Jesús Eduardo Quintanilla-López

The hold-up time in gas chromatography II. Validation of the estimation based on the concept of a zero carbon atoms alkane

Abstract The procedure of estimating the hold-up time in gas chromatography recently presented by Lebron-Aguilar et al. [J. Chromatogr. A, 760 (1997) 219] as the retention of an alkane of zero carbon atoms has been exhaustively tested with packed and capillary columns of various stationary phase types, between 30 and 150°C. Several permanent gases injected have shown that there is not a general rule to decide which is the least retained gas under all circumstances. Results of hold-up times obtained with the new procedure have been compared with those of other well established methods. The conclusion arrived at is that the retention of an n-alkane of n carbon atoms is best described by the expression: tR (n)=A+exp(B+ C nD), and the best estimation of the hold-up time is obtained from that expression for n=0.

Hold-up time in gas chromatography I. New approach to its estimation

Abstract A new procedure to reach a good estimate of the hold-up time value (tM) in gas chromatography is presented. The value of tM obtained lies close to the experimental retention time of neon, the gas which has shown the shortest retention time in the columns studied. The new method, based on the retention of n-alkanes, is easily applied with any personal computer and may be used with detectors which do not respond to permanent gases. The procedure is reliable, produces very reproducible hold-up times and the value of tM obtained may be safely used to calculate both chromatographic and thermodynamic parameters.

Characterization by the solvation parameter model of the retention properties of commercial ionic liquid columns for gas chromatography.

For the first time, four commercial ionic liquid columns (SLB-IL59, SLB-IL76, SLB-IL82 and SLB-IL100) for gas chromatography have been comprehensively evaluated in terms of efficiency, polarity and solvation properties. Grob tests and McReynolds constants showed that they were all high-efficiency columns of high polarity, but with low inertness to compounds with hydrogen bonding capabilities. The solvation parameter model was used to characterize the solvation interactions of the four columns in the 80-160°C temperature range. Results revealed that all the ionic liquids studied can be considered moderately hydrogen-bond acid and highly cohesive stationary phases, on which the dominant contributions to retention were the dipolar-type and hydrogen-bond base interactions, while π-π and n-π interactions were barely significant. The SLB-IL59 column provided the best separation of homologs, while the SLB-IL76 and SLB-IL100 columns had the most basic and the most acidic phases, respectively. A principal component analysis for the commonly used stationary phases in capillary GC showed that these commercial ionic liquid columns fill an empty area of the available selectivity space, which clearly enhances the separation capacity of this technique.

The hold-up time in gas chromatography1: III. Linearity of the plot of ln tR′ of the n-alkanes vs. carbon number questioned

Abstract Three arguments are presented to show that the plot of the logarithms of the adjusted retention times of n -alkanes eluted under isothermal conditions versus their chain length is not a straight line, as generally accepted, even for n -alkanes of seventeen carbon atoms. Comparison of values of t R ′ deduced by methods based on the linearity assumption (ln t R ′= mz + n ) and on a different approach [ t R = A +exp( B + Cz D )] shows that experimental retention times do not fit the first expression, independently of the value used for the hold-up time, even if the correlation coefficient of the fit is better than 0.99999.

Development of a new method using HILIC-tandem mass spectrometry for the characterization of O-sialoglycopeptides from proteolytically digested caseinomacropeptide

