Characterization Of Stationary Phases Research Articles

DETERMINATION OF THE FREE ENERGY OF ADSORPTION AND THE SURFACE AREA OF ADSORPTION OF SOME CARBOXAMIDE DERIVATIVES BY NORMAL PHASE THIN-LAYER CHROMATOGRAPHY

The free energy of adsorption (S°), and the surface area of adsorption (AS) on silica and alumina surfaces of 17 carboxamide fungicides was determined by thin-layer chromatography (TLC) using silica and alumina stationary phases. The relationships between the measured physicochemical parameters of analytes and the relationships between these parameters and 29 computed physicochemical characteristics were calculated by linear regression analysis and the Fisher's test. It was found that the adsorption characteristics of stationary phases are significantly different while the similar parameters of carboxamide derivatives showed marked similarities. It was further established that in the majority of cases the computed and measured physicochemical parameters are not correlated. This finding indicates that the free energy of adsorption and the surface area of adsorption can be used as new independent variables in both Quantitative Structure Activity Relationship (QSAR) and Quantitative Structure Retention Relationship (QSRR) studies.

High performance liquid chromatography of slightly soluble brominated indigoids from Tyrian purple

Attempts at identification of mollusc species producing Tyrian purple from archaeological material are usually done with high performance liquid chromatography in the reversed-phase system, but the peaks obtained are often wide and asymmetric. This is due to the low solubility of the indigoids and their brominated derivatives in the mobile phase, especially 6,6′-dibromoindigotin, which is soluble in only few, particular solvents. Our study focused on improving both symmetry and peak height for more precise quantification. The influence of various factors was evaluated: stationary phase characteristics, mobile phase composition, elution gradient parameters and temperature on the peak shape of the main components of Tyrian purple. The best results were obtained using highly retentive, but moderately bonded ODS stationary phases (about 2.8 μmol m −2), percolated with gradient of acetonitrile with acidified aqueous mobile phases (0.1% strong acid) at elevated temperatures (70 °C). The upper quantification limit for 6,6′-dibromoindigotin was improved by over 350%, between standard and optimised systems. Using them, the detection and quantification of trace Tyrian purple components (less than 0.15%) aside from major indigoids becomes possible. Consequently, for the first time, the new analogues of brominated and unbrominated indirubins were found in the shellfish purple from Hexaplex trunculus.

Selectivity of calixarene-bonded silica phases in HPLC: Description of special characteristics with a multiple term linear equation at different methanol concentrations

Retention and selectivity characteristics of different calixarene-, resorcinarene- and alkyl-bonded stationary phases are examined by analyzing a set of test solutes covering the main interactions (hydrophobic, steric, ionic, polar) that apply in HPLC. Therefore Dolan and Snyder's multiple term linear equation has been adapted to fit the properties of calixarene-bonded columns. The obtained parameters are used to describe retention and selectivity of the novel Caltrex(®) phases and to elucidate underlying mechanisms of retention. Here, differences of stationary phase characteristics at different methanol concentrations in the mobile phases are examined. Both selectivity and retention were found to depend on the methanol content. Differences of these dependencies were found for different stationary phases and interactions. The differences between common alkyl-bonded and novel calixarene-bonded phases increase with increasing methanol content.

Characterization of stationary phases by a linear solvation energy relationship utilizing supercritical fluid chromatography

Supercritical fluid chromatography was utilized in combination with the Abraham model of linear solvation energy relationship to characterize 11 different HPLC stationary phases. System constants were determined at one supercritical fluid chromatography condition for each stationary phase. The results indicate that several types of silica columns, including type B silica, type C silica, and fused core silica, are very similar in their retention behavior. Several aromatic stationary phases were characterized and it was found that, in contrast to the other phases studied, all of the aromatic stationary phases had positive contributions from the dispersion/cavity (v) term of the linear solvation energy relationship. Several aliphatic phases were characterized and there were several linear solvation energy relationship constants that differentiated the phases from each other, mainly the polar terms (dipolarity and hydrogen bonding). One stationary phase, a fused core pentafluorophenyl (PFP) phase, had very poor regression quality. The column volume of this phase was lower than the others in the study, which may have had some impact on the results of the regression.

