Characterization Of Stationary Phases Research Articles

Theoretical calculation of the parameters of the three-parameter chromatographic phase characterization method I. Dispersion forces parameter – generalized charge

In this work we studied the flavonoid composition of tangerine skins using 10 kinds of tangerines from the selectivity of separation in gas chromatography is determined by the nature of the stationary phase. The authors previously proposed a model of intermolecular interactions and a theoretical method of three-parameter characterisation of stationary phases in liquid chromatography based on it. They were applied to quantify the ability of molecules to participate in dispersion and dipole-dipole interactions and hydrogen bonds. The method proved to be efficient to describe the properties of stationary phases based on hydrocarbons, polyethylene glycol, polysiloxanes, and ionic liquids. The properties of stationary phases and analyte molecules are described by two selectivity characteristics: polarity and hydrophilicity, which can be calculated as a direct problem using the structural formula of the substance, or as an inverse problem using experimental data in the form of the Kovacs retention indices or Rohrschneider and McReynolds constants. No contradictions have been found between the characteristics calculated by the two methods. Using the proposed method, the relationship between the molecular weight of the polymer molecule and the values of selectivity characteristics was revealed. We proposed a selectivity map as a convenient and illustrative way to classify the stationary phases. Backed by the principle of similarity of properties, it can be used to determine the most selective stationary phase for a given analyte, without any experiments. The aim of this work was to determine the generalised charge as the first and key parameter of the three-parameter characterisation method. The main tool was the theory of generalised charges developed earlier in the laboratory of sorption methods of GEOKHI RAS. This theory, derived from fundamental principles, describes van der Waals interactions in the form of a Lennard-Jones potential, using the characteristics of molecules determined from their molecular structure. Previously, it successfully described nonpolar chromatographic systems. In the present study, we defined generalised charges, showed their relation to physical and experimental values, and provided calculation formulas for isolated molecules and for liquid phases. We presented the results of a detailed calculation of the generalised charges of substances of different classes, including gas chromatographic stationary phases.

High-performance liquid chromatographic evaluation of strong cation exchanger-based chiral stationary phases focusing on stationary phase characteristics and mobile phase effects employing enantiomers of tetrahydro-ß-carboline and 1,2,3,4-tetrahydroisoquinoline analogs

In this study, we present results obtained on the enantioseparation of some cationic compounds of pharmaceutical relevance, namely tetrahydro-ß-carboline and 1,2,3,4-tetrahydroisoquinoline analogs. In high-performance liquid chromatography, chiral stationary phases (CSPs) based on strong cation exchanger were employed using mixtures of methanol and acetonitrile or tetrahydrofuran as mobile phase systems with organic salt additives.Through the variation of the applied chromatographic conditions, the focus has been placed on the study of retention and enantioselectivity characteristics as well as elution order. Retention behavior of the studied analytes could be described by the stoichiometric displacement model related to the counter-ion effect of ammonium salts as mobile phase additives. For the thermodynamic characterization parameters, such as changes in standard enthalpy Δ(ΔH°), entropy Δ(ΔS°), and free energy Δ(ΔG°), were calculated on the basis of van't Hoff plots derived from the ln α vs. 1/T curves. In all cases, enthalpy-driven enantioseparations were observed with a slight, but consistent dependence of the calculated thermodynamic parameters on the eluent composition. Elution sequences of the studied compounds were determined in all cases. They were found to be opposite on the enantiomeric stationary phases and they were not affected by either the temperature or the eluent composition.

Characterization of stationary phases in supercritical fluid chromatography including exploration of shape selectivity

