The Impact of Water as an Additive on the Elution of Some Basic Organic Compounds in Supercritical Fluid Chromatography.

In this study, water was used as an additive in the methanol-modified carbon dioxide-based eluent for the elution of some basic organic compounds from a hybrid silica column via supercritical fluid chromatography (SFC). The experiments were applied to sulfonamides, propranolol, and other organic nitrogen compounds involving aromatic rings from different classes of amine, pyrimidine, and purine with different pKa values (the pKa values for the studied analytes range from 4.6 to 10.4). The results revealed different responses to the different percentages of water addition. Adding 1~2% of water to the modifier (methanol) led to a positive effect manifested by more symmetrical peak shapes and reduced retention times for most compounds. The key factor for this improvement in the properties of chromatographic peaks is due to the adsorption of water on the silanol groups of the stationary phase, consequently resembling the phenomena observed in hydrophilic interaction liquid chromatography (HILIC). Moreover, the availability of hydrogen bond acceptor and donor sites in the analyte structure is an important factor to be considered when adding water as an additive to the modifier for improving the chromatographic peaks. However, introducing water in an amount higher than 3% resulted in perturbed chromatographic signals. It was also found that water as an additive alone could not successfully elute propranolol from the hybrid silica column with an acceptable peak shape; thus, the addition of a strong base such as amine salts was also necessary. The proposed use of a particular amount of water in the mobile phase could have a positive effect compared to the same mobile phase without water, improving the chromatographic peak properties of the elution of some basic organic compounds from the hybrid silica column.

Exploring the application limits of different hold-up time markers in supercritical fluid chromatography.

The study focuses on the application range of nitrous oxide as a hold-up time marker in supercritical fluid chromatography (SFC). This compound has been suggested a decade ago to be used as unretained marker, something that the field of SFC was missing for a long time, since its beneficial properties make it an ideal candidate as hold-up time marker. Determination of the hold-up volume and actual volumetric flow rates have always been problematic in SFC due to the compressibility of carbon dioxide and one part of this is the difficulty of hold-up time measurements. Depending on the mobile phase, different methods have been used to measure the hold-up time with varying results. Nitrous oxide and other molecules have been compared in different conditions, mobile phases and stationary phases. In all cases, nitrous oxide gave the lowest elution times. However, detection was difficult in mobile phases containing 10% or more of organic modifier, because most solvents mask the signal of nitrous oxide. Interestingly, the choice of stationary phase also had a slight effect on detection, while different pressure and temperature settings affected each compound in a different manner.

Retention mechanism on phosphodiester stationary phases in hydrophilic interaction liquid chromatography and purely aqueous mobile phase part II: Overloading with limited soluble samples.

The adsorption behaviour of caffeine and theophylline under hydrophilic interaction chromatography and purely aqueous conditions was investigated on four phosphodiester stationary phases. Solute adsorption isotherms were determined by frontal analysis or inverse method. The bi-Langmuir model was found to be the best choice to describe the behaviour of caffeine and theophylline adsorption in purely aqueous conditions, whereas the bi-Moreau model describes the adsorption phenomena in HILIC conditions. The results obtained demonstrate that the interaction of caffeine and theophylline with the stationary phase surface varies depending on the mobile phase composition. Both in pure aqueous mobile phase and in HILIC mode, the heterogeneity of the surface of the studied stationary phases is confirmed. In hydrophilic solutions, the sample molecules interact with the stationary phase only. In hydrophobic conditions, a lateral interaction occurs between caffeine or theophylline molecules, which are poorly soluble in acetonitrile-rich solvents. This confirms that the same compound on the same stationary phase can behave rather differently, depending on the mobile phase composition. Thus, the mobile phase may govern and control the retention mechanism.

Correctness of the van 't Hoff analysis for homogeneous or heterogeneous retention.

