Ein-einfaches Vollglas-Doppelsäulensystem als Identifizierungshilfe für gas-chromatographische Analysen

Zusammenfassung Im vorliegenden Bericht wird ein einfaches Vollglas-Doppelsäulensystem zur zweikanaligen gas-chromatographischen Analyse mit zwei unterschiedlichen Probenaufgabemöglichkeiten beschrieben. Am Beispiel einiger Analysen wird die Anwendbarkeit des Systems demonstriert. Hierbei werden Gemische, u.a. Kraftstoff und angereicherte Luftschadstoffe, auf zwei Trennsäulen mit unterschiedlich polaren Phasen aufgegeben. Die Detektion erfolgt simultan mit zwei FIDs. Zusätzlich zu den Retentionswerten erhält man aus den Peakverschiebungen, die mit substanzklassenspezifischen Retentionsindexinkrementen korrelieren, wichtige Hinweise zur Identifizierung.

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A simple all glass, double column system as an aid to identification in gas chromatographic analysis

Summary This paper describes a simple all glass, double column system with two different injection ports for two-channel gas chromatographic analysis. The versatility of the system is demonstrated by several examples. The analysis mixtures, including gasoline and air samples, are transferred onto two columns with phases of different polarity. Detection is achieved simultaneously by two FIDs. In addition to the retention values, the peak shifts corresponding to retention index increments, which are specific for substance classes, offer an important aid to identification.

     

Two-dimensional high performance liquid chromatography for the separation of complex mixtures of explosives and their by-products

Summary The insufficient ability of one dimensional HPLC to separate complex mixtures such as environmental samples can be overcome by using two dimensional systems combining columns with alternative/orthogonal selectivities. Such a system for the separation of complex mixtures of explosives, their by-products and degradation products from environmental samples was developed and evaluated. It makes use of the different retention characteristics of an alkyl modified silica and a safrol modified silica in the reversed phase mode. The high peak capacity of two dimensional systems predicted by theory was realized employing a flexible switching technique and utilizing differences in the elution strength of the mobile phases. Thus, peak compression on the head of the second column was exploited. The efficiency of the two dimensional system was demonstrated for the separation of a complex mixture of nitroaromatic reference compounds. Furthermore, the system was applied to separate and identify nitro and nitroamino organic compounds in a groundwater sample from a former ammunition plant. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok Hungary, September 3–5, 1997     

Estimation of chromatographic parameters on two silica-based columns connected in series under nonaqueous reversed phase liquid chromatographic conditions

Empirical equations were produced to relate important chromatographic parameters on two silica-based columns serially linked, in isocratic nonaqueous RP HPLC, to retention times and peak widths of the separated compounds on the individual columns. These equations were derived because the experimental data seemed to deviate from the values expected, applying basic chromatographic theoretical equations. The chromatographic parameters studied were retention time, peak width, resolution, number of theoretical plates, capacity factor, and separation factor. In addition, empirical linear relationships were produced for the estimation of the above mentioned parameters of the serial systems, in direct and reverse order, relating them to those obtained on each column, separately. The experimentally obtained values were in good agreement with those estimated by the derived equations.     

A simple,fast, and accurate thermodynamic‐based approach for transfer and prediction of gas chromatography retention times between columns and instruments Part I: Estimation of reference column geometry and thermodynamic parameters

The transfer of retention times based on thermodynamic models between columns can aid in separation optimization and compound identification in gas chromatography. Although earlier investigations have been reported, this problem remains unsuccessfully addressed. One barrier is poor predictive accuracy when moving from a reference column or system to a new target column or system. This is attributed to challenges associated with the accurate determination of the effective geometric parameters of the columns. To overcome this, we designed least squares‐based models that account for geometric parameters of the columns and thermodynamic parameters of compounds as they partition between mobile and stationary phases. Quasi‐Newton‐based algorithms were then used to perform the numerical optimization. In this first of three parts, the model used to determine the geometric parameters of the reference column and the thermodynamic parameters of compounds subjected to separation is introduced. As will be shown, the overall approach significantly improves the predictive accuracy and transferability of thermodynamic data (and retention times) between columns of the same stationary phase chemistry. The data required for the determination of the thermodynamic parameters and retention time prediction are obtained from fast and simple experiments. The proposed model and optimization algorithms were tested and validated using simulated and experimental data.     

