Chemometric studies of retention in capillary gas chromatographic separation of hydrocarbons in coupled columns

This paper describes how different multivariate analysis and classification methods can be used, to characterize the gas chromatographic separation of complex hydrocarbon mixtures in three columns coupled in series. Principal component analysis (PCA), correspondence factor analysis (CFA), and hierarchical ascending classification (HAC) were used as potential tools for evaluating the experiments on single columns and on column series. It has been demonstrated that: (1) multivariate analysis with PCA and CFA offers a powerful strategy to search for the main factors influencing the separation of hydrocarbons without a priori knowledge of the key factors of the separation. (2) With CFA the contribution of retention due to vapour pressure can be minimized. The use of retention indices, which use the n-alkanes as reference compounds, also helps to decrease the dominant focus on vapour pressure in favor of the more selectivity-based interaction forces. (3) CFA helps to analyze the degree of relevance of the chosen experimental design to the most important factors, controlling chromatographic selectivity.     

Comparison of theoretical and experimental models for characterizing solvent properties using reversed phase liquid chromatography

Theoretical and experimental quantitative structure–retention relationships (QSRR) models are useful for characterizing solvent properties and column selectivity in reversed phase liquid chromatography (RPLC). The chromatographic behavior of a model analyte, the herbicide atrazine, in a system derived from nine organic solvents and three chromatographic columns was used for developing QSRR models. Multiple linear regression (MLR) and partial least squares regression (PLSR) were used as statistical approaches. The similarities and differences between linear solvation energy relationships (LSER), and semi-empirical and theoretical molecular models were demonstrated. QSRR models show high predictive power, and can successfully predict retention factor (log k) for new solvents. The models are useful for solvent optimization and reducing time for method development in RPLC. The herbicide atrazine can be readily analyzed at a low level, and all three columns provided good resolution, high-performance and symmetrical peaks. The method is suitable for analysis of atrazine in water samples.     

Influence of column characteristics on retention and selectivity in RP-HPLC

Summary Eight commercially available reversed-phase (RP) columns of widely different characteristics were evaluated and compared using the linear solvation energy relationships (LSER). Retention factors of 32 solutes of different types were determined under isocratic conditions using an acetonitrile-water (30∶70) mobile phase. Stationary phase properties were compared by the fitting coefficients of the LSER-based regression equations which are characteristic of the individual stationary phases and represent the extent of various molecular interactions contributing to the retention process. The good agreement between the calculated and measured logk values for different type of compounds support the adequacy and applicability of the LSER model to describe chromatographic retention. Characterization of column performance for the separation of various type of compounds was established by the determination of the different selectivity factors representing hydrophobic selectivity, polar selectivity and specific selectivity.     

Prediction of internal standards in reversed-phase liquid chromatography. 1. Initial study on predicting internal standards for use with neutral samples based on linear solvation energy relationships

This paper describes the results of an initial study on the application of linear solvation energy relationships (LSERs) to the prediction of internal standard compounds in reversed-phase liquid chromatographic (RPLC) method development. Six neutral samples are separated on an Inertsil ODS(3) column by either acetonitrile-water or methanol-water mobile phases under either isocratic or linear gradient conditions. After the separation conditions are optimized, the desired positions for internal standard candidates are selected based on the "open windows" of the chromatograms. The compounds with the desired retention range are then predicted based on LSERs from a database consisting of more than 700 compounds with defined physicochemical properties. The prediction requires the use of LSER coefficients under the separation conditions for each sample. They are determined a priori by performing multivariable linear regression on the retention of 20 reference solutes against their physicochemical properties. It can be concluded from the study that LSER is an excellent approach to the selection of internal standard compounds for RPLC under either isocratic or gradient elution. The average prediction error is usually within 10%, but no more than 20%. Finally, LSER approach is fast and systematic, and will save a significant amount of time and resources during RPLC method development.     

溶质在两种反相色谱柱中的动力学色谱行为

研究了不同流速下4种溶质在两种不同反相色谱柱上的色谱图及它们的动力学色谱性质,进一步验证了溶质相对保留值(RRT)与流动相流速之间存在良好的双对数线性关系,并对溶质在流速改变时洗脱顺序发生改变的现象从动力学因素上进行了合理的解释;同时还发现色谱填料的孔径及拓扑学构型不同将导致溶质在其上具有不同的动力学色谱参数及动力学色谱行为。     

Quantitative structure-retention relationship studies using immobilized artificial membrane chromatography I: amended linear solvation energy relationships with the introduction of a molecular electronic factor

Linear solvation energy relationships (LSERs) amended by the introduction of a molecular electronic factor were employed to establish quantitative structure-retention relationships using immobilized artificial membrane (IAM) chromatography, in particular ionizable solutes. The chromatographic indices, log k(IAM), were determined by HPLC on an IAM.PC.DD2 column for 53 structurally diverse compounds, including neutral, acidic and basic compounds. Unlike neutral compounds, the IAM chromatographic retention of ionizable compounds was affected by their molecular charge state. When the mean net charge per molecule (delta) was introduced into the amended LSER as the sixth variable, the LSER regression coefficient was significantly improved for the test set including ionizable solutes. The delta coefficients of acidic and basic compounds were quite different indicating that the molecular electronic factor had a markedly different impact on the retention of acidic and basic compounds on IAM column. Ionization of acidic compounds containing a carboxylic group tended to impair their retention on IAM, while the ionization of basic compounds did not have such a marked effect. In addition, the extra-interaction with the polar head of phospholipids might cause a certain change in the retention of basic compounds. A comparison of calculated and experimental retention indices suggested that the semi-empirical LSER amended by the addition of a molecular electronic factor was able to reproduce adequately the experimental retention factors of the structurally diverse solutes investigated.     

