Optimization via liquid phase mixtures in capillary gas chromatography

The use of sequentially coupled columns to achieve a binary liquid phase mixture has been simplified by the availability of zero dead volume fittings compatible with fused silica columns. The extent to which the velocity gradient through the coupled column affects the “apparent” liquid phase ratio can be determined by graphic interpolation; the length ratio of the coupled column segments can then be adjusted so that the “apparent” liquid phase ratio actually experienced by the solutes agrees with the targeted value. The fact that the direction of flow through the coupled column affects the chromatographic dispersion suggests that accepted generalizations on flow optimization throughout the column may not be precisely correct.     

Optimization of capillary parameters for gas chromatography

An HETP equation for the capillary column is developed that takes into account the dependence of gaseous diffusion on pressure, the compressibility of the mobile phase, together with the unique relationship between mobile phase velocity, and the resistance to mass transfer in the stationary phase. The equation is used to develop a procedure for column optimization and expressions are derived that allow the optimum column radius and optimum column length to be calculated for a given fixed inlet pressure. It is shown that fast, simple separations are optimally achieved using relatively short small diameter columns. Conversely, optimum performance for the separation of complex mixtures requiring higher efficiencies requires the use of long columns with relatively large diameters.     

Capillary high-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic columns and carbon fiber electrospray ionization emitters

Monolithic columns having long hydrocarbon chains were prepared by in-situ polymerization in capillary fused silica tubing. The capillary columns were coupled with a newly developed carbon fiber electrospray ionization (ESI) emitter for proteomic analysis using sheathless capillary HPLC-ESI mass spectrometry (MS). The sample loading capacity and chromatographic performance of the styrene-based monolithic column, which was prepared by photo-polymerization of octylstyrene (OS) and divinylbenzene (DVB) were compared with that of the methacrylate-based monolithic column composed of lauryl methacrylate (LMA) and ethylene dimethacrylate (EDMA). The sample loading ability of tryptic digested protein in poly-OS (POS)-DVB column was higher than that of poly-LMA (PLMA)-EDMA column, possibly due to the irregular and rugluous surface offering a greater surface area of POS-DVB stationary phase. The POS-DVB column also provided better separation efficiency in the separation of high concentration (10 microg) of tryptic digested albumin bovine serum (BSA). Due to the successful interface of a highly efficient monolithic column and a stable, durable carbon fiber emitter, low femtomole levels of peptides were successfully separated and identified in the presence of large amounts of tryptic digested protein.     

Computer-aided selection and optimization of chromatographic columns and conditions for multicolumn analysis procedures

Clearly, the usefulness of a computer-aided column design program will depend on its ability to predict quickly and accurately, a design which will yield chromatograms closely approximating those obtained experimentally. Such a computer model for designing and specifying operating conditions for optimum performance of either single or serially coupled columns with different stationary phases is described herein. Tests have been performed in order to verify the accuracy of the model. In addition to single column optimization and the design of column combinations which can be used to achieve separations difficult or impossible on a single phase alone, the model has proven quite useful as an aid to the design and development of multicolumn analysis procedures that involve critical timing of valve-switching sequences.     

Characterization of High‐Pressure Liquid Chromatography Columns using Chromatographic Methods

Abstract

A great variety of columns for liquid chromatography (LC) are available in dimensions ranging from industrial scale to micro‐bore, nano‐bore, and capillary size, and on‐chip columns. The columns may be used in various liquid chromatography modes or in capillary electrochromatography, depending on the support materials and stationary phase chemistry. Every year many new column types are introduced on the market, with improved selectivity and efficiency, long lifetime, and mobile phase compatibility, intended for general use, for liquid chromatography/mass spectrometry (LC/MS) applications, proteomic research, or for the analysis of other specific sample types. Considerable improvement in pH, high‐temperature, and high‐pressure stability of new column types, together with advances in the instrumentation, enabled introduction of capillary, high‐temperature, and ultra‐high‐pressure HPLC into routine practice. Even though reversed‐phase mode is still by the most widely used in contemporary LC, applications of other separation modes (such as ion, normal‐phase, or high‐interaction liquid chromatography (HILC)) have become more frequent recently, because of unique separation selectivity for certain sample types.

