Analysis of seven chlorobiphenyl congeners by multidimensional gas chromatography

Chlorobiphenyl congeners (CBs) are used as indicator compounds in analysis performed to determine whether or not PCB concentrations in food products, waste mineral oil, and environmental samples comply with the maximum levels permitted by legislation. Seven of these compounds have been checked for coelution with other CB congeners by means of a multidimensional gas chromatographic method utilizing a combination of two narrow bore columns, one coated with a conventional non-polar stationary phase and the other with a liquid crystalline (smectic) stationary phase. Peaks of the relevant CBs have been transferred, by heart cutting, from the non-polar column to the liquid crystal column, on which unambiguous separation from possible coeluting CB congeners was obtained. It has been shown that if the seven congeners are analyzed solely on a single, non-polar capillary column the results obtained for two of them may be affected by coelution of other compounds.     

The effect of column characteristics on the minimum analyte concentration and the minimum detectable amount in capillary gas chromatography

The need for faster and more efficient separations of complex mixtures of organic compounds by gas chromatography has led to the development of small inner diameter open tubular columns. Owing to their decreased plate height, extremely narrow peaks are obtained. When differently sized columns with equal plate numbers are compared, injection of a fixed amount of a solute will give the highest detector signals for the smallest bore columns. When P is defined as the ratio of the column inlet and outlet pressures, it can be seen from theory that under normalized chromatographic conditions the minimum detectable amount (Q_º) for a mass flow sensitive detector increases proportionally to the square of the column diameter for P = 1. In the situation of greater interest in the practice of open tubular gas chromatography where P is large, a linear relationship is derived between Q_º and the column diameter. It is a widespread misunderstanding, however, that narrow bore capillary columns should be used for this reason in trace analysis. If a fixed relative contribution of the injection band width to the overall peak variance is allowed, a decreased plate height drastically restricts the maximum sample volume to be injected. It is shown that the minimum analyte concentration in the injected sample (C_º) is inversely proportional to the column inner diameter when a mass flow sensitive detector is used. For actual concentrations less than C_º, sample preconcentration is required. The effect of peak resolution and selectivity of the stationary phase in relation to C_º and Q_º will be discussed as well. The validity of the given theory is experimentally investigated. Minimum analyte concentrations and minimum detectable amounts are compared using columns with different inner diameter.     

Improved congener-specific GC analysis of chlorobiphenyls on coupled CPSil-8 and HT-5 columns

The congener-specific analysis of polychlorinated biphenyls (PCB) by high resolution gas chromatography on a 50 m × 0.25 mm fused silica column coated with a 0.26 μm film of 5% diphenyl polydimethylsiloxane (CPSil-8) has been significantly improved by series coupling with a 25 m × 0.22 mm column coated with a 0.10 μm film of 1,2-dicarba-closo-dodecarborane polydimethylsiloxane (HT-5). Using helium as carrier gas, a total of 64 congeners in technical PCB mixtures could be analyzed as resolved peaks by ECD (86 by MS) with the CPSil-8 column, and 84 by ECD (108 by MS) with the combination. The high upper temperature limit for these stationary phases (>300°C) enabled fast temperature programming and rapid analysis (60 min).     

The optimization of capacity and efficiency when coupling fused silica open tubular columns in gas chromatography

Summary There are a number of parameters which have to be chosen depending on the analysis being done in gas chromatography. While the choice of stationary phase material is based on the solutes to be separated, the thickness is dependent on the concentration and the volatility of the components to be analyzed. This study undertakes a coupled column phase ratio optimization by connecting a short piece of a particular column prior to a normal length of an analytical column. Various columns of different dimensions (phase ratio), but of the same stationary phase material (methyl silicone), are coupled together by a deactivated glass press-fit connector, and the efficiency and capacity are measured. The coupling of fused silica open tubular columns is optimized in efficiency by matching or decreasing the phase ratio of successive columns. Capacity optimization is accomplished by increasing the phase ratio of consecutive columns. Capacity and efficiency optimization are opposing each other; therefore, if some efficiency can be sacrificed a substantial increase in capacity is possible.     

