Experimental designs for modeling retention patterns and separation efficiency in analysis of fatty acid methyl esters by gas chromatography-mass spectrometry

The retention behavior of components analyzed by chromatography varies with instrumental settings. Being able to predict how changes in these settings alter the elution pattern is useful, both with regards to component identification, as well as with regards to optimization of the chromatographic system. In this work, it is shown how experimental designs can be used for this purpose. Different experimental designs for response surface modeling of the separation of fatty acid methyl esters (FAME) as function of chromatographic conditions in GC have been evaluated. Full factorial, central composite, Doehlert and Box-Behnken designs were applied. A mixture of 38 FAMEs was separated on a polar cyanopropyl substituted polysilphenylene-siloxane phase capillary column. The temperature gradient, the start temperature of the gradient, and the carrier gas velocity were varied in the experiments. The modeled responses, as functions of chromatographic conditions, were retention time, retention indices, peak widths, separation efficiency and resolution between selected peak pairs. The designs that allowed inclusion of quadratic terms among the predictors performed significantly better than factorial design. Box-Behnken design provided the best results for prediction of retention, but the differences between the central composite, Doehlert and Box-Behnken designs were small. Retention indices could be modeled with much better accuracy than retention times. However, because the errors of predicted tR of closely eluting peaks were highly correlated, models of resolution (Rs) that were based on retention time had errors in the same range as corresponding models based on ECL.     

Comparative study of differential flow and cryogenic modulators comprehensive two-dimensional gas chromatography systems for the detailed analysis of light cycle oil

The modulator is the key point of comprehensive two-dimensional gas chromatography (GC×GC). This interface ensures the sampling and transfer of the sample from the first to the second dimension. Many systems based on different principles have been developed. However, to our knowledge, almost only cryogenic modulators are used in the petroleum industry. Nevertheless cryogenic fluids represent some disadvantages in term of safety, cost and time consuming. This paper reports a comparative study between differential flow and cryogenic liquid modulators for the detailed analysis of hydrocarbons in middle distillates type light cycle oil (LCO). Optimization of geometrical dimensions of a set of columns was carried out on the differential flow modulator system in order to reproduce the quality of separation of cryogenic modulation. Then a comparative study was investigated on sensibility and resolution (separation space and peak capacity) between the two systems.     

A new method for chemical identification based on orthogonal parallel liquid chromatography separation and accurate molecular weight confirmation

Recent advances in the theory and application of orthogonal LC separation have allowed for the establishment of a more effective method for the chemical identification of target compounds in complex samples, especially structurally similar compounds. In this study, a new chemical identification method based on orthogonal parallel separation and accurate molecular weight confirmation was developed. An orthogonal separation system consisting of an XTerra MS C(18) column, a home-made Click OEG column, and a Click CD column was established for separation and identification. In addition, 82 flavonoids were selected as references, to be used for the construction of a library. Retention times of each reference flavonoid on each column and accurate molecular weights were recorded and imported into a searchable library as "tags" for the unknown screening. For the method validation, two complex mixtures, fractions of Dalbergia odorifera T. Chen and Scutellaria baicalensis Georgi, specifically, were separated and identified. In total, nine compounds were unequivocally identified by retention time and confirmation of accurate molecular weight, demonstrating that this method is suitable and efficient for the chemical identification of complex samples.     

Studies on the effect of column angle in figure-8 centrifugal counter-current chromatography

