Retention mechanisms in super/subcritical fluid chromatography on packed columns

Whereas the retention rules of achiral compounds are well defined in high-performance liquid chromatography, on the basis of the nature of the stationary phase, some difficulties appear in super/subcritical fluid chromatography on packed columns. This is mainly due to the supposed effect of volatility on retention behaviours in supercritical fluid chromatography (SFC) and to the nature of carbon dioxide, which is not polar, thus SFC is classified as a normal-phase separation technique. Moreover, additional effects are not well known and described. They are mainly related to density changes of the mobile phase or to adsorption of fluid on the stationary phase causing a modification of its surface. It is admitted that pressure or temperature modifications induce variation in the eluotropic strength of the mobile phase, but effects of flow rate or column length on retention factor changes are more surprising. Nevertheless, the retention behaviour in SFC first depends on the stationary phase nature. Working with polar stationary phases induces normal-phase retention behaviour, whereas using non-polar bonded phases induces reversed-phase retention behaviour. These rules are verified for most carbon dioxide-based mobile phases in common use (CO(2)/MeOH, CO(2)/acetonitrile or CO(2)/EtOH). Moreover, the absence of water in the mobile phase favours the interactions between the compounds and the stationary phase, compared to what occurs in hydro-organic liquids. Other stationary phases such as aromatic phases and polymers display intermediate behaviours. In this paper, all these behaviours are discussed, mainly by using log k-log k plots, which allow a simple comparison of stationary phase properties. Some examples are presented to illustrate these retention properties.     

The Influence of Reversed-Phase n-Alkyl Chain Length on Protein Retention,Resolution and Recovery: Implications for Preparative HPLC

Abstract

High-performance liquid chromatography (HPLC) of proteins on reversed-phase columns of varying n-alkyl chain length (C₂ to C₂₂ was studied using a trifluoroacetic acid/2-propanol mobile phase system. Protein resolution was influenced by chain length but retention times for proteins, unlike those of small molecules, were relatively constant, independent of chain length or carbon loading. Loading capacities were found to be affected by chain length, and aspects of protein interaction with stationary phase are discussed.     

混合色谱填料的研究进展

随着科学技术的发展,人们需要分离分析的样品越来越复杂,尤其是多肽、蛋白质类生物样品的复杂性使得单一模式色谱难以满足分离分析的要求。混合模式色谱因其独特的分离性能,可以在一次分离中获得与多维色谱相当的分离效果,而且可以避免多维色谱系统结构复杂、流动相兼容性差、分析时间长等问题,成为近年来的研究热点之一。混合模式色谱的研究重点是色谱固定相的设计与开发。混合模式色谱固定相包括反相/离子交换混合固定相、反相/亲水混合固定相、亲水/离子交换混合固定相、两性离子交换混合固定相及三相混合固定相。本文综述了近年来混合模式色谱填料的研究及应用进展,并对混合模式色谱及固定相的发展前景进行了展望。     

Stationary and mobile phases in hydrophilic interaction chromatography: a review

Hydrophilic interaction chromatography (HILIC) is valuable alternative to reversed-phase liquid chromatography separations of polar, weakly acidic or basic samples. In principle, this separation mode can be characterized as normal-phase chromatography on polar columns in aqueous-organic mobile phases rich in organic solvents (usually acetonitrile). Highly organic HILIC mobile phases usually enhance ionization in the electrospray ion source of a mass spectrometer, in comparison to mobile phases with higher concentrations of water generally used in reversed-phase (RP) LC separations of polar or ionic compounds, which is another reason for increasing popularity of this technique. Various columns can be used in the HILIC mode for separations of peptides, proteins, oligosaccharides, drugs, metabolites and various natural compounds: bare silica gel, silica-based amino-, amido-, cyano-, carbamate-, diol-, polyol-, zwitterionic sulfobetaine, or poly(2-sulphoethyl aspartamide) and other polar stationary phases chemically bonded on silica gel support, but also ion exchangers or zwitterionic materials showing combined HILIC-ion interaction retention mechanism. Some stationary phases are designed to enhance the mixed-mode retention character. Many polar columns show some contributions of reversed phase (hydrophobic) separation mechanism, depending on the composition of the mobile phase, which can be tuned to suit specific separation problems. Because the separation selectivity in the HILIC mode is complementary to that in reversed-phase and other modes, combinations of the HILIC, RP and other systems are attractive for two-dimensional applications. This review deals with recent advances in the development of HILIC phase separation systems with special attention to the properties of stationary phases. The effects of the mobile phase, of sample structure and of temperature on separation are addressed, too.     