Characterization of column quality is not a simple task, because a number of factors should be taken into account, that affect the selectivity, efficiency and resolution of sample separation and the reproducibility of chromatographic data. These include the type of the support, the arrangement and density of the stationary phase on the adsorbent surface, the homogeneity of the chromatographic bed, etc. Various physicochemical techniques are used for characterization of the properties of column packings however, most of them are suitable for bulk materials only and cannot be directly applied for commercial columns without damaging them. Not to destroy the columns, often precious and expensive, practicing chromatographers can apply chromatographic methods to characterize columns and evaluate their analytical suitability under real‐life conditions, where the intermolecular interactions between the analytes, the stationary phase, and the mobile phases affect the retention. The present review reports various chromatographic tests and strategies available for column evaluation.     

Chromatographic separation of simulants of nerve and blister agents by combining one‐ and two‐channel columns with different stationary phases

A two‐channel gas chromatography column and a single‐channel column were made by deep reactive‐ion etching technology. The two short columns were coated with different stationary phases, and then linked without a modulator. This is to aim at increasing the sample capacity and achieving a higher separation efficiency in complex environments. The results show that the capacity of the connected column is approximately 4 and 1.5 times larger than that of the single‐ and two‐channel columns, respectively. The linked column was utilized to separate a six‐component mixture, composed of three simulants of nerve and blister agents and three interfering vapors. The results demonstrate that the combined column has a remarkably higher separation efficiency than the individual columns, and an acceptable resolution is achieved although the total length of the linked column is only 1.5 m.     

Performance of open tubular columns as a function of tube diameter and liquid phase film thickness

Summary Wall-coated, open-tubular (capillary) columns prepared from different diameter tubing, with different liquid phase film thickness, are compared with each other and with packed and support-coated open-tubular columns. The comparison is based on the variation of the phase ratio and the capacity factor, and includes column efficiency (HETP, theoretical plate number), resolution, retention time, and sample capacity. Problems associated with the evaluation of the sample capacity are outlined. The influence of temperature on column performance is discussed in detail. Finally, the possibilities of short, wide-bore open-tubular columns prepared with a thick liquid-phase film are demonstrated.Parts of this paper were presented at the 35th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlantic City NJ, March 5–9, 1984, and at the 20th International Symposium on Advances in Chromatography, New York NY, April 16–18, 1984.     

A New Definition of the Stationary Phase Volume in Mixed-Mode Chromatographic Columns in Hydrophilic Liquid Chromatography

Polar columns used in the HILIC (Hydrophilic Interaction Liquid Chromatography) systems take up water from the mixed aqueous–organic mobile phases in excess of the water concentration in the bulk mobile phase. The adsorbed water forms a diffuse layer, which becomes a part of the HILIC stationary phase and plays dominant role in the retention of polar compounds. It is difficult to fix the exact boundary between the diffuse stationary and the bulk mobile phase, hence determining the column hold-up volume is subject to errors. Adopting a convention that presumes that the volume of the adsorbed water can be understood as the column stationary phase volume enables unambiguous determination of the volumes of the stationary and of the mobile phases in the column, which is necessary for obtaining thermodynamically correct chromatographic data in HILIC systems. The volume of the aqueous stationary phase, Vex, can be determined experimentally by frontal analysis combined with Karl Fischer titration method, yielding isotherms of water adsorbed on polar columns, which allow direct prediction of the effects of the composition of aqueous–organic mobile phase on the retention in HILIC systems, and more accurate determination of phase volumes in columns and consistent retention data for any mobile phase composition. The n phase volume ratios of 18 columns calculated according to the new phase convention strongly depend on the type of the polar column. Zwitterionic and TSK gel amide and amine columns show especially strong water adsorption.     

有机-无机杂化硅胶基质整体柱的制备及其电色谱性能

采用溶胶-凝胶法,以四乙氧基硅烷和苯基三乙氧基硅烷作为反应单体,通过酸碱两步催化在毛细管中进行原位缩聚反应,制备了新型有机-无机杂化硅胶基质毛细管整体柱,制备过程简单。整体柱基质中均匀分布的苯基基团可直接用于反相毛细管电色谱的分离,因而不需要对基质再进行衍生化。优化了整体柱的制备条件,采用扫描电镜和压汞法对整体柱的微观结构和孔径分布进行了表征。分别考察了溶胶-凝胶初始反应液中水的用量对柱床结构的影响和两种单体的配比对材料孔径分布的影响。研究了稠环芳烃类化合物在整体柱上的保留行为,用所制备的整体柱分离了7种苯酚类化合物,平均柱效达100000塔板/m。     

Optimization of selectivity using sequentially coupled capillary columns

This investigation lays groundwork for the development of an optimization system for sequentially coupled capillary column systems. Three methods for calculating effective capacity factors were obtained from the literature and were tested to determine suitability for optimization use, using four test analytes and two dissimilar columns. This work concentrates on the development of a method for the estimation of optimum column temperatures in sequentially coupled systems.     