The performance of the figure-8 column configuration in centrifugal counter-current chromatography was investigated by changing the angle between the column axis (a line through the central post and the peripheral post on which the figure-8 coil is wound) and the centrifugal force. The first series of experiments was performed using a polar two-phase solvent system composed of 1-butanol-acetic acid-water (4:1:5, v/v) to separate two dipeptide samples, Trp-Tyr and Val-Tyr, at a flow rate of 0.05 ml/min at 1000 rpm. When the column angle was changed from 0° (column axis parallel to the centrifugal force) to 45° and 45° to 90° (column axis perpendicular to the centrifugal force), peak resolution (Rs) changed from 1.93 (Sf=37.8%) to 1.54 (Sf=30.6%), then to 1.31 (Sf=40.5%) with the lower mobile phase and from 1.21 (Sf=38.8%) to 1.10 (Sf=34.4%), then to 0.99 (Sf=42.2%) with the upper mobile phase, respectively, where the stationary phase retention, Sf, is given in parentheses. The second series of experiments was similarly performed with a more hydrophobic two-phase solvent system composed of hexane-ethyl acetate-methanol-0.1M hydrochloric acid (1:1:1:1, v/v) to separate three DNP-amino acids, DNP-glu, DNP-β-ala and DNP-ala, at a flow rate of 0.05 ml/min at 1000 rpm. When the column angle was altered from 0° to 45° and 45° to 90°, Rs changed from 1.77 (1st peak/2nd peak) and 1.52 (2nd peak/3rd peak) (Sf=27.3%) to 1.24 and 1.02 (Sf=35.4%), then to 1.69 and 1.49 (Sf=42.1%) with the lower mobile phase, and from 1.73 and 0.84 (SF=41.2%) to 1.44 and 0.73 (Sf=45.6%), then to 1.21 and 0.63 (Sf=55.6%) with the upper mobile phase, respectively. The performance of figure-8 column at 0° and 90° was also compared at different flow rates. The results show that Rs was increased with decreased flow rate yielding the highest value at the 0° column angle with lower mobile phase. The overall results of our studies indicated that a 0° column angle for the figure-8 column enhances the mixing of two phases in the column to improve peak resolution while decreasing the stationary phase retention by interrupting the laminar flow of the mobile phase.     

A study on retention "projection" as a supplementary means for compound identification by liquid chromatography-mass spectrometry capable of predicting retention with different gradients, flow rates, and instruments

Using current data analysis techniques, even the most advanced LC-MS instrumentation can identify only a small fraction of compounds found in typical biological extracts. Augmenting MS information with HPLC retention information allows many more to be identified. In fact, our calculations indicate that a quadrupole MS is able to identify more compounds than an FTICR-MS when the quadrupole spectrum is augmented with retention information. Unfortunately, retention information is extremely difficult to harness for compound identification. Here, we demonstrate the first use of isocratic data measured on one LC-MS to "project" gradient retention on to different LC-MS systems. Using 35 chemically diverse solutes chosen to encompass the full range of reversed-phase alkylsilica interactions, and using experimental conditions typical of metabolomics experiments, gradient retention was projected from one instrument to another with only 1.2-2.6% error-enough accuracy to considerably improve compound identification. Besides accounting for nonlinear relationships of retention versus solvent composition as well as dead time versus solvent composition, accounting for the precise shape of the gradient profile (not just the dwell volume) improved projection accuracy on one instrument by up to 4 fold whereas flow rate non-idealities likely caused considerable error on the other instrument. Thus, these two factors must be taken into account to accurately project retention on diverse instrumentation.     

Retention prediction of a set of amino acids under gradient elution conditions in hydrophilic interaction liquid chromatography

The analysis of amino acids presents significant challenges to contemporary analytical separations. The present paper investigates the possibility of retention prediction in hydrophilic interaction chromatography (HILIC) gradient elution based on the analytical solution of the fundamental equation of the multilinear gradient elution derived for reversed‐phase systems. A simple linear dependence of the logarithm of the solute retention (ln k) upon the volume fraction of organic modifier (φ) in a binary aqueous‐organic mobile is adopted. Utility of the developed methodology was tested on the separation of a mixture of 21 amino acids carried out with 14 different gradient elution programs (from simple linear to multilinear and curved shaped) using ternary eluents in which a mixture of methanol and water (1:1, v/v) was the strong eluting member and acetonitrile was the weak solvent. Starting from at least two gradient runs, the prediction of solute retention obtained under all the rest gradients was excellent, even when curved gradient profiles were used. Development of such methodologies can be of great interest for a wide range of applications.     