Use of Centrifugal Partition Chromatography and Proteins in The Preparative Separation of Amino Acid Enantiomers

Abstract

The growth of analytical methodologies for the separation of enantiomers has been impressive. Attention is now turning to the large scale separation of enantiomers. Often scaling-up sensitive analytical separations is ineffective and inefficient. Centrifugal partition chromatography (CPC) may be a viable alternative for the preparative separation of racemic mixtures in some cases. The use of proteins as chiral selectors in CPC is examined. Attention was focused on proteins that previously were used as bonded phases in analytical LC columns. The enzymatic properties of α-chymotrypsin allowed it to be used as a bioreactor in conjunction with CPC. When proteins are used as components of the stationary or mobile phase there can be problems with denaturation. However, when used in external incubation processes or as column bioreactors coupled with CPC, effective gram-scale separations can be performed. Tryptophan methyl ester was used as a model compound to evaluate this approach.     

Arginine as an effective additive in gel permeation chromatography

A major problem in gel permeation chromatography (GPC) or size exclusion chromatography is non-specific binding of applied proteins to the column matrix (stationary phase). We have tested an aqueous arginine solution as the GPC mobile phase on silica-based and polymer-based columns, using mouse monoclonal antibody and recombinant human activin, interleukin-6, basic fibroblast growth factor, and interferon-gamma as model proteins. We observed that addition of arginine to the mobile phase improves separation of the proteins and their soluble aggregates from the GPC columns, which suggests that arginine is an effective additive for the GPC mobile phase.     

Supercritical fluid chromatographic solvatochromic modifier polarity studies

To understand the chromatographic process as a whole, whether it be for gas chromatography (GC), liquid chromatography (LC), or supercritical fluid chromatography (SFC), one needs to know the chemical and physical nature of the mobile and stationary phases and also the interactions that take place between analytes (solutes) and the two phases. An approach towards Investigating the ways that stationary and mobile phases contribute to chromatographic retention Involves exploring the effects of solvent polarity on the strength of the mobile phase. In SFC this could involve determining the polarity of several different modifier/carbon dioxide mobile phases. In this paper, the use of a solvatochromic indicator to learn more about the effects of SFC modifier/mobile phase polarity will be investigated and discussed using several different modifiers and a diolmodified silica column.     

Preparation of weak cation exchange packings for chromatographic separation of proteins using "click chemistry'

"Click chemistry" is defined as a class of robust and selective chemical reactions affording high yields and is tolerant to a variety of solvents (including water), functional groups, and air. In this study, click chemistry was used as an effective strategy for coupling three alkyne-carboxylic acids onto the azide-silica to obtain three novel stationary phases of weak cation exchange chromatography, which were characterized with FTIR and elemental analysis. Six kinds of standard proteins, such as myoglobin, RNase A, RNase B, cytochrome C, α-chymotrypsin A, and lysozyme, were separated completely with the three novel weak cation exchange chromatography stationary phases. Compared with commercial weak cation exchange chromatography columns, the three kinds of novel weak cation exchange chromatography packings prepared by click chemistry approach have better resolution and selectivity. The mass recovery of more than 97% was obtained for all the tested proteins, and the bioactivity recovery of lysozyme on the prepared column was determined to be 96%. In addition, lysozyme was purified successfully from egg white with the novel weak cation exchange chromatography column by one step. The purity was more than 97% and a high specific activity was achieved to be 81 435 U/mg. The results illustrate the potential of click chemistry for preparing stationary phase for ion-exchange chromatography.     