Applications of packed and capillary supercritical fluid chromatography in the separation of tocochromanols

Tocochromanols consisting of tocopherols and tocotrienols, is collectively known as vitamin E. Similarity in their structures, physical and chemical properties rendered the tocochromanols to be subject of chromatography interest. Supercritical fluid chromatography is a highly efficient tool for the separation and analysis of tocochromanols. Separation and analysis of tocochromanols using supercritical fluid chromatography had been carried out in the past using capillary or packed columns. Each of these techniques offer their own advantages and drawbacks. Besides being used for analysis, packed column supercritical fluid chromatography found applications as a purification and content enrichment tool. Emergence of new equipment and stationary phase technologies in recent years also helped in making supercritical fluid chromatography a highly efficient tool for the separation and analysis of tocochromanols. This paper gives an insight into the use of capillary and packed columns in supercritical fluid chromatography for the separation and/or analysis of tocochromanols. The types of stationary phase used, as well as chromatographic conditions are also discussed.     

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.     

Characterization of the efficiency of microbore liquid chromatography columns by van Deemter and kinetic plot analysis

The efficiency of miniaturized liquid chromatography columns with inner diameters between 200 and 300 μm has been investigated using a dedicated micro‐liquid chromatography system. Fully porous, core–shell and monolithic commercially available stationary phases were compared applying van Deemter and kinetic plot analysis. The sub‐2 μm fully porous as well as the 2.7 μm core–shell particle packed columns showed superior efficiency and similar values for the minimum reduced plate heights (2.56–2.69) before correction for extra‐column contribution compared to normal‐bore columns. Moreover, the influence of extra‐column contribution was investigated to demonstrate the difference between apparent and intrinsic efficiency by replacing the column by a zero dead volume union to determine the band spreading caused by the system. It was demonstrated that 72% of the intrinsic efficiency could be reached. The results of the kinetic plot analysis indicate the superior performance of the sub‐2 μm fully porous particle packed column for ultra‐fast liquid chromatography.     

Synthetic ceramic clay material as stationary phase in packed column supercritical fluid chromatography

Summary A novel inorganic synthetic clay material (SC) has been evaluated as the stationary phase in packed-column, supercritical fluid chromatography (SFC). The molecular recognition capability of the SC stationary phase in SFC for polycyclic aromatic hydrocarbons has been evaluated using carbon dioxide and carbon dioxide modified with methanol as the mobile phase. This recognition derives from the layer structure of the SC material which acts as a slit to distinguish non-planar solutes from the molecular-molecular interaction between solute and stationary phase and leads to smaller retention for non-planar solutes. The recognition capability is also dependent on the SFC conditions such as column pressure and column temperature.     

Preparation of a monolithic column with a mixed‐mode stationary phase of reversed‐phase/hydrophilic interaction for capillary liquid chromatography

A monolithic capillary column with a mixed‐mode stationary phase of reversed‐phase/hydrophilic interaction chromatography was prepared for capillary liquid chromatography. The monolith was created by an in‐situ copolymerization of a homemade monomer N,N‐dimethyl‐N‐acryloxyundecyl‐N‐(3‐sulfopropyl) ammonium betaine and a crosslinker pentaerythritol triacrylate in a binary porogen agent consisting of methanol and isopropanol. The functional monomer was designed to have a highly polar zwitterionic sulfobetaine terminal group and a hydrophobic long alkyl chain moiety. The composition of the polymerization solution was systematically optimized to permit the best column performance. The columns were evaluated by using acidic, basic, polar neutral analytes, as well as a set of alkylbenzenes and Triton X100. Very good separations were obtained on the column with the mixed‐mode stationary phase. It was demonstrated that the mixed‐mode stationary phase displayed typic dual retention mechanisms of reversed‐phase/hydrophilic interaction liquid chromatography depending on the content of acetonitrile in the mobile phase. The method for column preparation is reproducible.     

Temperature dependence of the resolution of diastereomeric isoprenoids in capillary GC. Experiments with 50 μm, 100 μm,and 250 μm i.d. Columns

The resolution of the diastereoisomers of norpristane, pristane, and phytane was studied as a function of the column internal diameter and/or the residence time of the compounds in the column. Increasing the residence time in the column by operating the column at a lower temperature program rate enhances the resolution more than reducing the internal diameter of the column. Practical experience with ultra narrow bore columns is also presented.     