Repeatability and reproducibility of retention data and band profiles on reversed-phase liquid chromatography columns: I. Experimental protocol

A procedure is described for the determination of the short-term and long-term repeatabilities and of the column-to-column and lot-to-lot reproducibilities of the retention and profile characteristics of the peaks obtained with a number of different reversed-phase liquid chromatography (RPLC) C₁₈-bonded silica columns of several commercial brands. Data characterizing the retention, the steric selectivity, the hydrogen bonding capacity, the hydrophobic interaction selectivity, the column efficiency and the peak asymmetry will be acquired for all the probe compounds. These include 30 neutral, acidic and basic compounds distributed into five groups to be eluted under as many different sets of chromatographic conditions. The data will be obtained using an HP 1100 liquid chromatograph. The compounds and experimental conditions selected were similar to those previously used by different authors for comparison purposes. Careful attention will be paid to minimization of external error contributions by adhering to a strict operational procedure. The precision expected will be high because preliminary results gave standard deviations of around 0.04% for the separation factors, below 0.15% for the retention times and around 1% for the column efficiency in short-term repeatability experiments performed with one column.     

Quantitative Structure-Retention Relationship Studies with Biopartitioning Micellar Chromatography Systems by Amended Linear Solvation Energy Relationships in Consideration of Electronic Factor

Linear solvation energy relationship (LSER) amended by the introduction of a molecular electronic factor was employed to establish quantitative structure-retention relationship of biopartitioning micellar chromatography (BMC) system. The chromatographic indices, log k, were determined by LC on a C18 column for sixty-five structurally diverse compounds, including neutral (32), acidic (19) and basic (14) compounds. Two micellar mobile phases composed of 0.04 mol L^?1 polyoxyethylene (23) lauryl ether (Brij35) were adjusted by phosphate buffer to pH 7.4 and pH 6.5, respectively. When the mean net charge per molecule (δ) was introduced into LSER as the sixth variable, the LSER regression coefficients and predictive capability were significantly improved. However, the δ coefficients of the amended LSER were quite different for acidic and basic compounds, indicating that the molecular electronic factor had a markedly different impact on the retention of acidic and basic compounds in the studied BMC system. This may attribute to the extra interaction for ionized compounds with the free silanol groups in the stationary phase. The comparison of calculated and experimental retention indices suggested that the amended LSER could reproduce adequately the retention of the structurally diverse solutes investigated in BMC.     

Comparison of Retention Indices of Some Monosubstituted Benzenes Calculated by Different Mathematical Methods in RP-LC

In reversed phase liquid chromatography, the retention indices of benzene and nine mono substituted benzenes with different functionality based on the alkan-2-ones and alkyl aryl ketones retention index standards have been determined by the application of two new mathematical adaptation methods, viz. a multiparametric least-squares regression iterative method based on the determination of the adjusted retention times and a local cubic interpolation method directly using the total retention times. The two methods were applied to two types of columns. The first group includes four octadecyl-C₁₈ columns with different packing materials obtained from different manufacturers, while the second comprises an octyl-C₈ column. The retention indices have been extensively studied using either methanol–water or acetonitrile–water mobile phase systems. The influences of the concentration of the organic modifier in the mobile phase (methanol or acetonitrile), the column temperature, and the column packing material on retention indices of the set of the ten monoaromatics studied were also investigated. The calculated multiparametric retention indices values, those obtained by the local cubic interpolation and Kováts’ methods are compared. Good agreement was observed between the retention indices calculated by the three methods.     

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.     

Study of peak shape and efficiency in butyl acrylate-based monolithic columns for capillary electrochromatography

The study and modelling peak shape of in capillary electrochromatography (CEC), obtained using butyl acrylate (BA)-based monolithic columns, is described in this article. A modified-Gaussian model, which is a function of conventional experimental parameters: retention time (t_R), peak height (H₀) and standard deviation (σ₀) at the peak maximum, and left (A) and right (B) halfwidths, was used to describe the peaks of thiourea and several polyaromatic hydrocarbons compounds, which were eluted under several applied voltages. A mean relative error below 2% in the prediction of peak shape is obtained. Based on peak parameters, an easy and reliable estimation of global chromatographic performance, efficiency and peak capacity of BA-based monoliths was also considered. A comparison in terms of chromatographic performance of BA-based monoliths in CEC with CE mode and RPLC microparticulate columns was also performed.     

