect of ionic liquid additives to mobile phase on separation and system efficiency for HPLC of selected alkaloids on different stationary phases

The silica-based stationary phases with favorable physical characteristics are the most popular in liquid chromatography. However, there are several problems with silica-based materials: severe peak tailing in the chromatography of basic compounds, non-reproducibility for the same chemistry columns, and limited pH stability. Ionic liquids (ILs) as mobile phase components can reduce peak tailing by masking residual free silanol groups. The chromatographic behavior of some alkaloids from different classes was studied on C18, phenyl, and pentafluorophenyl columns with different kinds and concentrations of ionic liquids as additives to aqueous mobile phases. Ionic liquids with different alkyl substituents on different cations or with different counterions as eluent additives were investigated. The addition of ionic liquids has great effects on the separation of alkaloids: decrease in band tailing, increase in system efficiency, and improved resolution. The retention, separation selectivity, and sequence of alkaloid elution were different when using eluents containing various ILs. The increase of IL concentration caused an increase in silanol blocking, thus conducted to decrease the interaction between alkaloid cations and free silanol groups, and caused a decrease of alkaloids retention, improvement of peak symmetry, and increase of theoretical plate number in most cases. The effect of ILs on stationary phases with different properties was also examined. The different properties of stationary phases resulted in differences in analyte retention, separation selectivity, peak shape, and system efficiency. The best shape of peaks and the highest theoretical plate number for most investigated alkaloids in mobile phases containing IL was obtained on pentafluorophenyl (PFP) phase.     

Effect of steric exclusion on the separation of proteins by hydrophilic size-exclusion chromatography

On the basis of studying the retention and band broadening of proteins on the TSK SW column, diffusion coefficients (Ds) of solute in stationary phase were obtained which elucidate the hydrodynamic process of chromatographic resolution of proteins by hydrophilic size-exclusion chromatography (SEC). After calculating the correlation between Ds and the molecular weight of the solute, the molecular dimensions of proteins in the process of chromatographic separation can be predicted. Deviations in diffusion coefficient of a protein from the calculated value reflect differences of measured molecular dimensions from molecular volumes predicted from the calibration curve of the SEC column. This study illustrates a convenient method for estimating the purity of proteins by SEC. Deviations from 2 lambda dp (where dp is the particle diameter) in the intercept of the theoretical plate height (H) versus flow-rate (U) curve from the band broadening equation H = CsU + 2 lambda dp + f(alpha M)T (where CsU represents mass transfer resistance caused by solute diffusion in the stationary phase and f(alpha M)T an added term for polydisperse solutes as proposed by Knox and McLennan [Chromatographia, 10 (1977) 75]) reflect impurities in the proteins.     

Determination of the enrichment of isotopically labelled molecules by mass spectrometry

A general method for the determination of the enrichment of isotopically labelled molecules by mass spectrometry (MS) is described. In contrast to other published procedures, the method described here takes into account and corrects for measurement errors such as the contribution at M ? 1 due to loss of hydrogen or lack of spectral resolution and provides an uncertainty value for the determined enrichment. The general procedure requires the following steps: (1) evaluation of linearity in the mass spectrometer by injecting the natural abundance compound at different concentration levels, (2) determination of the purity of the mass cluster using the natural abundance analogue, (3) calculation of the theoretical isotope composition of the labelled compound using different tentative isotope enrichments, (4) calculation of ‘convoluted’ isotope distributions for the labelled compound taking into account the purity of the mass cluster determined with the natural abundance analogue and (5) comparison of the isotope distributions measured for the labelled compound with those calculated for different isotope enrichments using linear regression. The method was applied to a series of commercially available ¹³C‐ and ²H‐labelled compounds and to a suite of singly ¹³C‐labelled β₂‐agonist prepared in‐house both by gas chromatography (GC)–MS, GC–tandem MS (MS/MS) and liquid chromatography–MS/MS with satisfactory results. It was observed that the main uncertainty source for the isotope enrichment was the uncertainty in the purity of the measured cluster as determined with the natural abundance compound. Copyright © 2014 John Wiley & Sons, Ltd.     