Theory of Fast Capillary Gas Chromatography Part 4: Column Performance vs.Liquid Film Thickness

The negative effect of the liquid stationary phase film thickness on the column efficiency is strongest for peaks with retention factors, k, in the vicinity of k = 0.3–0.4 and rapidly diminishes with the departure of k from that region. Additionally, at the high pressure drop required for fast analysis of complex mixtures, the negative effect of the same film thickness diminishes with the increase in column length, regardless of the k values. In practice, it is recommended to ignore the film thickness and optimize the columns as thin film ones regardless of their actual film thickness. Accounting for the film thickness results only in a modest improvement in the resolution for a few affected peaks – those with k = 0.3–0.4. However, this improvement comes at the cost of a substantial increase in analysis time, and should be used only as the action of the last resort.     

Separation of mixtures of solvents with wide bore capillary columns connected in series

The gas chromatographic separation of the components of a complex mixture of industrial solvents, not possible on a single stationary phase owing to coelution of some of the compounds, has been achieved on two serially connected wide bore capillary columns of different polarity. The analysis of a mixture of twenty five compounds was optimized using the serial gas chromatography theory of Purnell and Williams. The capacity factors of sample components measured with the two columns coupled in series were found to agree with those predicted by theory, regardless of the order in which they were connected.     

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.     

Siloxane/silarylene copolymers as stationary phases for capillary gas chromatography part I: Evaluation of silanol terminated dimethyl substituted polymers

The thermal stability of silicones can be improved on replacement of certain of the oxygen atoms in the polymer backbone by phenyl groups. Such a polymer has been synthesized and evaluated for use as stationary phase in fused silica capillary gas chromatography; the polymer was dimethyl substituted and silanol terminated. A selectivity was provided by the phenyl groups in the backbone. For comparative purposes, a silanol-terminated dimethylpolysiloxane has also been evaluated. Both stationary phases gave columns of highest separation efficiency and the supporting fused silica surface was deactivated by the stationary phases on thermal treatment. Further, low column bleeding was observed at the maximum temperature tested, 370°C. The phenyl-containing phase could be immobilized to 60% by heat treatment, but the pure dimethylpolysiloxane was 10% immobilized. The influence on immobilization of factors such as nature of the supporting surface, stationary phase silanol content, reaction temperature and atmosphere in the column during reaction has been studied.     

Retention times of fifty one chlorobiphenyl congeners on seven narrow bore capillary columns coated with different stationary phases

Retention times of fifty one important chlorobiphenyl congeners have been determined on seven narrow bore GC columns coated with different stationary phases. The retention times of the chlorobiphenyls relative to tetrachloronaphthalene were plotted for different column combinations; the plots generated can be used to choose an optimum column combination for the multidimensional gas chromatographic separation of the chlorobiphenyls.     

Chiral separation on achiral stationary phases with different functionalities using β-cyclodextrin in the mobile phase and application to bioanalysis and coupled columns

Summary -cyclodextrin was used in the mobile phase as chiral selector for separating the enantiomers of terbutaline, chlorthalidone and oxazepam. The effect on chiral resolution using e.g. hydrophobic, polar or cation exchanging stationary phases was investigated. Both the chiral separation factor and retention level were affected by the concentration of methanol and -cyclodextrin. The stationary phase had no effect on the chiral separation only on the level of retention. By tuning the concentration of -cyclodextrin and methanol in the mobile phase chiral separation could be obtained on most stationary phases. By changing the stationary phase while adjusting the mobile phase composition to maintain the chiral selectivity, improvements of the selectivity towards e.g. endogenous compounds can be obtained when separating enantiomers in complex matrixes as biological fluids. Further improvement on selectivity can be obtained if coupled columns are used. This is examplified for separation of chlorthalidone and terbutaline enantiomers in biological fluids by coupling an achiral column to another achiral column and using a mobile phase containing -cyclodextrin on the last column.     

Polarizable biphenyl polysiloxane stationary phase for capillary column gas chromatography

A crosslinkable biphenylmethylpolysiloxane stationary phase was synthesized for capillary column gas chromatography and compared with methyl, phenyl, and cyanopropyl polysiloxane stationary phases for the separation of isomeric polycyclic aromatic compounds. While the new phase gave similar separations of nonpolar isomers when compared to the nonpolar phases, separations of polar isomers were greatly improved because of the induced polarity of the biphenyl group of the stationary phase by the solute molecules. This polarizable stationary phase offers a unique selectivity which is not available in other stationary phases.     

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.     

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.     

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.