Tandem LC columns for the simultaneous retention of polar and nonpolar molecules in comprehensive metabolomics analysis

The tandem use of hydrophilic interaction LC columns with RP columns in series configuration has resulted in the retention of both polar and nonpolar components in complex biological samples (mouse serum) in a single analysis. This approach successfully coupled various columns with orthogonal separation characteristics, employed a single solvent gradient program compatible with the two columns and used ESI coupled to a TOF mass spectrometer for detection. Ion suppression, a common problem in ESI, was virtually eliminated for components eluting with apparent capacity factors >0.7. Retention time reproducibility with the tandem columns performed over three days with over 100 injections was comparable to that observed for single columns alone. This method was applied to the analysis of a pooled mouse serum sample and afforded highly reproducible data for up to 3000 mass spectral features. This approach was implemented with a conventional LC–MS system and should find broad applicability in the comprehensive analysis of complex mixtures containing a wide range of compound polarities.     

Effects of pH in reversed-phase liquid chromatography

The effects of concomitant variations in pH and organic modifier concentration on retention, efficiency and peak symmetry are considered for reversed-phase liquid chromatography (RPLC) on octadecyl-modified silica (ODS) columns. A number of factors are discussed, which make the systematic exploitation of pH effects in RPLC more complicated than the optimization of solvent composition. If the pH is varied, a second factor (usually the concentration of organic modifier) will need to be varied simultaneously to maintain retention (capacity factors) in the optimum range. When pH is considered as a parameter in RPLC, not only its effects on retention, but also the variations in efficiency (plate count) and peak shape (asymmetry) need to be considered. These parameters turn out to vary drastically between individual solutes and between different experimental conditions. The results of a study involving a number of acidic, basic and neutral solutes, two different ODS columns and mixtures of either methanol or acetonitrile with aqueous buffers are reported. In the earlier part of the study, using methanol as the organic modifier, reproducible data for retention, peak width and peak symmetry were obtained and these data are reported. In the later part of the study, using acetonitrile, a gradual change in retention as a function of time was observed, this effect coinciding with a decrease in column efficiency. It is concluded that ODS columns are subject to considerable degradation during studies in which the pH is varied. Although this effect can be described mathematically, the preferred solution is thought to be the use of pH-stable columns.     

Improving peptide identification using an empirical peptide retention time database

Peptide retention time (RT) is independent of tandem mass spectrometry (MS/MS) parameters and can be combined with MS/MS information to enhance peptide identification. In this paper, we utilized peptide empirical RT and MS/MS for peptide identification. This new approach resulted in the construction of an Empirical Peptide Retention Time Database (EPRTD) based on peptides showing a false‐positive rate (FPR) ≤1%, detected in several liquid chromatography (LC)/MS/MS analyses. In subsequent experiments, the RT of peptides with FPR >1% was compared with empirical data derived from the EPRTD. If the experimental RT was within a specified time range of the empirical value, the corresponding MS/MS spectra were accepted as positive. Application of the EPRTD approach to simple samples (known protein mixtures) and complex samples (human urinary proteome) revealed that this method could significantly enhance peptide identification without compromising the associated confidence levels. Further analysis indicated that the EPRTD approach could improve low‐abundance peptides and with the expansion of the EPRTD the number of peptide identifications will be increased. This approach is suitable for large‐scale clinical proteomics research, in which tens of LC/MS/MS analyses are run for different samples with similar components. Copyright © 2008 John Wiley & Sons, Ltd.     