Rapid quality control analysis of (13)C-enriched substrate synthesis by isotope ratio mass spectrometry

There is a growing interest in the use of (13)C-enriched substrates to investigate metabolic processes in humans. The non-invasive nature of (13)C breath tests makes them attractive to clinicians, particularly because they can be safely used in children. The availability of suitable (13)C-enriched substrates can limit the application of this biotechnology. We have used isotope ratio mass spectrometry to assay the chemical purity and isotopic enrichment of substrates that were synthesised to study gut transit and colonic fermentation. Lactose ureide and lactose [(13)C]ureide were synthesised by acid-catalysed condensation of lactose and urea or (13)C urea, respectively. Glucose ureide and glucose [(13)C]ureide were synthesised by similar methods but required an additional purification step to remove urea of crystallisation. Substrates were analysed by standard analytical techniques and combustion isotope ratio mass spectrometry for carbon and nitrogen content and (13)C-enrichment. Monitoring the C/N ratio proved to be a sensitive assay of chemical purity. Analysis of the percentage composition of C and N (and hence O + H) suggested that lactose ureide crystallises as the dihydrate. It was synthesised with approximately 99% chemical purity and with the theoretical enrichment. Glucose ureide was synthesised with approximately 98% chemical purity but with lower than theoretical enrichment.     

Determination of phenylalanine isotope ratio enrichment by liquid chromatography/time- of-flight mass spectrometry

The application of time-of-flight mass spectrometry to isotope ratio measurements has been limited by the relatively low dynamic range of the time-to-digital converter detectors available on commercial LC/ToF-MS systems. Here we report the measurement of phenylalanine isotope ratio enrichment by using a new LC/ToF-MS system with wide dynamic range. Underivatized phenylalanine was injected onto a C18 column directly with 0.1% formic acid/acetonitrile as the mobile phase. The optimal instrument parameters for the time-of-flight mass spectrometer were determined by tuning the instrument with a phenylalanine standard. The accuracy of the isotope enrichment measurement was determined by the injection of standard solutions with known isotope ratios ranging from 0.02% to 9.2%. A plot of the results against the theoretical values gave a linear curve with R2 of 0.9999. The coefficient of variation for the isotope ratio measurement was below 2%. The method is simple, rapid, and accurate and presents an attractive alternative to traditional GC/MS applications.     

Separation of vanadium isotopes by ion-exchange chromatography

Cation-exchange displacement chromatography of VO2+ was carried out for studying vanadium isotope effects in carboxylate ligand-exchange systems. The heavier isotope 51V was enriched in the carboxylate complex solution. The isotope separation coefficients epsilon(= alpha-1) for 50V/51V were 2.2 x 10(-4) and 2.4 x 10(-4) for citrate and lactate systems at 298 K, respectively. These values are much larger than those obtained in a previous study on the malate system. The existence of binuclear complexes of VO2+ may create the conditions for larger isotope fractionation. From the viewpoint of the process development of isotope separation, the heights equivalent to a theoretical plate of these processes were analyzed and found to be very small in each system due to the homogeneous, small and highly porous resin used. Citrate may be better than the other tested systems for the vanadium isotope separation.     

Band broadening of DNA fragments isolated by polyacrylamide gel electrophoresis in capillary electrophoresis

Polyacrylamide gel electrophoresis (PAGE) is used frequently for isolation and purification of DNA fragments. In the present study, DNA fragments extracted from polyacrylamide gels showed significant band broadening in capillary electrophoresis (CE). A pHY300PLK (a shuttle vector functioning in Escherichia coli and Bacillus subtilis) marker, which contained nine fragments ranging from 80 to 4870 bp, was separated by PAGE, and each fragment was isolated by phenol/chloroform extraction and ethanol precipitation. After extraction from the polyacrylamide gel, the peaks of the isolated DNA fragments exhibited band broadening in CE, where a linear poly(ethylene oxide) was used as a sieving matrix. The theoretical plate numbers of the DNA fragments contained in the pHY300PLK marker were >10⁶ for all the fragments before extraction. However, the DNA fragments extracted from the polyacrylamide gel showed decreased theoretical plate numbers (5–20 times smaller). The degradation of the theoretical plate number was significant for middle sizes of the DNA fragments ranging from 489 to 1360 bp, whereas the largest and smallest fragments (80 and 4870 bp) had no obvious influence. The band broadening was attributed to contamination of the DNA fragments by polyacrylamide fibers during the separation and extraction process.     