Achiral packed column supercritical fluid chromatography (SFC) has shown an important regain of interest in academic and industrial laboratories in the recent years. In relation to this increased concern, major instrument manufacturers have designed some stationary phases specifically for SFC use. SFC stationary phases have been widely examined over the last two decades, based on the use of linear solvation energy relationships (LSER), which relate analyte retention to its properties and to the interaction capabilities of the chromatographic system. The method provides some understanding on retention mechanisms (normal phase, reversed phase or mixed-mode) and the possibility to compare stationary phases on a rational basis, especially through a spider diagram providing a visual classification. The latter can be used as a primary tool to select complementary stationary phases to be screened for any separation at early stages of method development, before optimization steps.In this context, the characterization of the 14 columns from the Shim-pack UC series (Shimadzu Corporation, Kyoto, Japan), which are dedicated to SFC and more broadly to unified chromatography (UC), was performed, using the LSER methodology. As in previous works, seven descriptors, including five Abraham descriptors (E, S, A, B, V) and two descriptors describing positive and negative charges (D− and D+) were first employed to describe interactions with neutral and charged analytes. Secondly, two more descriptors were introduced, which were previously employed solely for the characterization of enantioselective systems and expressing shape features of the analytes (flexibility F and globularity G). They brought additional insight into the retention mechanisms, showing how spatial insertion of the analytes in some stationary phases is contributing to shape separation capabilities and how folding possibilities in flexible molecules is unfavorable to retention in other stationary phases.

Recent applications of retention modelling in liquid chromatography.

Recent applications of retention modelling in liquid chromatography (2015–2020) are comprehensively reviewed. The fundamentals of the field, which date back much longer, are summarized. Retention modeling is used in retention‐mechanism studies, for determining physical parameters, such as lipophilicity, and for various more‐practical purposes, including method development and optimization, method transfer, and stationary‐phase characterization and comparison. The review focusses on the effects of mobile‐phase composition on retention, but other variables and novel models to describe their effects are also considered. The five most‐common models are addressed in detail, i.e. the log‐linear (linear‐solvent‐strength) model, the quadratic model, the log–log (adsorption) model, the mixed‐mode model, and the Neue–Kuss model. Isocratic and gradient‐elution methods are considered for determining model parameters and the evaluation and validation of fitted models is discussed. Strategies in which retention models are applied for developing and optimizing one‐ and two‐dimensional liquid chromatographic separations are discussed. The review culminates in some overall conclusions and several concrete recommendations.

Predicting intraparticle diffusivity as function of stationary phase characteristics in preparative chromatography

Diffusion inside pores is the rate limiting step in many preparative chromatographic separations and a key parameter for process design in weak interaction aqueous chromatographic separations employed in food and bio processing. This work aims at relating diffusion inside porous networks to properties of stationary phase and of diffusing molecules. Intraparticle diffusivities were determined for eight small molecules in nine different stationary phases made from three different backbone materials. Measured intraparticle diffusivities were compared to the predictive capability of the correlation by Mackie and Meares and the parallel pore model. All stationary phases were analyzed for their porosity, apparent pore size distribution and tortuosity, which are input parameters for the models. The parallel pore model provides understanding of the occurring phenomena, but the input parameters were difficult to determine experimentally. The model predictions of intraparticle diffusion were of limited accuracy. We show that prediction can be improved when combining the model of Mackie and Meares with the fraction of accessible pore volume. The accessible pore volume fraction can be determined from inverse size exclusion chromatographic measurements. Future work should further challenge the improved model, specifically widening the applicability to greater accessible pore fractions (> 0.7) with corresponding higher intraparticle diffusivities (Dp/Dm > 0.2). A database of intraparticle diffusion and stationary phase pore property measurements is supplied, to contribute to general understanding of the relationship between intraparticle diffusion and pore properties.

Liquid chromatographic resolution of natural and racemic Cinchona alkaloid analogues using strong cation- and zwitterion ion-exchange type stationary phases. Qualitative evaluation of stationary phase characteristics and mobile phase effects on stereoselectivity and retention

