Updating chromatographic predictions by accounting ageing for single and tandem columns

The most efficient optimization methodologies in liquid chromatography are based on the modeling and prediction of the chromatographic behavior for each compound in the sample. However, when the column suffers some ageing after the modeling process, predictions may differ significantly from the actual separation. Repeating the modeling is especially troublesome when several columns are involved, as is the case of coupled columns. We propose a shortcut to correct the time and peak profiles in these situations, after evaluating the effects of ageing. The original models are corrected by introducing parameters accounting for column ageing, obtained using the data of a small subset of compounds from those used to model the brand‐new column. The ageing parameters are fitted from the discrepancies between the data predicted with the original retention models for the brand‐new column and the experimental data measured for the aged column. The approach was developed and tested to predict the chromatographic behavior of 15 sulfonamides, analyzed with individual and tandem columns, using isocratic and gradient elution. Chromatograms more in line with the aged column performance were predicted. The agreement between predictions and experimental data in the aged columns was excellent.     

Modeling the effects of column packing quality and residence time changes on protein monomer/aggregate separation

The packing quality of chromatography columns used for the purification of protein therapeutics is routinely monitored to ensure consistent and reproducible performance. In this work, we used established chromatography models to determine the effect of column packing quality and fluid residence time on the separation of protein therapeutic monomer and aggregate species using a hydrophobic interaction chromatography adsorbent (Phenyl Sepharose Fast Flow). The relationship between the number of theoretical plates, fluid residence time, and column separation performance was quantified using modeling simulations. The simulations showed the separation depended on both the fluid residence time and the number of theoretical plates. However, when the number of theoretical plates was increased to ≥150, the simulations predicted that the separation performance of the column was not significantly improved. The approach described here could be used as a method to quantify acceptable height equivalent of a theoretical plate values for columns, and serve as a tool to understand how column packing quality impacts a given chromatographic separation prior to column scale-up, as well as during the monitoring of column lifetime in the manufacturing of large scale protein therapeutics.     

Chromatographic characterization of molecularly imprinted polymers

Recent efforts in the investigation of chromatographic characterization of molecularly imprinted polymers (MIPs) have focused mainly on the nature of heterogeneous binding sites. More data on the thermodynamics than on the kinetic features of MIP columns have been published. The present article addresses the sources of peak broadening and tailing, which are the main drawbacks often associated with imprinted polymers in chromatography for practical applications. With use of the theory of nonlinear chromatography, the peak properties of a MIP column, including the retention and peak broadening and tailing, can be well interpreted. Efforts to improve chromatographic efficiency using MIPs prepared by approaches different from the conventional method, including covalent imprinting and the format of uniformly sized spherical microbeads, are reviewed and discussed. This review leads to the conclusion that nonlinear chromatography theory is useful for characterizing chromatographic features of MIP columns, since a MIP is essentially an affinity-based chromatographic stationary phase. We expect more theoretical and experimental studies on the kinetic aspects of MIP columns, especially the factors influencing the apparent rate constant, as well as the analysis of the influences of mobile-phase composition on the chromatographic performance. In addition to revealing the affinity interaction by molecular recognition, slow nonspecific interactions which may be inherited from the imperfect imprinting and may be involved in the rebinding of the template to MIPs also need to be characterized. Figure The peak broadening and tailing associated often with molecularly imprinted polymers (MIPs) in column chromatography for practical applications can be well characterized by the theory of nonlinear chromatography.     

Comparison of different types of stationary phases for the analysis of soy isoflavones by HPLC