Evaluation of Alkyl Bonded Silica and Solvent Phase Modifiers for the Efficient Elution of Basic Drugs on HPLC

Abstract

Nine representative drugs were used to evaluate the effects of alkyl bonded stationary phases containing type A and type B silica and the effects of an amine modifier on the efficiency of high performance liquid chromatographic elution of basic and acidic drugs. The theoretical plate count and asymmetry factor of the eluted peaks were compared to that of acetophenone as a reference to the maximal efficiency of each system evaluated. Zorbax^R C₈ was used as the stationary phase prepared from type A silica and Zorbax RX^R was used as the stationary phase prepared from the type B silica. The theoretical plate count and asymmetry factor of acetophenone was observed to be the same on both columns when analyzed in an acidic aqueous/acetonitrile mobile phase. An improvement in the efficiency and peak shape of the amine containing compounds was observed using the Zorbax RX^R stationary phase as compared to the efficiency and peak shape of these compounds on the Zorbax^R C₈ stationary phase. Interestingly, the acidic compounds salicylic acid and mefenamic acid showed better peak shapes on the Zorbax RX^R column than on the Zorbax C₈. For all drugs studied the theoretical plate count and asymmetry factor was better on both the Zorbax^R C₈ and the Zorbax RX^R stationary phases when the amine modifier triethylamine was used in the mobile phase. Except for salicylic acid, the theoretical plate count and asymmetry factor for each drug was similar on the Zorbax^R C₈ and the Zorbax RX^R columns when the amine modifier     

Boronic acid lectin affinity chromatography (BLAC). 3. Temperature dependence of glycoprotein isolation and enrichment

In this paper, the effect of temperature is investigated on the performance of glycoprotein enrichment by boronic acid lectin affinity chromatography (BLAC). Wheat germ agglutinin and m-aminophenyl boronic acid containing stationary phases were evaluated individually and in a mixed mode using an automated liquid handling robot with an integrated 96-well plate temperature controller. Glycoaffinity enrichment of the model proteins of ribonuclease B and trypsin inhibitor was investigated in the presence of the non-glycosylated proteins of myoglobin (neutral) and lysozyme (basic) at a wide temperature range of 5–65 °C. Our results revealed that glycoaffinity micropartitioning at the temperature of 25 °C provided the highest recovery rate for glycoprotein enrichment. We have also found that a large amount of lysozyme was present in the elution fractions of the m-aminophenyl boronic acid containing micropartitioning columns due to ion-exchange mechanism occurring between the positively charged protein and the negatively charged stationary phase at the operation pH. On the other hand, at high temperature (65 °C), non-specific interactions with the agarose carrier prevailed, evidenced by the presence of myoglobin in the eluate.     

Online preconcentration using monoliths in electrochromatography capillary format and microchips

Online preconcentration and separation of analytes using an in situ photopolymerized hexyl acrylate-based monolith stationary phase was evaluated using electrochromatography in capillary format and microchip. The band broadening occurring during the preconcentration process by frontal electrochromatography and during the desorption process by elution electrochromatography was studied. The hexyl acrylate-based monolith provides high retention for neutral analytes allowing the handling of large sample volumes and its structure allows rapid mass transfer, thus reducing the band broadening. For moderately polar analytes such as mono-chlorophenols that are slightly retained in water, it was shown that enrichment factors up to 3500 can be obtained by a hydrodynamic injection of several bed volumes for 120 min under 0.8 MPa with a decrease in efficiency of 50% and a decrease of 30% for the resolution between 2- and 3-chlorophenol. An 8 min preconcentration time allows enrichment factors above 100 for polyaromatic hydrocarbons. The interest of these monoliths when synthesized in microchip is also demonstrated. A 200-fold enrichment was easily obtained for PAHs with only 1 min as preconcentration time, without decrease in efficiency.     