Liquid chromatographic (LC) and subcritical fluid chromatographic (SFC) resolution of the basic natural and synthetic Cinchona alkaloid analogues has been studied. Focus has been placed on the employment of four enantiomerically structured chiral strong cation-exchangers and four chiral diastereoisomeric Cinchona alkaloid and cyclohexyl aminosulfonic acid-based zwitterionic ion-exchangers. Except for the novel, recently synthesized racemic quinine the other investigated pairs of basic analytes are diastereomeric, but often called “pseudoenantiomeric” compounds of quinine and quinidine, cinchonidine and cinchonine, 9‑epi‑quinine and 9‑epi‑quinidine. As expected, the elution order of the resolved racemic quinine was reversed for all the eight investigated enantiomeric and (pseudo)enantiomeric pairs of chiral stationary phases, whereas this was not necessarily the case for the diastereomeric pairs of the Cinchona alkaloid related analytes. Varying the type and composition of the protic (methanol) and non-protic (acetonitrile) but polar bulk solvents in combination with organic salt additives in the mobile phase the overall retention and stereoselectivity characteristics could be triggered, leading to well performing LC and SFC systems.Thus the retention behavior of the basic analytes on both the chiral cation-exchangers and the diastereomeric zwitterionic ion-exchangers, used as cation-exchangers, could be described by the stoichiometric displacement model related to the counter-ion effect of the mobile phase additives. In addition, it became obvious that the non-protic acetonitrile compared to methanol as bulk solvent lead to a significant increase in retention, which can be associated with an increased electrostatic interaction of the charged sites due to a smaller solvation shell of the solvated cationic and anionic species. Based on the chromatographic results of the systematically selected chiral analytes and stationary phases attempts were undertaken to interpret qualitatively the observed stereoselectivity phenomena.

Characterisation of Gas-Chromatographic Poly(Siloxane) Stationary Phases by Theoretical Molecular Descriptors and Prediction of McReynolds Constants.

Retention in gas–liquid chromatography is mainly governed by the extent of intermolecular interactions between the solute and the stationary phase. While molecular descriptors of computational origin are commonly used to encode the effect of the solute structure in quantitative structure–retention relationship (QSRR) approaches, characterisation of stationary phases is historically based on empirical scales, the McReynolds system of phase constants being one of the most popular. In this work, poly(siloxane) stationary phases, which occupy a dominant position in modern gas–liquid chromatography, were characterised by theoretical molecular descriptors. With this aim, the first five McReynolds constants of 29 columns were modelled by multilinear regression (MLR) coupled with genetic algorithm (GA) variable selection applied to the molecular descriptors provided by software Dragon. The generalisation ability of the established GA-MLR models, evaluated by both external prediction and repeated calibration/evaluation splitting, was better than that reported in analogous studies regarding nonpolymeric (molecular) stationary phases. Principal component analysis on the significant molecular descriptors allowed to classify the poly(siloxanes) according to their chemical composition and partitioning properties. Development of QSRR-based models combining molecular descriptors of both solutes and stationary phases, which will be applied to transfer retention data among different columns, is in progress.

Characterization of stationary phases based on monosubstituted benzene retention indices using correspondence factor analysis and linear solvation energy relationships in RPLC

In reversed-phase liquid chromatography (RPLC), the comparison of experimental results obtained from different columns is a complex problem. A correspondence factor analysis (CFA) and a linear solvation energy relationship (LSER) were applied on retention data to characterize second-order intermolecular interactions responsible for retention on a set of RPLC columns. Seven octadecyl-C18 columns with different packing materials are obtained from different manufacturers and one octyl-C8 column. The retention data were determined under isocratic conditions using a methanol–water (65:35, v/v) mobile phase. The chromatographic retention indices based on alkan-2-ones and alkyl aryl ketones retention index scales are calculated using a multiparametric least-squares regressions iterative method. The CFA and LSER results permitted to highlight that the retention indices were appropriate for studying the second-order retention mechanisms on the eight chromatographic systems investigated and exhibited the best reproducibility. Although many earlier studies have reported the use of chemometric methods to characterize chemical factors affecting retention in RPLC using retention factors as retention parameters, this is the first study based on retention indices.

Organic solvent modifier and temperature effects in non-aqueous size-exclusion chromatography on reversed-phase columns

Common reversed-phase columns (C18, C4, phenyl, and cyano) offer inert surfaces suitable for the analysis of polymers by size-exclusion chromatography (SEC). The effect of tetrahydrofuran (THF) solvent and the mixtures of THF with a variety of common solvents used in high performance liquid chromatography (acetonitrile, methanol, dimethylformamide, 2-propanol, ethanol, acetone and chloroform) on reversed-phase stationary phase characteristics relevant to size exclusion were studied. The effect of solvent on the elution of polystyrene (PS) and poly(methyl methacrylate) (PMMA) and the effect of column temperature (within a relatively narrow range corresponding to typical chromatographic conditions, i.e., 10°C–60°C) on the SEC partition coefficients KSEC of PS and PMMA polymers, were also investigated. The bonded phases show remarkable differences in size separations when binary mixtures of THF with other solvents are used as the mobile phase. The solvent impact can be two-fold: (i) change of the polymeric coil size, and possible shape, and (ii) change of the stationary phase pore volume. If the effect of this impact is properly moderated, then the greatest benefit of optimized solute resolution can be achieved. Additionally, this work provides an insight on solvent-stationary phase interactions and their effects on column pore volume. The only effect of temperature observed in our studies was a decreased elution volume of the polymers with increasing temperature. SEC partition coefficients were temperature-independent in the range of 10°C–60°C and therefore, over this temperature range elution of PS and PMMA polymers is by near-ideal SEC on reversed-phase columns. Non-ideal SEC appears to occur for high molar mass PMMA polymers on a cyano column when alcohols are used as mobile phase modifiers.