Nowadays, there are new technologies in high-performance liquid chromatography columns available enabling faster and more efficient separations. In this work, we compared three different types of columns for the analysis of main soy isoflavones. The evaluated columns were a conventional reverse phase particle column, a fused-core particle column, and a monolithic column. The comparison was in terms of chromatographic parameters such as resolution, asymmetry, number of theoretical plates, variability of retention time, and peak width. The lower column pressure was provided by the monolithic column, although lower chromatographic performance was achieved. Conventional and fused-core particle columns presented similar pressure. Results also indicate that direct transfer between particle and monolithic columns is not possible requiring adjustment of conditions and a different method optimization strategy. The best chromatographic performance and separation speed were observed for the fused-core particle column. Also, the effect of sample solvent on the separation and peak shape was evaluated and indicated that monolithic column is the most affected especially when using higher concentrations of acetonitrile or ethanol. Sample solvent that showed the lowest effect on the chromatographic performance of the columns was methanol. Overall evaluation of methanol and acetonitrile as mobile phase for the separation of isoflavones indicated higher chromatographic performance of acetonitrile, although methanol may be an attractive alternative. Using acetonitrile as mobile phase resulted in faster, higher resolution, narrower, and more symmetric peaks than methanol with all columns. It also generated the lower column pressure and flatter pressure profile due to mobile phase changes, and therefore, it presents a higher potential to be explored for the development of faster separation methods.     

Recent developments in microcolumn liquid chromatography.

An overview of the most recent developments in microcolumn liquid chromatography (LC) is presented. A short theoretical discussion on chromatographic dilution and extracolumn bandbroadening is given and also the recent progress and advances in column technology and instrumentation are reviewed. However, the emphasis of this review is on miniaturized sample clean-up, sample introduction techniques and on both established and more recent detection techniques for microcolumn LC. The hyphenation of miniaturized LC columns with other techniques, specifically on multidimensional chromatography and the coupling of microcolumn LC to mass spectrometry is discussed in detail. Both the on-line and automated off-line interfacing to other separation and detection techniques will also be addressed. Finally, a number of typical microcolumn LC applications are presented in order to demonstrate the potential of microcolumn LC methods in a variety of scientific areas.     

开管离子色谱柱的制备与表征

近年来,开管离子色谱(OTIC)的研究已取得重大进展,尤其是兼容OH^-淋洗液的OTIC柱的成功研制使OTIC梯度洗脱得以实现。该文介绍了各种OTIC柱的制备和表征方法,涵盖早期的二氧化硅开管柱和新近出现的有机聚合物基质开管柱,对静电乳胶附聚的OTIC柱的一系列表征方法进行了系统阐述,包括开管柱柱容量的实验测定和理论计算方法、固定相相同时填充柱与开管柱之间的联系、柱效改善方法以及开管柱均一性表征等。     

Study of open-tubular micro-capillary liquid chromatography

Capillary columns in gas chromatography, first proposed by Golay [1,2], have become well established [3] which have extremely high column efficiencies and separabilities. Everyone could imagine it to be a great tool if such a kind of high efficiency column could be established in liquid chromatography (LC). Microcapillary liquid chromatography (MCLC) may be one way of achieving high-efficiency in terms of theoretical plates. Few papers were published in this field before 1976 [4–6], but more recently, after 1977 [7–15], this field has become one of topical interest in liquid chromatography. Temporarily there are two microcapillary liquid chromatography, that is: ?open-tubular”? [7,8,10–15] and ?packed”? [9] MCLC. In this presentation we focus on open-tubular microcapillary liquid chromatography and a review contemporary developments of ours and others.     

Implications of column peak capacity on the separation of complex peptide mixtures in single- and two-dimensional high-performance liquid chromatography

Column peak capacity was utilized as a measure of column efficiency for gradient elution conditions. Peak capacity was evaluated experimentally for reversed-phase (RP) and cation-exchange high-performance liquid chromatography (HPLC) columns, and compared to the values predicted from RP-HPLC gradient theory. The model was found to be useful for the prediction of peak capacity and productivity in single- and two-dimensional (2D) chromatography. Both theoretical prediction and experimental data suggest that the number of peaks separated in HPLC reaches an upper limit, despite using highly efficient columns or very shallow gradients. The practical peak capacity value is about several hundred for state-of-the-art RP-HPLC columns. Doubling the column length (efficiency) improves the peak capacity by only 40%, and proportionally increases both the separation time and the backpressure. Similarly, extremely shallow gradients have a positive effect on the peak capacity, but analysis becomes unacceptably long. The model predicts that a 2D-HPLC peak capacity of 15,000 can be achieved in 8 h using multiple fraction collection in the first dimension followed by fast RP-HPLC gradients employing short, but efficient columns in the second dimension.     