Retention and column efficiency in reversed phase liquid chromatography as a function of pH for optimization purposes

Summary The effects of pH on both the solute retention and the peak shape of ionogenic compounds are studied in order to propose accurate models for pH optimization purposes. Several mathematical models (theoretical and empirical) for describing the variation of the retention factor versus pH are compared within different pH ranges. Limits of such models used for optimizing the pH by requiring only 3 preliminary experimental runs, are discussed in terms of deviations (≤±5%) of predicted retention times from experimental retention times. An original procedure is developed for selecting the most convenient retention model, from a given set of three retention data. This set is also applied to modeling the variation of both peak width and peak asymmetry with mobile phase pH conditions. Such a procedure is demonstrated as helpful for the separation of ionogenic solutes by considering mobile phase pH as an additional variable that can be useful during optimization procedures.     

Alternative methods for verifying the results of the mass spectrometric identification of peptides in shotgun proteomics

Database search is the most popular approach used for the identification of peptides in contemporary shotgun proteomics; it utilizes only mass spectrometric data. In this work, we introduce three criteria for the verification of peptide identification; these are based on the analysis of data orthogonal to tandem mass spectra. The first one utilizes chromatographic retention times of peptides. The development of such approaches has been hindered by the relatively low accuracy of retention time prediction algorithms. In this work, we suggest the use of two independent models of the liquid chromatography of peptides, which increase the reliability of the results. The second criterion utilizes the mean number of missed tryptic cleavages per peptide. The third one results from the analysis of the difference between theoretical and experimentally measured peptide masses. The proposed criteria were applied to the tandem mass spectra of tryptic peptides from rat kidney tissue, which were processed by the Mascot search engine. All the criteria showed that Mascot significantly overestimated the reliability of an identification. This conclusion was supported by the PeptideProphet algorithm.     

Comments on the Implementation of a Simple Peak Capacity Optimization Procedure and Comparison of Poly(styrene–divinylbenzene) and RP-18 Silica Monolithic Capillary Columns of 5-cm for the Analysis of Protein Digests

For the analysis of protein digests, the peak capacity in reversed-phase liquid chromatography is of paramount importance. A univariate method to maximize the peak capacity as developed by Wang et al. (Anal Chem 78:3406–3416, 20) has been applied and tested for a monolithic RP-18 silica capillary column. In their method, using model peptides representing a bovine serum albumin digest, the gradient time and temperature are kept constant while the flow rate and eluent strength are varied. Despite our criticism on the fixed starting conditions, a long gradient time leading to an unnecessary long analysis time and a high temperature leading to possible degradation products in the chromatogram, and the peak capacity as the only optimization parameter this fast and simple optimization strategy turns out to be applicable to capillary monolithic columns. Furthermore, the influence of the peak capacity on a second optimization parameter, the MS protein identification score, is examined. The procedure is also used to enhance the performance of two popular types of monolithic capillary LC columns (silica-C18 and poly(styrene–divinylbenzene)) of the same length for the analysis of protein digests. Comparison of both columns show that the calculated chromatographic parameters, like productivity and peak capacity, and identification score for both columns are about the same. For a more complicated nine-protein digest the performance of the silica monolith is slightly better.     

Dual column capillary gas chromatographic system for the in situ analysis of volatile organic compounds on a cometary nucleus

Two Wall Coated Open Tubular capillary columns, coated with poly(cyanopropylphenyl-dimethyl)siloxane and poly(diphenyl-dimethyl)siloxane stationary phases, have been selected for use in the COmetary SAmpling and Composition space experiment for the separation and identification of the wide range of volatile organic compounds which could be present in cometary nuclei. This article presents the main characteristics of the tandem column system for the analysis of solutes of cometary interest within the constraints of space instrumental operating conditions. The high efficiency of the columns is demonstrated and the influence of the operating conditions on their separation properties are investigated. The studied columns exhibit complementary retention pattern: their use in a dual column system makes it possible to achieve the separation and the identification of the compounds of interest. Finally, the good analytical behavior of the columns when analyzing samples which include large amounts of water, the main presumed volatile in comets, is demonstrated. The presented results thus show the suitability of the selected tandem columns system for the desired analyses, and their performance on adaptation to in-situ cometary chemical investigation.     