Chromatographic behavior of selected antibiotic drugs supported by quantitative structure-retention relationships

As antibiotic drugs have a wide variety of applications, there is a growing demand for their selective determination, a task for which chromatographic methods seem to be appropriate. With this end in view, chromatographic measurements were performed with the use of six different HPLC columns (ACE 5C18, IAM.PC.DD.2, octadecyl, phenyl, alkylamide and cholesteryl-alkylamide) to determine the logkw of selected antibiotic drugs (amoxicillin, cefatoxime, ciprofloxacin, fluconazole, gentamicin, clindamycin, linezolid and metronidazole). The retention behavior of the analytes was investigated as a function of different binary hydro-organic mobile phases containing 10–45% (v/v) acetonitrile. The studied compounds were separated under isocratic conditions. The best results of separation of the tested biologically active compounds were obtained on the commercially available C18 column. Special attention was dedicated to the study of interactions among the stationary phase, mobile phase and the analytes. Another goal was to selecting the best column for separation of the tested biologically active compounds. Finally, QSRR models together with stationary phase characterization provided reliable information on the properties and characteristics of studied columns.

Chromatographic methods enabling the characterization of stationary phases and retention prediction in high-performance liquid chromatography and supercritical fluid chromatography.

In the scope of the present review, the current status of high-performance liquid chromatography/ultra-high performance liquid chromatography and supercritical fluid chromatography is briefly provided. These techniques and their retention mechanisms are compared. Various alternative approaches utilized for the determination and description of the retention processes in these two systems are mapped. Two frequently used concepts, linear-free energy relationships, and hydrophobic subtraction models, used for the characterization of the retention interactions, are discussed. Principles and selected applications of the both methods are also covered. Then the models applied for the prediction of retention behavior of solutes on stationary phases are outlined. The procedures utilized for the sorbent/column classification are also covered. Simple chromatographic tests frequently used for the basic characterization and mutual comparison of stationary phases are summarized and briefly commented on. The importance of a statistical evaluation of complex retention data obtained from the chromatographic measurements is outlined. Finally, computer simulations aiming at the facilitation of the quest to optimize separation conditions for a given mixture of analytes are touched upon.

Method development for impurity profiling in SFC: The selection of a dissimilar set of stationary phases

Supercritical fluid chromatography (SFC) is drawing considerable interest as separation technique in the pharmaceutical industry. The technique is already well established in chiral separations both analytically and on a preparative scale. The use of SFC as a technique for drug impurity profiling is examined here. To define starting conditions in method development for drug impurity profiling, a set of dissimilar stationary phases is screened in parallel. The possibility to select a set of dissimilar columns using the retention factors (k-values) for a set of 64 drugs measured on 27 columns in SFC was examined. Experiments were carried out at a back-pressure of 150bar and 25°C with a mobile phase consisting of CO2 and methanol with 0.1% isopropylamine (5–40% over 10min) at a flow rate of 3mL/min. These k-values were then used to calculate correlation coefficients on the one hand and to perform a principal component analysis on the other. The Kennard and Stone algorithm, besides dendrograms and correlation-coefficient colour maps were used to select a set of 6 dissimilar stationary phases. The stationary phase characterization results from this study were compared to those from previous studies found in the literature. Retention mechanisms for compounds possessing different properties were also evaluated. The dissimilarity of the selected subset of 6 stationary phases was validated using mixtures of compounds with similar properties and structures, as one can expect in a drug impurity profile.

Genome-wide analysis of Sphingomonas wittichii RW1 behaviour during inoculation and growth in contaminated sand.