A study of the efficiency of monolithic silica gel capillary columns for gas chromatography

The efficiency and dynamic characteristics of seven silica-gel-based monolithic capillary columns were analyzed by separating on them a mixture of five light hydrocarbons. For helium carrier gas flowing at an optimum velocity, the height equivalent to a theoretical plate was found to be 0.15–0.20 mm, values comparable to those typical of packed capillary columns. An analysis of the Van Deemter curves for the columns under study demonstrated that the main contribution to the smearing of the chromatographic zone comes from the diffusional processes in the mobile phase while the mass transfer between the mobile and stationary phases plays only a minor role. At the same time, the parameter A in the Van Deemter equation, which characterizes the degree of column packing uniformity, was found to be negative. This result contradicts the classical theory of chromatography and calls for further studies of monolithic capillary columns.     

Contribution to the time balance in gas-liquid chromatography new definition equations of the retention times and retention volumes

The numerous research groups and researchers, as well as IUPAC, that during the last half century have dealt with different theoretical and practical problems in gas-liquid chromatography (GLC), including its nomenclature, have failed in giving an exact definition equation of the net retention time. Using our earlier results and starting from a time balance of GLC we have solved this problem by introducing the so-called acceleration time, t(ac), in the absence of which, the theoretical plate number concept, including the stationary phase transfer, is misinterpreted. The measurements were carried out both on support coated and on wall-coated open tubular columns with apolar and polar stationary phases. Different relationships of t(ac) with some solute properties and the column temperature for a series of n-alkanes on an apolar stationary phase under isothermal conditions were tested. The results obtained are presented in different tables and mathematical relationships.     

基于微机电系统的微型气相色谱柱研究进展

气相色谱柱是气相色谱仪的关键部件,主要用于混合气体组分的分离。与传统气相色谱柱相比,基于微机电系统（MEMS）技术设计制作的微型气相色谱柱具有重量轻、体积小、功耗低、分离快速等优点,便于集成到便携式气相色谱仪中,适应了目前气相色谱仪微型化的发展趋势。该文综述了MEMS微型气相色谱柱的研究进展,首先阐述了MEMS微型气相色谱柱理论基础,随后对MEMS微型气相色谱柱沟道布局及柱内结构、固定相支撑层及固定相制备等方面进行了综述,最后对其发展趋势进行了展望。     

Probing the kinetic performance limits for ion chromatography. I. Isocratic conditions for small ions

The first use of the kinetic plot method to characterise the performance of ion-exchange columns for separations of small inorganic anions is reported. The influence of analyte type (mono- and divalent), particle size (5 and 9 μm), temperature (30 and 60 °C) and maximum pressure drop upon theoretical extrapolations was investigated using data collected from anion-exchange polymeric particulate columns. The quality of extrapolations was found to depend upon the choice of analyte, but could be verified by coupling a series of columns to demonstrate some practical solutions for ion chromatography separations requiring relatively high efficiency. Separations of small anions yielding 25–40,000 theoretical plates using five serially connected columns (9 μm particles) were obtained and yielded deviations of <15% from the kinetic plot predictions. While this approach for achieving high efficiencies results in a very long analysis time (t₀ = 21 min), separations yielding approximately 10,000 theoretical plates using two serially connected columns (t₀ < 5 min) were shown to be more practically useful for isocratic separations when compared to use of a single column operated at optimum linear velocity (t₀ > 10 min).     