Retention mechanism assessment and method development for the analysis of iohexol and its related compounds in hydrophilic interaction liquid chromatography

Hydrophilic interaction liquid chromatography (HILIC) has emerged in recent years as a valuable alternative to reversed-phase liquid chromatography in the analysis of polar compounds. Research in HILIC is divided into two directions: the assessment of the retention mechanism and retention behavior, and the development of HILIC methods. In this work, four polar neutral analytes (iohexol and its related compounds A, B, and C) were analyzed on two silica and two diol columns in HILIC mode with the aim to investigate thoroughly the retention mechanisms and retention behavior of polar neutral compounds on these four columns. The adsorption and partition contribution to the overall HILIC retention mechanism was investigated by fitting the retention data to linear (adsorption and partition) and nonlinear (mixed-retention and quadratic) theoretical models. On the other hand, the establishment of empirical second-order polynomial retention models on the basis of D-optimal design made possible the estimation of the simultaneous influence of several mobile-phase-related factors. Furthermore, these models were also used as the basis for the application of indirect modeling of the selectivity factor and a grid point search approach in order to achieve the optimal separation of analytes. After the optimization goals had been set, the grids were searched and the optimal conditions were identified. Finally, the optimized method was subjected to validation.

Synthesis and characterization of novel polar-embedded silica stationary phases for use in reversed-phase high-performance liquid chromatography

We have developed a series of new C₁₀ dipeptide stationary phases via a simple and effective synthetic method. The preparation of the new phases involves the synthesis of silanes and the surface modification of silica. Chromatographic evaluations of these columns were performed using the Engelhardt, Tanaka, and Neue test mixtures. The applicability of these new stationary phases was also evaluated using a series of diagnostic probes including acids, bases or neutral compounds and several generic applications. These new C₁₀ dipeptide stationary phases showed excellent hydrolytic stability over a wide pH range. Like other existing amide-embedded columns, these new stationary phases exhibit higher retention for polar and hydrophilic compounds and different selectivity as compared to conventional C₁₈ columns. These new phases are compatible with 100% aqueous mobile phases, and also provide high column efficiency and good peak shapes for both acidic and basic compounds.     

Comprehensive two-dimensional liquid chromatography with parallel gradients for separation of phenolic and flavone antioxidants

Various combinations of PEG-silica, phenyl-silica and C18 columns in a single-column or serial (tandem) arrangement in the first dimension and a monolithic Chromolith column in the second dimension were tested for comprehensive two-dimensional (2D) LCxLC separation of phenolic and flavone natural antioxidants. The combinations of different stationary phase chemistries provided low selectivity correlations between the first-dimension and the second-dimension separation systems. Improvement in system orthogonality, bandwidths suppression, more regular band distribution over the whole 2D retention plane and increased peak capacity in different 2D setups was achieved by using gradients with matching profiles running in parallel in the two dimensions over the whole 2D separation time range. Instead of two sampling loops, two alternating trapping XTerra columns were used for sample fraction transfer from the first-dimension column to the second dimension. Stronger retention on the XTerra columns in comparison to PEG-silica or phenyl-silica columns in the first dimension allowed using focusing of sample fractions in narrow zones on the top of a trapping column and back-flushing into the second dimension in a very low volume of the mobile phase. This fraction transfer modulation provided significant bandwidth suppression in the second dimension. 2D systems with optimized stationary phase selectivity, parallel gradients and fraction transfer modulation using two trapping columns were applied for the analysis of natural antioxidants in beer and wine samples.     

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.     