High-performance liquid chromatography of substituted p-benzoquinones and p-hydroquinones. II. Retention behavior, quantitative structure-retention relationships and octanol-water partition coefficients

The retention behavior of methoxy-substituted p-benzoquinones and the corresponding hydroquinones in reversed-phase chromatography was examined on octylsilica and two octadecylsilica stationary phases and with five hydroorganic mobile phases containing acetonitrile, methanol or tetrahydrofuran and additionally in most cases (NH3OH)3PO4 used as a reducing and buffering agent. The retention order of benzoquinones and hydroquinones was the same on each stationary phase with either methanol or acetonitrile as the organic modifier. On the other hand, minor differences in the retention order were observed with the various stationary phases. In all cases, satisfactory quantitative structure-retention relationships (QSRRs) were found and the data suggest that the differences in the retention behaviour of octadecylsilicas used in this study are silanophilic interactions which, together with solvophobic interaction contribute to the retention of these eluites. Further analysis showed that QSRRs of sterically crowded molecules must take into account reduced surface area available for binding. The retention data obtained with use of aqueous tetrahydrofuran as mobile phase failed to give rise to satisfactory QSRRs. This was attributed to selective solvation of eluite by tetrahydrofuran and/or nearly equipotent binding of eluite and tetrahydrofuran to stationary phase.     

Reversed-phase liquid chromatography and argentation chromatography of the minor capsaicinoids

An investigation of the liquid chromatography of the minor capsaicinoids in a commercial capsaicinoid mixture is reported. Twelve stationary phases including C₈, C₁₈, C₃₀, phenyl, and cation-exchange chemistries were examined in combination with isocratic aqueous methanol and aqueous acetonitrile mobile phases. A phenyl stationary phase and aqueous acetonitrile mobile phase baseline-resolved 7 of 11 capsaicinoids, and selected ion chromatograms (LC–ESI-MS) demonstrated this was the most effective reversed-phase separation. Argentation chromatography with an alkyl or phenyl column and aqueous silver nitrate–methanol mobile phase revealed the presence of the 6-ene-8-methyl and 6-ene-9-methyl homocapsaicin isomers and the absence of 7-ene-9-methyl homocapsaicin. A mixed phenyl–cation-exchange stationary phase (charged with silver ion) enabled unique and useful separations of the capsaicinoids.     

β-环糊精流动相添加剂在手性分离中的应用综述

介绍了β-环糊精的基本性质,综述了β-环糊精及其衍生物作为流动相添加剂在高效液相色谱和高效毛细管电泳手性分离中的应用,并探讨了其作为手性流动相添加剂的特点.指出β-环糊精是良好的手性识别体,不仅可作为色谱手性固定相,还可作为流动相添加剂,用于手性对映体的拆分.     

Monolithic stationary phases for liquid chromatography and capillary electrochromatography

A monolithic stationary phase is the continuous unitary porous structure prepared by in situ polymerization or consolidation inside the column tubing and, if necessary, the surface is functionalized to convert it into a sorbent with the desired chromatographic binding properties [J. Chromatogr. A 855 (1999) 273]. Monolithic stationary phases have attracted considerable attention in liquid chromatography and capillary electrochromatography in recent years due to their simple preparation procedure, unique properties and excellent performance, especially for separation of biopolymers. This review summarizes the preparation, characterization and applications of the monolithic stationary phases. In addition, the disadvantages and limitations of the monolithic stationary phases are also briefly discussed.     