Characterization of stationary phases for reversed‐phase chromatography

Retention characteristics of seven silica-based chromatographic columns (Bidentate C18, Silica-hydride, Spherisorb ODS-1, Resolve C18, XTerra C18, XBridge Phenyl and Blaze C8 Polydentate) were compared. The columns differ in the character of the support, including silica type B, hydrosilated silica, and hybrid silica gel with incorporated methyl groups, or ethylene bridges; further in the type of chemically bonded stationary phase, including C(18), C(8) and phenyl ligands, endcapped and non-endcapped with single point, bidentate and polydentate multi-point attachment to the adsorbent surface. Special attention was focused on hydrophobic and polar selectivity, silanol activity and anion-exclusion. The columns were compared and classified on the basis of the results of the classical column performance tests, retention of homologous n-alkylbenzenes over a broad composition range of aqueous-organic mobile phases and of naphthalene sulphonic acids in aqueous mobile phases without organic solvents.

The use of scanning contactless conductivity detection for the characterisation of stationary phases in micro-fluidic chips

The use of scanning capacitively coupled contactless conductivity detection for the evaluation of the structural homogeneity and density of both packed and monolithic stationary phases in micro-fluidic chips is presented here for the first time.

Influence of Stationary Phase Properties on the Retention of Ketones and Alcohols in RP-HPLC Mode: A New Test for the Stationary Phase Characterization

Two types of octadecyl stationary phases, monomeric and polymeric, were characterized using a simple test based on aliphatic alcohol and ketone retentions. Chromatographic measurements in reversed phase systems (acetonitrile/water, methanol/water) permitted reporting changes of the separation selectivity. For the tested compounds, shape selectivity is different in systems with packings with different surface morphologies and depends also on the type of modifier used in the mobile phase. Separation selectivity was also investigated at three different temperatures: 20°C, 40°C, and 60°C. Standard enthalpy and entropy change calculations confirmed the heterogeneity of surface coverage density and conformational changes of chemically bonded silica gel ligands for various solvents. Presentation of differences between stationary phases, surface architecture, and the application of simple compounds and chromatographic systems for this purpose was the aim of the paper.

Characterisation of stationary phases in supercritical fluid chromatography with the solvation parameter model: V. Elaboration of a reduced set of test solutes for rapid evaluation

Characterisation of chromatographic systems with the solvation parameter model provides satisfactory information on the main non-ionic interactions developed in a chromatographic system. The procedure requires the analysis of a large number of compounds to warrant the relevance and the accuracy of the calculated models, and even if retention time is lower in supercritical fluid chromatography (SFC) than in HPLC (3–5 times lower), a decrease in the time required for that procedure would favour the use of this model in method development. Consequently, in order to establish a rapid testing procedure that would provide equivalent information, nine key solutes were carefully selected among the hundred we classically use. The separation factors calculated between these key solutes, taken two by two to establish new equations, allow the calculation of the model coefficients. The normal testing procedure is thus reduced from one or two days down to 2 h. Precision and accuracy of the models provided are assessed through back-calculation of the coefficients that served for the establishment of the procedure, then through calculation of the coefficients of 13 new SFC systems. The applicability of the rapid testing procedure in SFC is evidenced with three examples: the elaboration of a system map, by varying the modifier concentration in the mobile phase, and the comparison of six ODS phases bonded on the same silica base. The simplified procedure presented here does not pretend to characterize the chromatographic systems as precisely as the complete testing procedure does, but is only aimed at rapidly evaluating the chromatographic retention characteristics when operating parameters are varied.

Stationary phase characterization and method development

The properties of stationary phases and their characterization methods are reviewed. New and significant developments have occurred in the last few years, and new methods for stationary phase characterization have become available. The characterization methods are discussed, and the differences between the different methods are pointed out. In addition, method development approaches are reviewed, with special emphasis on recent developments that employ multiple parameters in parallel. Also, the renewed interest of temperature as a tool in method development is surveyed.

Evaluation of the influence of residual silanol groups on the total retention in reversed-phase chromatography

It was demonstrated that the characteristics of stationary phases for reversed-phase chromatography can be compared by analyzing the retention of several compounds belonging to a homologous series during elution with one and the same composition of mobile phase or the retention of a nonpolar compound during elution with several mobile phases of different compositions. The slope of the corresponding linear correlations can be used to characterize the hydrophobicity of the columns (stationary phases) under study. To analyze the polar interactions between the sorbate and the stationary phase it is advantageous to study the retention of a polar substance in several (at least in two) mobile phases of different compositions. The degree of displacement of the linear correlation relative to that for nonpolar substances can be considered a measure of the influence of residual silanol groups on the total retention characteristics.