It is already known that the physical meaning of the numerical values of the calculated enthalpy (ΔH) and entropy (ΔS) change in chromatography via the van 't Hoff plot analysis is rather questionable. In the former work of these authors, it has been demonstrated that by serially coupling two reversed-phase columns of the same inner diameter but of different retention mechanism, the obtained thermodynamic values are not related to the numerical results obtained on the individual columns respectively. Since surface heterogeneity of the stationary phase is intrinsically present in chiral chromatography (enantioselective and non-selective sites), the calculation of ΔH and ΔS should be revisited in that field. In this study, more details of the pressure dependence were investigated. Using special POPLC columns, the effect of the column length on the calculated thermodynamic values was investigated. The calculated values differ by 30-400% when the column length is increased from 4 cm to 12 cm. The dependence of the calculated results on the applied flow rate was already highlighted earlier, and here the emphasis was put on the instrument on which the separation was performed. It turns out that there is a difference when using a Shimadzu HPLC system compared to using a Waters Acquity UPLC system. Because the Eyring method serves as an alternative route to calculate the thermodynamic values from chromatographic data, the differences obtained by the two methods were also investigated.

The impact of placement, experimental conditions, and injections on mass flow measurements in supercritical fluid chromatography.

In supercritical fluid chromatography (SFC), the variation of pressure, temperature and volumetric flow-rate is most noticeable when the mobile phase contains only neat carbon dioxide. This can be explained by the compressibility of CO2 and introduces several difficulties to the work of chromatographers. The only flow parameter that is considered to be constant across the SFC system is the mass flow-rate. It has been shown that the Coriolis flow meter (CFM) provides different types of information depending on its placement in the instrument. Therefore, the goal of this paper is to investigate several factors affecting the variation of mass flow-rate in SFC, including four different configurations around the column, four sets of experimental conditions along with two columns and a zero-volume union. The effect of disturbances introduced by injections are studied as well. The results show different mass flow-rates when taken at the inlet or the outlet of the column. In addition, different columns produced different tendencies of variations. Study of the injections showed that the initial severe drop of mass flow is reduced when the averages are taken until the elution times of the chosen compounds. Additional testing related to possible leaks and CFM calibration showed that even if all standard operating procedures are strictly followed, reproducibility of the mass-flow rate can still be an issue.

The adsorption of methanol on reversed phase stationary phases in supercritical fluid chromatography.

The surface excess isotherms of methanol from carbon dioxide on reversed phase stationary phases under two different operational conditions - which can be considered subcritical and supercritical conditions depending on the molar fraction of CO2 in methanol - were determined using the minor disturbance peak method. The shapes of the surface excess isotherms were very similar in subcritical and supercritical conditions for the same column. To verify the influence of the sample solvent on the separation efficiency, two solvents methanol and heptane were used as sample solvents for alkylbenzene samples for the separation on the studied columns with pure carbon dioxide mobile phase. The separation efficiency was determined by calculating the number of theoretical plates. On the embedded amide stationary phase with methanol as a sample solvent the efficiency has increased due to the displacement effect of methanol on the solutes which are retained less than methanol. Then the efficiency for the rest of solutes, which coincide with the elution of the methanol peak tail has decreased as a result of the tag-along effect. The surface adsorbent heterogeneity has been discussed; the bonded ligands on the stationary phase surface demonstrated adsorption a big amount of CO2, while methanol could adsorb with small amount on the residual silanols on the surface of stationary phase and the embedded (amide) polar group in the bonded phase.

Separation of enantiomers of chiral basic drugs with amylose- and cellulose- phenylcarbamate-based chiral columns in acetonitrile and aqueous-acetonitrile in high-performance liquid chromatography with a focus on substituent electron-donor and electron-acceptor effects.