Effect of stationary and mobile phases on hydrophobic interaction chromatography of proteins and peptides

A number of different stationary phases designed for hydrophobic interaction chromatography have been examined to assess their efficiency and resolving capability with respect to protein and peptide mixtures. A packing with an ether-bonded phase was substantially less hydrophobic than those with propyl- or phenyl-bonded surface chemistry. While the overall efficiencies of most columns were broadly similar with respect to most proteins, some proteins did chromatograph with enhanced efficiency on specific packings. The elution order of individual proteins was, with one or two exceptions, similar for all columns tested using comparable mobile phases. It differed, however, substantially from orders obtained with conventional reversed-phase alkyl-bonded phases and from the elution orders obtained when the hydrophobic packings were used in a reversed-phase mode, i.e. with an organic modifier gradient. Varying the salt used in the mobile phase and its pH under hydrophobic interaction conditions (high ionic strength) changed overall retentivities and also altered specific retention orders, thus offering possibilities of selective resolution of some mixtures.     

Reversed-phase chromatography of proteins with diphenyl-silica column and hydro-organic eluents containing two organic solvents

High-performance liquid chromatography of proteins with silica bonded diphenyl stationary phase and hydro-organic mobile phases with linear gradient elution has been carried out with binary organic modifiers. The use of a mixture of 2-propanol and 1-butanol facilitated elution at total organic modifier concentrations significantly lower than with the use of 2-propanol alone. Furthermore higher protein recovery and retention of biological activity was obtained with the binary organic modifier because the increase in eluent strength with binary organic modifier was greater than that of the denaturing strength of the eluent. The use of a short, 3 cm long, column, relatively high flow-rates and steep gradients was also advantageous in attenuating protein denaturation. The results were interpreted within the framework of a theoretical treatment of the combined effect of the retention process and denaturation reaction simultaneously occurring in the column.     

Selectivity optimization in liquid chromatography via stationary phase tuning

In striving for the best possible separation, the selectivity of stationary phases as an optimization parameter is often underestimated although there are many ways to influence this powerful tool. This review serves to provide an insight into the various ways of adapting the selectivity of a separation in liquid chromatography. Approaches via temperature and flow rate tuning are discussed as a basis followed by focusing on the stationary phase as the superior optimization parameter. Highly selective stationary phases hereby provide an advantage for groups of similar analytes. For more complex mixtures, separations can be improved using mixed-mode technologies where different retention mechanisms are combined. Serial coupling, mixed-bed columns, and stationary phase optimized selectivity liquid chromatography provide solutions to various degrees. Finally, the advantages of stationary phase tuning over adaption of mobile phase and/or temperature are presented in terms of optimum application range.     

High-performance liquid chromatography of nucleic acid constituents: Chromatographic examination of novel stationary phases

Summary Silica-bonded stationary phases were developed for the separation of nucleic acid constituents and their properties investigated with homologous oligoriboadenylic acids in electrostatic interaction chromatography and with alkylbenzenes in reversed-phase chromatography. Analysis of retention data confirmed the stratified molecular structure of the surface which consist of a layer of propyl chains anchoredvia siloxane bridges to the silica surface proper and of polar moieties attached to the hydrocarbonaceous functions. The polar top layer contains weak cationic and/or hydrophobic binding sites, is strongly hydrated in contact with aqueous eluents and bars the access by large biopolymers to the hydrocarbonaceous sublayer. In reversed-phase chromatography of small non polar molecules with hydro-organic eluents, however, this layer is accessible and engenders a retentive behavior typical for weak hydro-carbonaceous bonded phases. As a result the stationary phases, depending on the nature of the sample and the mobile phase, exhibit the properties of "soft" phases for the chromatography of biopolymers under mild elution conditions and those of "hard" phases for the separation of small non-polar molecules under conditions generally employed in reversed-phase chromatography. The retention of nucleic acid constituents on most of the stationary phases investigated subject to a dual mechanism as a result of the interplay of electrostatic and hydrophobic interactions between the eluites and the binding sites on the stationary phase surface. Siliceous stationary phases having surface morphology described above are suitable for the separation of nucleic acid constituents having widely ranging molecular weights up to 3 × 10⁶ Daltons provided the support has appropriate pore dimensions. This is demonstrated by the separation of mixtures arising from digesting t-RNA^pha or polyadenylic acids as well as those of ribosomal RNA’s and different forms of the plasmid pBR322 DNA. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