An accurate and easy procedure to obtain isothermal Kováts retention indices in gas chromatography

Isothermal Kováts retention indices may be used in GC for identification purposes, but they are also useful in characterisation of stationary phases and for studying structural and physico-chemical properties of both the analyte and the stationary phase. They are currently reported as whole numbers with an accuracy of one index unit for non-polar stationary phases. The method recommended for their calculation uses a linear regression model, although non-linear models have also been applied with good results. In both cases, a computer programme and the retention times of a series of n-alkanes that elute correctly and resolved from the other compounds are needed, conditions which cannot always be fulfilled. However, it is possible to calculate retention indices with an accuracy of 0.1 retention index units (0.2 units for packed columns) with the help of only three n-alkanes using just a pocket calculator or a computer spreadsheet. The main requirement is that at least one of the n-alkanes has a retention index differing by no more than one hundred retention index units from that of the analyte being investigated. Examples are given for different stationary phases.

Characterization of stationary phases for gas chromatography by 29Si nuclear magnetic resonance spectroscopy : III. Carborane–siloxane copolymers

Five bis(dimethylsilyl)- m-carborane–siloxane polymers with methyl, phenyl, and 2-cyanoethyl ligands were characterized by 1H, 11B, 13C, and 29Si nuclear magnetic resonance (NMR) spectroscopy. All relevant chemical shifts are reported, whereas signal assignment was confirmed by 2D NMR spectroscopy. The chemical composition of the polymers was calculated from the 1H and 29Si NMR spectra. Only 29Si NMR spectroscopy was able to quantify the methoxy end group, from which the average molecular weights were calculated. The copolymer Dexsil 300 turned out to have a regular microstructure, whilst the terpolymers Dexsil 400 and Dexsil 410 have only partly regular sequences. 11B NMR spectroscopy confirmed the m-carborane structure and revealed some low molecular weight impurities.

Characterisation of stationary phases in subcritical fluid chromatography with the solvation parameter model IV: Aromatic stationary phases

The purpose of the present work was to systematically study the chromatographic behaviour of different aromatic stationary phases in a subcritical fluid mobile phase. We attempted to assess the chemical origin of the differences in retention characteristics between the different columns. Various types of aromatic stationary phases, all commercially available, were investigated. The effect of the nature of the aromatic bonding on interactions between solute and stationary phases and between solute and carbon dioxide–methanol mobile phase was studied by the use of a linear solvation energy relationship (LSER): the solvation parameter model. This study was performed to provide a greater knowledge of the properties of these phases in subcritical fluid chromatography, and to allow a more rapid and efficient choice of aromatic stationary phase in regard of the chemical nature of the solutes to be separated. Charge transfer interactions naturally contribute to the retention on all these stationary phases but are completed by various other types of interactions, depending on the nature of the aromatic group. The solvation vectors were used to compare the different phase properties. In particular, the similarities in the chromatographic behaviour of porous graphitic carbon (PGC), polystyrene-divinylbenzene (PS-DVB) and aromatic-bonded silica stationary phases are evidenced.

Stationary phases in the screening of drug/impurity profiles and in their separation method development: Identification of columns with different and similar selectivities

The classification or characterization of stationary phases based on chromatographic parameters, in general, requires different test solutes/mixtures and several mobile phases. To simplify the classification/characterization of reversed-phase liquid chromatographic columns, to be used in separating drug/impurity profiles, a new test procedure was proposed. It consists of injecting two mixtures of relatively similar active substances applying a standard gradient. The aim was to evaluate from this approach the selectivity differences and overall separation quality of newly tested columns compared to that in an earlier selected set of eight stationary phases. The selectivity differences of the columns were evaluated by correlation coefficient-based weighted-average-linkage dendrograms and color maps. Derringer's desirability functions were used to rank similar stationary phases according to their overall separation quality. Four columns of 27 examined were, for instance, considered different from the earlier selected eight and could be added to the selection. A number of tested stationary phases might be considered as alternatives for some from the initial set. For three columns the newly tested stationary phases did not contain alternatives.