Analysis of channel-geometry effects on separation efficiency in rectangular-capillary electrochromatography columns

A three-dimensional random walk model was developed to evaluate the impact of column geometry on separation efficiency in chromatography systems driven by electroosmotic flow. Contributions of injection plug length, cross-sectional area of channels, and aspect ratio of rectangular channels were examined in these simulation studies. Sample plug length had no impact on efficiency until it exceeded roughly 0.4% of the channel length. Plate height increased rapidly with increasing k' as expected, almost doubling in going from k'=0.25 to 0.35. Channel geometry also had a major effect on efficiency. Plate height increased sharply in rectangular channel columns until the channel aspect ratio reached 4-8. But the effect of channel depth was even more dramatic. Minimum plate height (Hmin) was roughly half that of the channel depth in ideal cases. Hmin in a 10x2 microm channel was at 1.6 mm s(-1). Rectangular channels comparable to those obtained by microfabrication are equivalent to packed column capillary electrochromatography columns in all cases.     

Use of electrokinetic measurements for characterization of columns used in capillary electrochromatography

The separation mechanism in capillary electrochromatography (CEC) is a hybrid differential migration process, which entails the features of both high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE), i.e., chromatographic retention and electrophoretic migration. The focus of this paper is on the use of electrokinetic data, such as current, electroosmotic flow (EOF) and column efficiency measurements, that are readily available, for an improved understanding of CEC separations. A framework is presented here for the use of this data for evaluation of a variety of performance parameters including, conductivity ratio, interstitial EOF mobility, porosity, and zeta potential. This framework is applied for characterization of two monolithic columns with different chemistry that were manufactured in-house. The above-mentioned performance parameters were calculated for the two columns and it is found that the poly(VBC-EGDMA-SWNT) monolithic column with the GPTMS-PEI coating offers a significantly improved flow distribution in comparison to the poly(VBC-EGDMA) monolithic column. This observation is confirmed by performing separation of peptides on the two columns and height equivalent of a theoretical plate (HETP) measurements on the resulting peaks. It is shown that following our approach leads to an improved understanding of the separations achieved with the columns and to better column design.     

A theoretical basis for parameter selection and instrument design in comprehensive size-exclusion chromatography x liquid chromatography

A novel approach for the selection of the operational parameters (linear velocity, column length) for a comprehensive 2D-LC system is discussed. Starting point for the calculations is a given second dimension ((2)D) separation and a desired peak capacity for the 2D system. Using the theory developed here the optimum settings for the first dimension ((1)D) column can be derived. Theory clearly indicates that the choice of the (1)D conditions is basically limited to just one set of column lengths and linear velocities. The new method is tested on a comprehensive two-dimensional liquid chromatography system which uses size-exclusion chromatography (SEC) followed by reversed phase liquid chromatography (RPLC). A novel LC/LC interface, using a six-port valve rather than storage loops, joins the two chromatographic dimensions. From a theoretical comparison of continuous low flow and stop-flow operation the latter method was found to be an attractive mode of interfacing. The common idea that stop-flow operation results in additional band broadening is shown to be incorrect. The new interface design operated in the stop-flow mode permits the use of conventional analytical diameter HPLC columns, 7.8mm for SEC and 4.6mm for RPLC. The reversed phase chromatography utilizes a monolithic C-18 modified silica column, which produces fast and efficient analyses. As test samples complex mixtures of peptides were analyzed.     

Probing the kinetic performance limits for ion chromatography. II. Gradient conditions for small ions

A gradient kinetic plot method is used for theoretical characterisation of the performance of polymeric particulate anion exchange columns for gradient separations of small inorganic anions. The method employed requires only information obtained from a series of isocratic column performance measurements and in silico predictions of retention time and peak width under gradient conditions. Results obtained under practically constrained conditions provide parameters for the generation of high peak capacities and rapid peak production for fast analysis to be determined. Using this prediction method, a maximum theoretical peak capacity of 84 could be used to achieve separation of 26 components using a 120 min gradient (R_s > 1). This approach provides a highly convenient tool for development of both mono- and multidimensional ion chromatography (IC) methodologies as it yields comprehensive understanding of the influence of gradient slope, analysis time, column length and temperature upon kinetically optimised gradient performance.     