Effect of extra-column volume on practical chromatographic parameters of sub-2-μm particle-packed columns in ultra-high pressure liquid chromatography

Effects of extra-column volume on apparent separation parameters were studied in ultra-high pressure liquid chromatography with columns and inlet connection tubings of various internal diameters (id) using 50-mm long columns packed with 1.8-μm particles under isocratic conditions. The results showed that apparent retention factors were on average 5, 11, 18, and 41% lower than those corrected with extra-column volumes for 4.6-, 3.0-, 2.1-, and 1.0-mm id columns, respectively, when the extra-column volume (11.3 μL) was kept constant. Also, apparent pressures were 31, 16, 12, and 10% higher than those corrected with pressures from extra-column volumes for 4.6-, 3.0-, 2.1-, and 1.0-mm id columns at the respective optimum flow rate for a typical ultra-high pressure liquid chromatography system. The loss in apparent efficiency increased dramatically from 4.6- to 3.0- to 2.1- to 1.0-mm id columns, less significantly as retention factors increased. The column efficiency was significantly improved as the inlet tubing id was decreased for a given column. The results suggest that maximum ratio of extra-column volume to column void volume should be approximately 1:10 for column porosity more than 0.6 and a retention factor more than 5, where 80% or higher of theoretically predicted efficiency could be achieved.     

Marker compounds for the determination of retention factors in EKC

EKC and its sub‐techniques, such as MEKC and microemulsion EKC, have attracted wide interest in recent years. Investigations on this topic have covered several analytical applications, but attention has also been paid more and more to basic studies. This review provides an overview of the different approaches to calculating retention factors, which express the ratio of the amount of sample component in the pseudostationary and mobile phases. Special attention is given to the selection of markers for the determination of the electrophoretic mobility or migration time of a marker describing the behavior of the pseudostationary phase in EKC. Introduction of a hydrophobic marker is by far the most common approach, but the use of a homologous series of compounds is also quite popular. In addition, other possible approaches found in the literature will be described.     

Prediction of liquid chromatographic retention for differentiation of structural isomers

A liquid chromatography (LC) retention time prediction software, ACD/ChromGenius, was employed to calculate retention times for structural isomers, which cannot be differentiated by accurate mass measurement techniques alone. For 486 drug compounds included in an in-house database for urine drug screening by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOFMS), a retention time knowledge base was created with the software. ACD/ChromGenius calculated retention times for compounds based on the drawn molecular structure and given chromatographic parameters. The ability of the software for compound identification was evaluated by calculating the retention order of the 118 isomers, in 50 isomer groups of 2–5 compounds each, included in the database. ACD/ChromGenius predicted the correct elution order for 68% (34) of isomer groups. Of the 16 groups for which the isomer elution order was incorrectly calculated, two were diastereomer pairs and thus difficult to distinguish using the software. Correlation between the calculated and experimental retention times in the knowledge base tested was moderate, r² = 0.8533. The mean and median absolute errors were 1.12 min, and 0.84 min, respectively, and the standard deviation was 1.04 min. The information generated by ACD/ChromGenius, together with other in silico methods employing accurate mass data, makes the identification of substances more reliable. This study demonstrates an approach for tentatively identifying compounds in a large target database without a need for primary reference standards.     

Zur Vorausberechnung von Bruttoretentionszeiten bei temperaturprogrammierter Gaschromatographie mit Hilfe isotherm bestimmter Retentionsindices und einer Anpassung an experimentelle Retentionszeiten

Zusammenfassung Durch mathematische Modellierung und Parameterschätzung lassen sich aus isotherm bestimmten Retentionsindices und ihrem Temperaturgang sowie aus wenigstens drei Stützwerten einer aktuellen temperaturprogrammierten Ánalyse Bruttoretentionszeiten weiterer Komponenten vorausberechnen. Am Beispiel einer mit Methylsilikonöl OV-1 imprägnierten Glaskapillarsäule wird gezeigt, daß für 19 Parametersätze mit Variation von Trägergasvordruck, isothermer Vorlaufzeit, Temperaturanstieg und isothermer Nachlauftemperatur die Bruttoretentionszeiten von 28 Komponenten einer Benzinprobe mit mittleren relativen Gesamtfehlern zwischen 1,3% und 3,8% je Analyse erhalten werden konnten.

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Prediction of retention times in temperature-programmed gas chromatography from isothermally determined retention indices and adjustment using experimental values of retention times

Summary Mathematical modelling and parameter estimation from three or more reference values in an actual temperature-programmed gas chromatographic analysis allows the prediction of retention times of the other components from a knowledge of their isothermally determined retention indices and the temperature profile. It is shown using a glass capillary column coated with OV-1 for measuring 19 sets of parameters obtained by varying the carrier gas inlet pressure, pre-program time, rate of temperature rise and past-program time, that the retention times of 28 components of a gasoline sample can be predicted with a mean error lying between 1,3% and 3.8% for each of these 19 analyses.