The efficacy of inoculation of single pure bacterial cultures into complex microbiomes, for example, in order to achieve increased pollutant degradation rates in contaminated material (that is, bioaugmentation), has been frustrated by insufficient knowledge on the behaviour of the inoculated bacteria under the specific abiotic and biotic boundary conditions. Here we present a comprehensive analysis of genome-wide gene expression of the bacterium Sphingomonas wittichii RW1 in contaminated non-sterile sand, compared with regular suspended batch growth in liquid culture. RW1 is a well-known bacterium capable of mineralizing dibenzodioxins and dibenzofurans. We tested the reactions of the cells both during the immediate transition phase from liquid culture to sand with or without dibenzofuran, as well as during growth and stationary phase in sand. Cells during transition show stationary phase characteristics, evidence for stress and for nutrient scavenging, and adjust their primary metabolism if they were not precultured on the same contaminant as found in the soil. Cells growing and surviving in sand degrade dibenzofuran but display a very different transcriptome signature as in liquid or in liquid culture exposed to chemicals inducing drought stress, and we obtain evidence for numerous ‘soil-specific' expressed genes. Studies focusing on inoculation efficacy should test behaviour under conditions as closely as possible mimicking the intended microbiome conditions.

Quantitative structure–retention relationships of ionic liquid cations in characterization of stationary phases for HPLC

In this report chemometric methodology was utilized in comprehensive column comparative characterization in order to define patterns of similarity and dissimilarity between stationary phases under investigation. A quantitative structure–retention relationship approach was adopted to obtain statistically significant molecular-descriptor-based models describing the retention mechanism of eight ionic liquids in two home-made and five commercially available chromatographic columns. Consequently their retention mechanism was also studied and discussed. Principal component analysis was adopted to reduce the dimensionality of experimental data in order to characterize different stationary phases based on the behavior of ionic liquids. QSRR models together with stationary phase characterization provided reliable information on the properties and characteristics of the studied columns.

Characterization of stationary phases based on polysiloxanes thermally immobilized onto silica and metalized silica using supercritical fluid chromatography with the solvation parameter model

Stationary phases (SP) prepared by immobilization of polymers on silica or metalized silica are interesting for use in reversed-phase high-performance liquid chromatography (RP-HPLC) due to better protection of residual silanols or Lewis acid sites, as well as ease of preparation. On the other hand, there are no previous reports of the use of such phases in packed column supercritical fluid chromatography (SFC). Fourteen different SPs based on polysiloxanes (phenyl, C1, C8, C14, C18) thermally immobilized onto different supports (silica, titanized silica, zirconized silica) and one chemically bonded and end-capped C18 stationary phase having a doubly zirconized silica support, were characterized with identical SFC conditions (CO2 with 10% of methanol, 25°C, 15.0MPa backpressure). Characterization was achieved based on retention factors measured for 85 test compounds and linear solvation energy relationships (LSER), namely the solvation parameter model based on Abraham descriptors. The polysiloxane SP were compared to a commercial C18-bonded silica (Kromasil C18), and to the native silica and metalized silica supports to better unravel the effects of stationary phase chemistry on chromatographic behavior. Together, 19 stationary phases were compared. The results highlight the variety of polarities provided by the column set. The polysiloxane backbone contributes significantly to the SP polarity as evidenced by the comparison to a classical alkylsiloxane-bonded SP. Metalization also appears to modify the polar characteristics of the SP, particularly toward basic compounds. Sample applications illustrate the applicability of these SP in SFC to the analysis of compounds of interest to the pharmaceutical and cosmetic industries.

Application of the zeta potential for stationary phase characterization in ion chromatography

Two series of homemade stationary bonded phases for ion chromatography were investigated according to their zeta potential. One set of dendrimer anion exchanger was synthesized on the polymer support whereas the second material was prepared on the silica gel. The zeta potential data in water environment as well as buffered water solution were obtained. The influence of the length of anion-exchanger chains, the type of the support of the modified surface, and charge distribution on these data was investigated. Additionally, the zeta potential was correlated with retention factor of inorganic ions to describe their influence on the retention mechanism in ion chromatography.