Classification and replacement test of HPLC systems using principal component analysis

Theoretical plate numbers and symmetry factor values have been measured for three solutes (benzoic acid, N, N-dimethyl-aniline and Vancomycin) in various chromatographic systems (stationary phases and different mobile phase compositions) with the aim of grouping the systems and studying the possibilities for replacement of columns and eluent compositions. The mobile phases consisted of acetonitrile or methanol modifier and KH 2PO 4–H 3PO 4 or triethylamine–H 3PO 4 aqueous buffer. The organic content of mobile phases was adjusted so that the log ks (logarithms of retention factor) of tested compounds vary in the range of 0.1–1.3 in case of all chromatographic systems and test compounds. Plate numbers and symmetry factors at all retention factor values (between log k = 0.1–1.3 with 0.1 units) were subjected to principal component analysis (PCA). The input data were arranged in 13 × 28 data-matrices in case of all compounds separately for plate numbers and symmetry factors. Two principal components were retained in the model, which carry not less than 98% of the total variance. Principal component analysis showed which chromatographic systems (stationary and mobile phases) gave the same (similar) chromatographic parameters using three different kinds of compounds. Replacement of chromatographic systems is possible on the basis of measured chromatographic parameters using principal component analysis. The monofunctional test compounds provide only approximate information on the usefulness, classification and replacement of HPLC systems, if the aim is the separation of multifunctional solutes. Although the theoretical plate number (column efficiency) is negatively correlated with the symmetry factor, both are necessary for proper classification and characterization of stationary phases.

NMR Investigations of Stationary Phases

AbstractFor Abstract see ChemInform Abstract in Full Text.

NMR investigations of stationary phases

AbstractAn overview of the application of solid‐state and suspended‐state NMR spectroscopy for the characterization of stationary phases is given. Solid‐state 29Si NMR spectroscopy reveals valuable information about the variety and quantity of the surface species formed in the modification reaction. Solid‐state 13C NMR spectroscopy gives an insight into the dynamic properties of the immobilized ligands and ligand organization. With the help of 1H and 13C suspended‐state NMR spectroscopy, investigations can be performed in the presence of a mobile phase. Thus, the structural and dynamic properties of the stationary phase under real‐life chromatographic conditions can be derived from the NMR investigations. These findings can be directly correlated with the resulting chromatographic separation behaviour.

High-performance liquid chromatographic stationary phases based on poly(methyloctylsiloxane) immobilized on silica: II. Chromatographic evaluation

This work describes the chromatographic characterization of stationary phases prepared by deposition of poly(methyloctylsiloxane) (PMOS) on silica followed by immobilization using one of several different processes: thermal treatments (120 or 220 °C for 4 h), microwave irradiation (495 W for 15 min), γ radiation (dose of 80 kGy) or self-immobilization. This evaluation was based on the chromatographic parameters of several test solutes. The stationary phases immobilized at 220 °C and which underwent self-immobilization were not appropriate for chromatographic use but the other immobilized phases presented chromatographic performances similar in most respects to a commercial phase (Rainin C 8) while the peak characteristics of the basic probe were significantly better with these phases.

Trends and progress in the characterization of stationary phases for reversed-phase liquid chromatography

The state-of-the-art and progress in the characterization of reversed-phase liquid chromatographic (RPLC) columns are discussed. Existing column tests are compared with tests under development. The validity of several test parameters, e.g. hydrophobicity and silanol activity, is also discussed. Solvent and eluent properties are treated with respect to retention and selectivity. Developments to improve the chemical stability of RPLC phases and thus column lifetime are reviewed. It is shown that substantial progress has been made in the manufacturing of RPLC columns, enabling their use over a wide variety of experimental conditions. In addition, general measures to significantly extend their lifespan are discussed. The substantial improvements in batch-to-batch and column-to-column repeatabilities achieved during the last two decades are also reviewed.

Gene expression and protein levels of the stationary phase sigma factor, RpoS, in continuously-fed Pseudomonas aeruginosa biofilms

Bacteria growing in biofilms experience gradients of environmental conditions, including varying levels of nutrients and oxygen. Therefore, bacteria within biofilms may enter distinct physiological states, depending on the surrounding conditions. In this study, rpoS expression and RpoS levels were measured as indicators of stationary phase growth within thick continuously-fed Pseudomonas aeruginosa biofilms. The level of rpoS expression in a 3-day-old biofilm was found to be three-fold higher than the average expression in stationary phase planktonic culture. RpoS levels in biofilms, indicated by immunoblot analysis, were similar to levels in stationary phase planktonic cultures. In planktonic cultures, oxygen limitation did not lead to increased levels of RpoS, suggesting that oxygen limitation was not the environmental signal causing increased expression of rpoS. These results suggest that bacteria within P. aeruginosa biofilms may exhibit stationary phase characteristics even when cultured in flow conditions that continually replenish nutrients.