Moment analysis of chromatographic behavior of superficially porous particles

Peak parking experiments were conducted to study the chromatographic behavior in a RPLC system consisting of a column packed with superficially porous C(18)-particles and a mixture of methanol and water (70/30, v/v). The values of the surface diffusion coefficient and the retention equilibrium constant of a column packed with superficially porous C(18)-particles were comparable to those of columns packed with a C(18)-silica monolith and full-porous C(18)-silica gel particles. The flow-rate dependence of HETP was hypothetically calculated by using moment equations to clarify the influence of the structural characteristics on the chromatographic behavior. The column efficiency of a column packed with the superficially porous particles is higher in the high flow-rate range than that with full-porous spherical particles. This is attributed to the smaller contribution of the intraparticulate mass transfer in the superficially porous particles to band broadening. The moment equations are effective for the quantitative analysis of chromatographic behavior of superficially porous particles.     

A collaborative study to investigate the retention reproducibility of barbiturates in HPLC with a view to establishing retention databases for drug identification

A collaborative study among 10 laboratories has been undertaken to investigate the interlaboratory reproducibility of retention measurements in a high performance liquid chromatographic (HPLC) system for separating barbiturates. The system involved an ODS-Hypersil column with an eluent of methanol:phosphate buffer (40:60, v/v) at pH 8.5; all laboratories used the same batch of packing material. The conventional methods of recording retention properties (retention times and capacity factors) gave poor interlaboratory reproducibility. Better results were obtained by expressing the retentions relative to a standard barbiturate (quinalbarbitone); relative adjusted retention times proved to be more effective than straightforward relative retention times. Retention indices based on the alkylarylketone scale were not as reproducible as the methods based on a single closely related compound. The best reproducibility was obtained using corrected capacity factors based on the retention of four barbiturates in a standard mixture. The results of the study are discussed with a view to selecting the best methods of recording retention in HPLC when considering the establishment of databases for drug identification.     

Comparison of Retention Indices of Some Explosives and Related Compounds Calculated by Different Mathematical Methods in Reversed-Phase Liquid Chromatography

In reversed phase liquid chromatography, the retention indices of some neutral and acidic explosives and related compounds (nitramines, nitroaromatics, aminoaromatics, and nitrophenols) based on the alkan-2-one, alkyl aryl ketone, and 1-nitroalkane retention index standards have been determined by the application of a new mathematical adaptation method, viz. a multiparametric least-squares regression iterative method. This method was applied to two types of columns. The first group includes six octadecyl-C₁₈ columns with different packing materials and obtained from different manufacturers, while the second group comprises one octyl-C₈ column. The retention indices have been extensively studied using either methanol-water or methanol-phosphate buffer mobile phase systems. The calculated multiparametric retention indices values were compared with those obtained by Guardino's, Grobler's, and Kovàts' methods. The influences of the concentration of the organic modifier in the mobile phase, aqueous mobile phase pH, and the column packing material on the retention indices of the explosives were also investigated. Good agreement was observed between the retention indices calculated by the use of the four mathematical methods for both neutral and acidic explosives.     

Retention and mass transfer characteristics in reversed-phase liquid chromatography using a tetrahydrofuran-water solution as the mobile phase.

The characteristics of the retention and the mass transfer kinetics in reversed-phase liquid chromatography (RPLC) were measured on a system consisting of a C18-silica gel and a tetrahydrofuran-water (50:50, v/v) solution. These parameters were derived from the first and the second moments of the elution peaks, respectively. Further information on the thermodynamic properties of this system was derived from the temperature dependence of these moments. Some correlations previously established were confirmed for this system, namely, an enthalpy-entropy compensation for both retention and surface diffusion and a linear free-energy relationship. These results are compared with those observed in other similar systems using methanol-water (70:30, v/v) and acetonitnile-water (70:30, v/v) solutions. The contribution of surface diffusion to intraparticle diffusion in C18-silica gel particles was shown to be important. The analysis of the thermodynamic properties of surface diffusion suggests that, in these three RPLC systems, its activation energy is lower than the isosteric heat of adsorption. The nature and the extent of the influence of the mobile phase composition on the parameters describing the retention and the mass transfer kinetics are different but the chromatographic mechanisms involved in RPLC systems appear similar, irrespective of the nature of the organic modifier in the mobile phase.     

Study of the influence of electronic effects on the retention of substitutedN-benzylideneanilines in normal-phase liquid chromatography

Summary A series of 72 substitutedN-benzylideneanilines (NBA) has been studied by normal-phase liquid chromatography by use of an experimental design based on variation of the composition of mobile phases prepared from heptane and three modifiers, tetrahydrofuran, 1-octanol and ethyl acetate, for each of which the specific interactions are different. Seven mobile phases were defined in the experimental design. The chromatographic data obtained are used to discuss the behavior of NBA by application of complementary chemometric methods—hierarchical ascending classification (HAC) and correspondence factor analysis (CFA). Although solute polarity has the greatest effect on retention, construction of HAC and CFA plots shows that solute behavior is also influenced by second-order electronic effects arising as a result of specific interactions between the solutes and the different modifier solvents. A quantitative structure-retention relationship has been established between Hammett's constants and the solutes projection on the first factorial axis of CFA.