Studies on the Characterization of Stationary Phase for Protein Retention on Mixed-mode Chromatography

Studies on the Characterization of Stationary Phase for Protein Retention on Mixed-mode Chromatography

Towards multimodal HPLC separations on humic acid-bonded aminopropyl silica: RPLC and HILIC behavior

The stationary phase characteristics of the material obtained through immobilization of humic acid (HA) to aminopropyl silica (APS) via amide-bond formation were investigated. The material was characterized in terms of elemental analysis, FTIR, thermogravimetric analyses, pH point of zero charge measurements, potentiometric titrations, and contact angle measurements. Amount of HA bonded to APS was determined from the elemental analysis results, and found as 170mgHA/gAPS. Stability of the material was studied in aqueous media at different pH values, and amount of HA released at pH=8 did not exceed 2% of the total immobilized HA. Stationary phase characteristics of the well-characterized material were investigated in an HPLC system by using some low-molecular weight polar compounds (i.e. some nucleosides and nucleobases) as test solutes. Effect of some experimental variables such as column conditioning, composition of mobile phase, and temperature on the chromatographic behavior of the studied compounds was studied. Role of ammonium solutions at different pH values on retentive properties of the species was also studied. Retention factors (k′) versus volume percentage of organic modifier exhibited a U-curve, which was evaluated as an indication for RPLC/HILIC mixed-mode behavior of the stationary phase. Orthogonality between RPLC and HILIC modes was analyzed through geometric approach, and found as 48.5%. Base-line separation for the studied groups of compounds was achieved under each studied mode, and some differentiations were observed in elution order of the compounds depending on the HPLC mode applied. Chromatograms recorded under RPLC and HILIC modes were compared with those recorded on APS under similar conditions, and thus the influence/importance of HA immobilization process was evaluated in detail. In light of the obtained results, immobilized HA is represented as a useful stationary phase for HPLC separations.

Comments on the paper “Characterization of stationary phases by a linear solvation energy relationship utilizing supercritical fluid chromatography” by C. R. Mitchell, N. J. Benz, S. Zhang

In a recent paper published by Mitchell et al. in this journal, some results obtained in supercritical fluid chromatography and interpreted with the solvation parameter model to characterize interactions for "novel stationary phases" were surprising to us. Indeed, we had already published results for most of the stationary phases reported, but, except for polar phases, our results were not in agreement with those, despite the use of identical mobile phases in both studies. These data were disturbing because they suggest that supercritical fluid chromatography is always a normal-phase mode, while we have shown that it is a reversed-phase mode when working with non-polar stationary phases. In the process of establishing the reason for the differences between our works, we examined several different factors. This paper deals with practice of linear solvation energy relationships: choice of dead-volume marker, choice of test-solutes to adequately probe the possible interactions and appropriate column length for characterization of chromatographic systems with highly eluting mobile phases are discussed. The importance of control experiments to validate retention models and confirm their accordance with the chromatographer's experience is evidenced. Recommendations for good linear solvation energy relationship practice are suggested in order to avoid the publication of results leading to erroneous conclusions.

Practical implications of the “Tanaka” stationary phase characterization methodology using ultra high performance liquid chromatographic conditions

The practical implications of performing column characterization protocols (i.e. Tanaka) and their resultant chromatographic selectivity parameters using small dimension columns (i.e. 50×2.1mm I.D.) at high pressures have been critically compared to those obtained using conventional LC methodology. Retention factors should be corrected for the system extra column volume even when determined on ultra high performance liquid chromatographic (UHPLC) systems with low system volumes. An increase in pressure resulted in a general increase in the retention factor for most analytes, the degree being dependent on the physico/chemical properties of each analyte and the chromatographic conditions employed. However, analytes chromatographed at pH values close to their pKa values exhibited a substantial decrease in retention factor. Performing the Tanaka and extended column characterization procedures at pressures that would be encountered during the characterization of small particle sizes packed into 50×2.1mm I.D. column formats at a constant linear velocity according to standard protocols, resulted in comparable chromatographic selectivity parameters to those determined using standard HPLC systems and column formats. However, due to the wide structural diversity of analytes employed in other popular column characterization protocols, it is imperative to demonstrate comparable results when small columns packed with small particle sizes are chromatographed at increased pressure and compared to standard column formats – otherwise erroneous comparisons and conclusions may be made.