Gas chromatographic analysis of high-molecular-mass polycyclic aromatic hydrocarbons II. Polycyclic aromatic hydrocarbons with relative molecular masses exceeding 328

Three columns were used for the gas chromatographic analysis of polycyclic aromatic hydrocarbons (PAHs) with relative molecular masses (M_r) up to 450. Two of the columns were commercially available, coated with a 50% methyltrifluoropropyl-substituted polysiloxane a 5% diphenyl-substituted methylpolysiloxane. The third column was laboratory made, coated with a biphenyl-substituted silarylene-siloxane copolymer. All three columns were utilized for the analysis of high-M_r PAHs as regards both thermal stability of the stationary phases, i.e., low bleeding rate, and chromatographic efficiency. The column coated with a trifluoropropyl-substituted stationary phase showed, however, a low separation efficiency, possibly owing to low solute stationary phase compatibility. The biphenyl-substituted stationary phase, on the other hand, showed a very high separation efficiency, but the retention of the PAHs was significantly higher on this column compared with the other two, leading to the demand for higher oven temperatures. Different retention mechanisms were observed on these columns, as shown by differences in the retention indices of the PAHs measured in a system using PAHs as retention index markers. A comparatively faster elution of non-planar PAHs was observed on the columns coated with the trifluoropropyl-substituted stationary phase and the biphenyl-substituted stationary phase compared with the column coated with the 5% diphenyl-substituted polymer. The usefulness of the columns for separations of high-M_r PAHs is demonstrated by gas chromatograms of carbon black extracts and a coal tar extract standard reference material.     

Advances in sol-gel based columns for capillary electrochromatography: sol-gel open-tubular columns

The development of sol-gel open-tubular column technology in capillary electrochromatography (CEC) is reviewed. Sol-gel column technology offers a versatile means of creating organic-inorganic hybrid stationary phases. Sol-gel column technology provides a general approach to column fabrication for microseparation techniques including CEC, and is amenable to both open-tubular and monolithic columns. Direct chemical bonding of the stationary phase to the capillary inner walls provides enhanced thermal and solvent stability to sol-gel columns. Sol-gel stationary phases inherently possess higher surface area, and thus provide an effective one-step alternative to conventional open-tubular column technology. Sol-gel column technology is applicable to both silica-based and transition metal oxide-based hybrid stationary phases, and thus, provides a great opportunity to utilize advanced material properties of a wide range of nontraditional stationary phases to achieve enhanced selectivity in analytical microseparations. A wide variety of stationary phase ligands can be chemically immobilized on the capillary inner surface using a single-step sol-gel procedure. Sol-gel chemistry can be applied to design stationary phases with desired chromatographic characteristics, including the possibility of creating columns with either a positive or a negative charge on the stationary phase surface. This provides a new tool to control electroosmotic flow (EOF) in the column. Column efficiencies on the order of half a million theoretical plates per meter have been reported for sol-gel open-tubular CEC columns. The selectivity of sol-gel stationary phases can be easily fine-tuned by adjusting the composition of the coating sol solution. Open-tubular columns have significant advantages over their packed counterparts because of the simplicity in column making and hassle-free fritless operation. Open-tubular CEC columns possess low sample capacity and low detection sensitivity. Full utilization of the analytical potential of sol-gel open-tubular columns will require a concomitant development in the area of high-sensitivity detection technology.     

Considerations of speed and efficiency in capillary gas chromatography

The analysis time for a given resolution is a complex function of stationary phase selectivity, column radius, and thickness of the stationary phase film. Variation of these parameters has a large effect not only on analysis time, but also on the column inlet pressure and other instrumental requirements. The minimum amount that can be reliably detected as well as the maximum sample capacity of a column are strongly related to the selected column dimensions.     