色谱柱选择对六味安消胶囊中大黄素和大黄酚含量测定的影响

以中国药典收载的六味安消胶囊中大黄素与大黄酚的含量测定方法为典型案例,揭示了色谱柱是影响HPLC质量控制方法测定结果准确性的关键风险因素。参加能力验证的155家实验室均采用方法规定的C₁₈柱,但由于不同品牌C₁₈柱间的选择性差异,有20家实验室的分析结果中大黄素峰与样品水解产物的色谱峰共流出,导致测定结果明显偏离正常值。统计结果表明,A型（早期开发、硅羟基残留较多）和E型（内嵌极性基团或以极性基团封尾）C₁₈柱更适于六味安消胶囊中大黄素与大黄酚的含量测定,这可能是因为大黄素和大黄酚结构中含有多个酚羟基,能与A型柱的残留硅羟基或E型柱内嵌/封尾的极性基团相互作用,从而增强其保留行为有关。建议相关实验室在制订HPLC质量控制方法时应特别注重色谱柱的耐用性考察,并尝试采用国外色谱柱分类数据库科学地指导色谱柱的选择,尽量采用难分离物质对作为系统适用性指标,完善质量标准,减少由于色谱柱选择不适宜导致测定结果不准确的风险。     

10°锥角台锥型液相色谱柱尺寸等比例放大和柱内谱带流型的可视化研究

摘要利用改进的可视化装置, 研究了10°锥角的台锥型液相色谱柱内的谱带流型与柱参数变化的关系. 将有机玻璃柱管加工成内圆台外方型的一体结构, 选择折射率一致的色谱固定相硅胶和流动相环己烷, 使整个色谱柱成为高度清晰的透明体, 能直接观察柱中彩色样品谱带的动态三维流型. 研究结果表明, 在实验条件范围内, 流动相流速对谱带流型无影响, 填料的形状和性质对塞子状流型有一定程度的影响. 比较了柱长为5cm和等比例放大后柱长为10cm锥型柱内的流型, 发现放大后的锥型柱内仍然保持塞子状流型, 总柱效等比例增加. 表明继续按比例放大成为工业规模色谱柱后仍能保持塞子状流型.     

Optimization of binary porogen solvent composition for preparation of butyl methacrylate monoliths in capillary liquid chromatography

Butyl methacrylate monolithic columns in 320 microm i.d. fused silica capillaries for reversed-phase capillary liquid chromatography were prepared by radical polymerization initiated thermally with azobisisobutyronitrile (AIBN). Polymerization mixture contained butyl methacrylate (BMA) as the function monomer and ethylene dimethacrylate (EDMA) as the crosslinking agent with 1,4-butanediol and 1-propanol as a binary porogen solvent. Ratio of 1,4-butanediol to 1-propanol in the porogen solvent was optimized regarding the monolithic column efficiency and performance. Total porosity, column permeability, separation impedance, Walters hydrophobicity index, retention factors, peak asymmetry factors, height equivalents to a theoretical plate and peak resolutions were used for characterization of the prepared monolithic columns. The polymerization mixture consisting of 17.8% of BMA, 21.8% of EDMA, 18.0% of 1,4-butanediol, 42.0% of 1-propanol and 0.4% AIBN generated monolithic columns of the best performance having a sufficient permeability and the lowest separation impedance. It was also demonstrated that monolithic columns of this composition exhibited good preparation reproducibility and an excellent pressure resistance when applied in capillary liquid chromatography.     

Mobile phase effects on the retention on polar columns with special attention to the dual hydrophilic interaction–reversed‐phase liquid chromatography mechanism,a review

Hydrophilic interaction liquid chromatography on polar columns in aqueous–organic mobile phases has become increasingly popular for the separation of many biologically important compounds in chemical, environmental, food, toxicological, and other samples. In spite of many new applications appearing in literature, the retention mechanism is still controversial. This review addresses recent progress in understanding of the retention models in hydrophilic interaction liquid chromatography. The main attention is focused on the role of water, both adsorbed by the column and contained in the bulk mobile phase. Further, the theoretical retention models in the isocratic and gradient elution modes are discussed. The dual hydrophilic interaction liquid chromatography reversed‐phase retention mechanism on polar columns is treated in detail, especially with respect to the practical use in one‐ and two‐dimensional liquid chromatography separations.