Capillary electrochromatography using columns packed with a supercritical fluid carrier

Summary Columns for capillary electrochromatography may be prepared by packing reversed phase silica-based material using a supercritical fluid carbon dioxide carrier. Procedures for the in-situ manufacture of frits and UV detection windows, and the wetting of columns are described. The columns were employed in two commercial instruments (and a home-built system), and their properties investigated during the separation of standard mixtures of test compounds. The columns are highly efficient and durable, with reduced plate heights of 1.0–1.4. The repeatability of retention time, peak area and peak height was measured. The influence of applied voltage and column temperature and of electrokinetic injection parameters was explored, along with other practical considerations.     

Liquid chromatographic retention and separation of phenols and related aromatic compounds on reversed phase columns

Summary Isocratic column liquid chromatographic systems with UV absorbance detection at 280 nm have been developed for the separation of 29 phenolics and related compounds.The selectivity was investigated on silica-, carbon- and polymer-based separation columns for the separation of phenolic type of components. The effects of various acetonitrile/buffer mixtures, and pH of the mobile phase, and their impact on the retention of the phenols was assessed. Tables of retention times on the four columns for the 29 phenols with two different acetonitrile/buffer mixtures, together with the retention times at three pHs from 6.5 to 2.3 with varying levels of organic modifier on the LiChrospher RP 18 column are presented.As an application, the analysis of real river water samples from the Ebro river is described using a solid phase extraction step prior to injection into the chromatographic system.     

Evaluation of a nitrogen-cooled, electrically heated cold trap inlet for high-speed gas chromatography

Gas chromatography has the potential to be a much faster method of separation than is usually realized. If column operating conditions are optimized for speed and injection band width is minimized, some simple separations can be completed in a few seconds. A prototype cryofocusing system for producing narrow injection bands with 0.25-mm i.d. columns is described here. The gas-cooled and electrically heated inlet produces injection bands with widths of about 10-20 ms. In the present study the system is evaluated using mixtures of common organics, including alkanes, aromatics, alcohols, ketones, and chlorinated hydrocarbons. Quantitative trapping and reinjection is achieved for all tested compounds. Coefficients of variation are less than 3% for peak area and less than 0.2% for retention time. Base-line separation of simple mixture is achieved with retention times of less than 10 s. By using the cold trap inlet with a low-dead-volume detector and a high-speed electrometer, the efficiency available from commercial capillary columns can be better utilized, and retention times for some routine separations may be reduced to a few seconds.     

Retention models for programmed gas chromatography

The models proposed by many authors for the prediction of retention times and temperatures, peak widths, retention indices and separation numbers in programmed temperature and pressure gas chromatography by starting from preliminary measurements of the retention in isothermal and isobaric conditions are reviewed. Several articles showing the correlation between retention data and thermodynamic parameters and the determination of the optimum programming rate are reported. The columns of different polarity used for the experimental measurement and the main equations, mathematical models and calculation procedures are listed. An empirical approach was used in the early models, followed by the application of thermodynamic considerations, iterative calculation procedures and statistical methods, based on increased computing power now available. Multiple column arrangements, simultaneous temperature and pressure programming, applications of two-dimensional and fast chromatography are summarised.     

Computer-assisted automatic peak recognition and result evaluation for analysis of chlorinated hydrocarbons in environmental samples

A data manipulation method has been developed for automatic peak recognition and result evaluation in the analysis of organic chlorinated hydrocarbons with dual-column gas chromatography. Based on the retention times of two internal standards, pentachlorotoluene and decachlorobiphenyl, the retention times of chlorinated hydrocarbons can be calibrated automatically and accurately. It is very convenient to identify the peaks by comparing the retention times of samples with the calibrated retention times calculated from the relative retention indices of standards. Meanwhile, with a suggested two-step evaluation method the evaluation coefficients and the suitable quantitative results of each component can be automatically achieved for practical samples in an analytical system using two columns with different polarities and two internal standards.