吸附固定相开管毛细管电色谱方法的建立（英文）

 首次将管壁吸附作用作为开管毛细管电色谱固定相制备的推动力,成功地建立了称为“吸附固定相开管毛细管电色谱”的一种新方法。原理是：选择合适的条件,让荷正电的化合物在毛细管管壁上充分吸附,直接用吸附层作为固定相。目前,已有数类化合物被用作固定相物质,其中包括阳离子表面活性剂如十六烷基三甲基溴化铵（CTAB）、碱性蛋白质如溶菌酶和细胞色素C、碱性小肽如赖氨酸-酪氨酸和赖氨酸-丝氨酸-酪氨酸、以及碱性氨基酸如L-赖氨酸。CTAB吸附固定相用于分离电中性化合物,其它吸附固定相用于手性分离。     

吸附固定相开管毛细管电色谱方法的建立（英文）

首次将管壁吸附作用作为开管毛细管电色谱固定相制备的推动力,成功地建立了称为“吸附固定相开管毛细管电色谱”的一种新方法。原理是：选择合适的条件,让荷正电的化合物在毛细管管壁上充分吸附,直接用吸附层作为固定相。目前,已有数类化合物被用作固定相物质,其中包括阳离子表面活性剂如十六烷基三甲基溴化铵（CTAB）、碱性蛋白质如溶菌酶和细胞色素C、碱性小肽如赖氨酸-酪氨酸和赖氨酸-丝氨酸-酪氨酸、以及碱性氨基酸如L-赖氨酸。CTAB吸附固定相用于分离电中性化合物,其它吸附固定相用于手性分离。所建立的方法重现性好（迁移时间次间RSD值小于2．3％）,柱效高（最高柱效可达590000／m,平均柱效在250000／m以上）,寿命较长（2～8℃下保存,至少30d）。与已有的固定相制备方法比较,所建立的方法具有简便、快速和稳定等优点。     

Automated interpretation of mass spectra of complex mixtures by matching of isotope peak distributions

Mass spectrometry is now firmly established as a powerful technique for the identification and characterization of proteins when used in conjunction with sequence databases. Various approaches involving stable-isotope labeling have been developed for quantitative comparisons between paired samples in proteomic expression analysis by mass spectrometry. However, interpretation of such mass spectra is far from being fully automated, mainly due to the difficulty of analyzing complex patterns resulting from the overlap of multiple peaks arising from the assortment of natural isotopes. In order to facilitate the interpretation of a complex mass spectrum of such a mixture, such as an MS spectrum of a stable-isotope-enriched ion species, we report on the development of a software application, 'Matching' (web accessible), that enables the automatic matching of theoretical isotope envelopes to multiple ion peaks in a raw spectrum. It is particularly useful for resolving the relative abundances of narrow-split paired peaks caused by enrichment with a stable isotope, such as 18O, 13C, 2H, or 15N.     

Phase optimized liquid chromatography as an instrument for steroid analysis

A new method using phase optimized LC (POPLC) for the analysis of steroids is described. The retention factors and the theoretical plate numbers of different steroids were determined for four different stationary phases. Based on these values, an optimal stationary phase composition and the resultant chromatogram have been calculated by an optimization software and compared to the measured data. It is demonstrated that these predicted data show only little differences to the experimental results. Furthermore, it is shown that the overall selectivity of the optimal stationary phase composition is much better than the selectivity of any individual stationary phase.     

Reducing Dimensionality in Principal Component Analysis – A Method Comparison

The retention time of 10 ring-substituted aniline derivatives was determined on six different reversed-phase stationary phases and the retention parameters log k, theoretical plate number (N) and asymmetry factor (F) were calculated for each analyte on each column. The similarities and differences among the stationary phase and analytes were assessed by principal component analysis coupled with nonlinear mapping and cluster analysis. Computations demonstrated that the retention characteristics of both the stationary phases and analytes markedly deviate from each other. Calculations indicated that the results considerably depend on the multivariate method applied, therefore, the employment of more than than one computation method and the critical comparison of the results is highly advocated.     

Changes in peak width and concentration at the inlet and outlet of a chromatographic column

The original plate model of chromatography is extended to the sorption process occurring at the column inlet and the desorption process at the column exit. At the column inlet it is shown that sufficiently wide feed bands undergo no change in concentration but a fall in band width, i.e., the volume of mobile phase occupied by the solute band is reduced. The reduction factor is (1 + k) where k is the mass distribution ratio (capacity factor). Narrower bands suffer partial reduction in both band width and concentration. On desorption at the outlet, however, the change is always in band width and not concentration. A perfect detector registers the true concentration-time profile of the band in the column if the solute mass fraction in the stationary phase is below 10^?3 at the column outlet. The risks of stripping the stationary phase at high solute concentrations in analytical and preparative or production gas chromatography are compared.