This work addresses the optimization of HILIC (hydrophilic interaction liquid chromatography)‐ESI‐MSn conditions for the comprehensive characterization of O‐glycopeptides from proteolytically digested caseinomacropeptide. O‐Glycopeptides were satisfactorily analysed on a zwitterionic HILIC column based on their glycan structure and amino acid sequence. The contribution of ionic interactions to the retention of charged glycopeptides was found to be substantial. Thus, O‐glycopeptides carrying neutral glycans were more retained than O‐sialoglycopeptides because negatively charged sialic acid residues were electrostatically repelled by the stationary phase. In addition, glycopeptides differing only in the position of the linkage of the sialic acid moiety could be separated. The same chromatographic behaviour was observed for model systems constituted by a synthetic tetrapeptide covalently conjugated to neutral and sialylated carbohydrates. Subsequent detection of caseinomacropeptide O‐glycopeptides was carried out on an electrospray ion trap tandem mass spectrometer at both positive and negative ionization modes. MS2 fragmentation at positive ionization mode was valid for determining the glycan structure as the resulting main fragments corresponded to Yn‐type ions derived from sequential glycosidic bond fragmentation, whilst the fragmentation of the peptide structure was preferably obtained through the formation of bn‐type ions at the MS3 stage, allowing the complete structure elucidation of the peptidic chain. Overall, the developed method allowed the identification and characterization of 41 O‐glycopeptides covering all the known glycosylation sites without any previous enrichment step. These results point out that HILIC coupled to multistage MS procedures can be a powerful technique for future glycoproteomic applications.

Feasibility of ultra-high performance liquid and gas chromatography coupled to mass spectrometry for accurate determination of primary and secondary phthalate metabolites in urine samples.

Phthalates (PAEs) are ubiquitous toxic chemical compounds. During the last few years, some phthalate metabolites (MPAEs) have been proposed as appropriate biomarkers in human urine samples to determine PAE human intake and exposure. So, it is necessary to have fast, easy, robust and validated analytical methods to determine selected MPAEs in urine human samples. Two different instrumental methods based on gas (GC) and ultra-high performance liquid (UHPLC) chromatography coupled to mass spectrometry (MS) have been optimized, characterized and validated for the simultaneous determination of nine primary and secondary phthalate metabolites in urine samples. Both instrumental methods have similar sensitivity (detection limits ranged from 0.03 to 8.89 pg μL(-1) and from 0.06 to 0.49 pg μL(-1) in GC-MS and UHPLC-MS(2), respectively), precision (repeatability, expressed as relative standard deviation, which was lower than 8.4% in both systems, except for 5OH-MEHP in the case of GC-MS) and accuracy. But some advantages of the UHPLC-MS(2) method, such as more selectivity and lower time in the chromatographic runs (6.8 min vs. 28.5 min), have caused the UHPLC-MS(2) method to be chosen to analyze the twenty one human urine samples from the general Spanish population. Regarding these samples, MEP showed the highest median concentration (68.6 μg L(-1)), followed by MiBP (23.3 μg L(-1)), 5cx-MEPP (22.5 μg L(-1)) and MBP (19.3μgL(-1)). MMP (6.99 μg L(-1)), 5oxo-MEHP (6.15 μg L(-1)), 5OH-MEHP (5.30 μg L(-1)) and MEHP (4.40 μg L(-1)) showed intermediate levels. Finally, the lowest levels were found for MBzP (2.55 μg L(-1)). These data are within the same order of magnitude as those found in other similar populations.

Measuring specific retention volumes in capillary gas chromatography with improved accuracy and precision

Abstract The specific retention volume (Vg), which has traditionally been determined in gas chromatography using long, heavily loaded packed columns, should produce better, more reliable values if capillary columns are used. However, the smaller dimensions of some of the parameters involved introduces additional sources of error which have been overcome with a recently proposed equation. A careful consideration of the sources of error involved has been carried out using both the traditional and the new equations. The conclusion drawn is that the new equation will produce reliable Vg values with expected errors (1%) lower than those expected from packed columns (2%) using the traditional equation.

Fast and simultaneous determination of endocrine disrupting compounds by ultra-high performance liquid chromatography-tandem mass spectrometry.