Effects of Blending Bare-Silica and Reversed-Phase on Retention of Neutral Compounds in Capillary Electrochromatography

Most commercially available instruments for capillary electrochromatography (CEC) have a fixed configuration and lack the flexibility to use shorter columns. Applying a blended stationary phase (a phase consisting of a given ratio of bare silica and reversed phase material) can simulate columns of different length in CEC. The goal of this work was to examine the effect of the degree of blending of reversed-phase columns (with bare silica) on the speed of the separation of neutral compounds in CEC. Optimum column packing mixture was determined from the variation of the solute retention factors as a function of the ratios of blending of reversed-phase and bare silica. By adjusting the column composition, solute retention factors and the analysis run time were halved when compared to a pure reversed-phase column of the same length. Stationary phase blending can be considered as an additional parameter to mobile phase variation, column temperature and applied electric field for the optimization of selectivity and analysis time. By adjusting the stationary phase composition, mobile phase composition, column temperature and applied electric field, the analysis run time of neutral components was decreased more than 75% when compared to a separation obtained on neat reversed-phase column of the same dimensions. The linear dependence of the retention factors as a function of the blend ratio (reversed phase/bare silica) offers a framework for designing a “blended” packed capillary column for CEC separations.     

Analysis of amino acids using serially coupled columns

Single conventional columns in reversed‐phase liquid chromatography are insufficient for analysing the isoindoles of primary amino acids due to their limited functionality. An interesting possibility for increasing the separation power is the combination of several columns of different nature, where the length is modified by coupling small segments. This approach may require a considerable investment to have multiple lengths for each stationary phase. However, the combination of only two columns of fixed length can be enough to resolve satisfactorily relatively complex mixtures, provided that an optimised gradient program is applied. In this work, a mixture of 19 primary amino acid isoindoles found in proteins was analysed. Four stationary phases were assayed: C18, pentafluorophenyl‐C18, C4 and cyano. The mixture of isoindoles was successfully resolved in practical times using a pentafluorophenyl‐C18 column coupled to a C4 column, in spite of the extremely poor performance obtained when each column is used isolatedly, independently of the length. The extreme diversity in the polarities of the isoindoles and the need of extrapolating the retention behaviour in certain regions of the solvent content domain makes the modelling of the retention behaviour of the isoindoles particularly difficult. Nevertheless, the predicted optimal separations were very satisfactory.     

超动态法快速制备毛细管色谱柱的研究

用超动态法快速制出了几种非极性毛细管柱,柱效、涂渍效率分别为3000～4500理论塔板/m和65～85%。研究了超动态法中涂渍压力、涂渍液浓度与容量因子的关系;考察了制柱重复性和液膜厚度均匀性;给出了涂渍一般口径和试涂大口径厚液膜毛细管柱的部分结果。     

Enantiomeric separation of α-phenylethylamine and its derivatives by series coupled open tubular columns

A series coupled fused-silica open tubular columns of a chiral and an achiral stationary phase were used to achieve the enantiomeric separation of a-phenylethylamine, o,m,p-methyl-and o,m,p-methoxy-a-phenylethylamine. A simplified equation describing the retention values of series coupled columns was derived, which relates the capacity factors of series coupled columns to the capacity factor of individual columns, their lengths, inlet and outlet pressure. Very good agreement was obtained both between calculated values by this equation and those by Krupcik's equation and between values measured experimentally and calculated theoretically. By means of this simplified equation a computer program was developed in locating the operating range of optimum amin, enantiomeric separation of all a-phenylethylamine and its derivatives were achieved in a single run.     

High speed capillary GC on 10 m × 100 μm i.d. FSOT Columns

The chromatographic performance of 10 m × 100 μm i.d. capillary columns, coated with different stationary phases, was studied. TZ-ū plots were measured at different temperature program rates. The polarity and selectivity are strongly influenced by the temperature program rate. The analysis time depends on the capacity ratio and the selectivity factor; stationary phase selection thus is an important aspect in high speed capillary GC. A number of applications is presented and the applicability of split injection is discussed.     

Optimization of peptide separations in reversed-phase HPLC: Isocratic versus gradient elution

Summary We have determined the interaction behaviour of peptides with hydrophobic stationary phases on analytical columns using isnocratic or shallow gradient elution for the purpose of developing procedures for rapid optimization of conditions for preparative reversed-phase chromatography of peptides. From our investigation of the separation of two closely related decapeptides (differing by one methyl group), in a 1:1 molar ratio on an analytical C8 column, we have found that shallow gradients of 0.1% acetonitrile/min appeared to be the best compromise between resolution and a practical run time for preparative peptide separations. Up to 20mg of the two-peptide mixture was efficiently resolved on the analytical column, with >97% recovery of homogeneous peptides.