蛋白折叠液相色谱法中流动相对重组人干扰素-γ质量回收率的影响

蛋白折叠液相色谱法(PFLC)用于变性蛋白质复性并同时纯化时对流动相组成及其洗脱条件的要求远较通常的液相色谱法高。用端基为PEG-200的高效疏水作用色谱固定相对重组人干扰素-γ(rhIFN-γ)进行纯化并同时复性,详细研究了流动相组成、梯度洗脱模式和流速对rhIFN-γ质量回收率和活性的影响。分别以3.0 mol/L (NH4)2SO4 +0.05 mol/L KH2PO4(pH 7.0)和0.05 mol/L KH2PO4(pH 7.0)为流动相A和B,采用35 min非线性梯度洗脱时,所得rhIFN-γ的质量回收率最高。     

Comparison of ceramic hydroxy- and fluoroapatite versus Protein A/G-based resins in the isolation of a recombinant human antibody from cell culture supernatant

A recombinant human antibody (IgG(1)-subtype) was produced in Chinese Hamster Ovary (CHO) cells. Alternatives to the established isolation by Protein A affinity chromatography were investigated. Neither an alternative elution agent (Arginine) nor an alternative affinity ligand (Protein G) resulted in an improvement in yield and/or purity. Subsequently, apatite stationary phases including a novel ceramic fluoroapatite material were tested. By applying a double gradient (first 0 to 1M NaCl, then 0.01 to 0.4M phosphate) the culture supernatant was separated into three fractions: the flow through, which contained no active antibody, the NaCl-eluate, which contained the antibody and no other discernible protein contaminants, and a fraction that eluted in the phosphate gradient and contained several proteins, but no active antibody. In case of the hydroxyapatite, retention of the antibody decreased and yield increased when the pH was raised from 6.0 to 8.2 (isoelectric point (pI) of the antibody: 8.3), to reach a yield of 71% at pH of 8.2. In case of the fluoroapatite, retention was also found to increase with increasing mobile phase pH, but the yields went through a maximum (of ca. 90%) at a mobile phase pH of 7.0. No traces of contaminants were seen in the corresponding gel. This is the first time that yields of 90% and such high purities have been reported as the result of a single chromatographic step for the antibody in question with either (Protein A) affinity or apatite chromatography.     

Computer simulation of non-linear preparative chromatography processes

Summary A theory of non-linear chromatography based on the mass balance equation is presented. The model is a system of partial differential equations and it includes axial dispersion in the mobile phase, flow in the mobile phase and mass transfer between phases. In the case of the injection of a mixture competition between components for places in the stationary or the mobile phase is taken into account. In the paper are discussed: numerical calculations of concentration and volume overloading and the effect of the mutual influence of the components of the injected mixture on the process of mass transfer between phases. Additionally, results of experiments for convex and concave adsorption isotherms are presented. In both cases the results obtained qualitatively confirm the theoretical analyses.     

乳状液对色谱过程的影响

运用特征线法和多流动相色谱的概念,从理论上研究了在非线性色谱过程中流动相为乳状液时其溶质浓度波的类型与特征。当流动相为乳状液时,溶质在乳状液的内相和外相之间发生分配并同时在固定相上进行吸附,此时其浓度波图像与流动相为均相时有很大不同。结合相应的算例分析并讨论了表面活性剂在乳状液内相和外相间的分配关系及其在固定相上的吸附等温线为Langmuir型时其浓度波的各种运动图像及形成机理,并与流动相为均相时的色谱过程作了比较。分析结果表明,多流动相的存在可能使简单波“陡峭化”而成为激波,或使激波溃散为简单波。