A rapid and sensitive analytical method for the simultaneous determination of thirteen endocrine disruptors (five phthalates, seven parabens, and bisphenol A) in a single chromatographic run has been developed for the first time. The separation method, based on ultra-high performance liquid chromatography (UHPLC), allows the separation of all compounds (including isobaric pairs) in less than 4.1 min. The fast polarity switching mode of the triple quadrupole mass spectrometer used enables the registration of positive (phthalates) and negative (parabens and BPA) ions in the same acquisition run. A Response Surface Methodology was used for the optimization of the method. The optimum elution program starts with 0.2 min in isocratic conditions (79.8% water; 20% acetonitrile, 0.2% ammonium formate 5mM at pH 10.2), then the content of acetonitrile is linearly increased in 2 min up to 42%, and later up to 98% in 1.1 min. The analytical characteristics of the developed method were satisfactory. The method is robust and showed a linear response with determination coefficients (R(2)) higher than 0.991 in the range 5.0-2000 pg on column (or higher) for all the compounds investigated. Instrumental intra- and inter-day precision (expressed as relative standard deviation) were lower than 12% for parabens and bisphenol A, and between 5.9% and 27% for phthalates. Instrumental detection and quantification limits (iLODs and iLOQs) were in the range of medium-high femtograms (270-1300 pg on column for iLODs). Finally, the suitability of the developed method was demonstrated through its application to the analysis of commercial personal care products (shower gels) without any sample treatment, only a simple dilution, being possible to determine the simultaneous presence of phthalates, parabens, and bisphenol A in almost all the gels analyzed.

Characterization and optimization by experimental design of a liquid chromatographic method for the separation of hydroxylated polychlorinated biphenyls on a polar-embedded stationary phase.

Traditionally, the determination of hydroxylated polychlorinated biphenyls (OH-PCBs) has been carried out by gas chromatography (GC). However, the gas chromatographic behavior and sensitivity of this type of hydroxylated compounds are not always satisfactory, hence a prior derivatization of the OH-PCBs must be performed. Therefore, the development of liquid chromatographic methods should prove to be a very interesting task aimed at dealing with the instrumental determination of OH-PCBs. Taking into account that octadecylsilane stationary phases are not the most adequate for the separation of isobaric compounds, an amide-type column has been tested. For the development of the method, the Response Surface Methodology was used, based on a Box-Wilson Central Composite experimental design. The initial content of methanol in the mobile phase, the gradient time, and the concentration and the pH value of the buffer were chosen as relevant experimental parameters. A global optimum was obtained by selecting the elution time, the sensitivity and the overall resolution as responses to optimize. The developed method for liquid chromatography presented a very good resolution and sensitivity, and a reasonably short analysis time. In addition, a retention study was conducted in order to survey the different interactions that take place in the separation process, showing that hydrogen bonding is the main interaction between OH-PCBs and the amide-type stationary phase. However, a substantial contribution of dispersion forces was present in methanol contents in the mobile phase below 65%. Besides, the pH value of the mobile phase was found to be the most important parameter to control the hydrogen bond forces and therefore, to regulate the OH-PCBs separation.

Analysis of iminosugars and other low molecular weight carbohydrates in Aglaonema sp. extracts by hydrophilic interaction liquid chromatography coupled to mass spectrometry.

A method by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry (HILIC-MS(2)) has been successfully developed for the simultaneous analysis of bioactive iminosugars and other low molecular weight carbohydrates in Aglaonema leaf extracts. Among other experimental chromatographic conditions, mobile phase eluents, additives and column temperature were evaluated in terms of retention time, resolution, peak width and symmetry provided for target carbohydrates. In general, narrow peaks (wh: 0.2-0.6min) with good symmetry (As: 0.9-1.3) and excellent resolution (Rs>1.8) were obtained for iminosugars using an acetonitrile:water gradient with 5mM ammonium acetate in both eluents at 55°C. Tandem mass spectra were used to confirm the presence of previously detected iminosugars in Aglaonema extracts and to tentatively identify for the first time others such as miglitol isomer, glycosyl-miglitol isomers and glycosyl-DMDP isomers. Concentration of total iminosugars varied from 1.35 to 2.84mgg(-1) in the extracts of the different Aglaonema samples analyzed. To the best of our knowledge, this is the first time that a HILIC-MS(2) method has been proposed for the simultaneous analysis of iminosugars and other low molecular weight carbohydrates of Aglaonema sp. extracts.