In this study, amylose- and cellulose-phenylcarbamate-based chiral columns with different chiral-selector (CS) chemistries were compared to each other for the separation of enantiomers of basic chiral analytes in acetonitrile and aqueous-acetonitrile mobile phases in HPLC. For two chemistries the amylose-based columns with coated and immobilized CSs were also compared. The comparison of CSs containing only electron-donating or electron-withdrawing substituents with those containing both electron-donating and electron-withdrawing substituents showed opposite results for the studied set of chiral analytes in the case of amylose and cellulose derivatives. Along with the chemistry of CS the focus was on the behavior of polysaccharide phenylcarbamates in acetonitrile versus aqueous acetonitrile as eluents. In agreement with earlier results, it was found that in contrast to the commonly accepted view, polysaccharide phenylcarbamates do not behave as typical reversed-phase materials for basic analytes either. In the range of water content in the mobile phase of up to 20-30% v/v the behavior of these CSs is similar to hydrophilic interaction liquid chromatography (HILIC)-type adsorbents. This means that with increasing water content in the mobile phase up to 20-30% v/v, the retention of analytes mostly decreases. The important finding of this study is that the separation efficiency improves for most analytes when switching from pure acetonitrile to aqueous acetonitrile. Therefore, in spite of reduced retention, the separation of enantiomers improves and thus, the HILIC-range of mobile phase composition, offering shorter analysis time and better peak resolution, is advantageous over pure polar-organic solvent mode. Interesting examples of enantiomer elution order (EEO) reversal were observed for some analytes based on the content of water in the mobile phase on Lux Cellulose-1 and Lux Amylose-2 columns.

The correctness of van 't Hoff plots in chiral and achiral chromatography.

van 't Hoff plots (logarithm of the retention factor, ln k, vs. the reciprocal of absolute temperature, 1/T) are commonly used in chromatographic studies to characterize the retention mechanisms based on the determined enthalpy (ΔH∘) and entropy (ΔS∘) change of analyte adsorption. In reversed phase liquid chromatography, the thermodynamic parameters could help to understand the retention mechanism. In chiral chromatography, however, the conclusions drawn based on van 't Hoff plots can be deceptive because several different types of adsorption sites are present on the surface of stationary phase. The influence of heterogeneity, however, cannot be studied experimentally. In this study, we employed two reversed phase columns with different retention mechanisms to show that by serially coupling the columns, the obtained thermodynamic parameters are not related to the results obtained on the respective individual columns. Furthermore, our results show that the experimental conditions - such as flow-rate or choice of instrument - will strongly influence the calculated enthalpy and entropy values.

Modeling the competitive adsorption of sample solvent and solute in supercritical fluid chromatography.

Uncommon retention behavior of a series of n-alkylbenzene homologues as well as the effect of different sample solvents on chromatographic efficiency were studied in supercritical fluid chromatography. After testing various columns, an alkylamide stationary phase was selected for detailed studies. The results showed that even a small amount of methanol originating only from the sample, overloaded the column and competitive adsorption was induced between the analytes and the sample solvent for adsorption on the stationary phase. This was indicated by the changes in column efficiency, retention and peak widths. The concentration of the analytes in the sample was negligible compared to the amount of methanol - but their adsorption was influenced by the solvent - while the adsorption of methanol remained unaffected by the n-alkylbenzenes. First, the competition was described by determining the single-component adsorption isotherms for both the analytes and their solvent, then competitive isotherms were calculated. Based on the peak profiles, bi-Langmuir and competitive bi-Langmuir isotherms were assumed. The solvent effect was modeled by a numerical method created in-house where the differential mass balance equation given by the equilibrium-dispersive (ED) model was integrated using the Rouchon algorithm. The experimental observations were confirmed by in silico experiments and additional cases involving two hypothetical analytes were studied as well.

The effect of column packing procedure on column end efficiency and on bed heterogeneity - Experiments with flow-reversal.

The effect of column end structure and bed heterogeneity of six commercially available reversed-phase chromatographic columns for fast liquid chromatography with different column packing materials - such as fully porous (Waters XBridge C18 with 1.7 µm particles) and superficially porous (Waters CORTECS C18 with 1.6 µm particles), with column dimension of 2.1 × 50, 100 or 150 mm were tested with flow-reversal method. The method includes arresting the flow when a non-retained marker (thiourea) has penetrated to a given distance into the column and then reversing the column. Hence, when the flow has been restarted, the sample is eluted at the same end of the column where it entered. The experiments showed that all columns are axially heterogeneous, and some differences could be observed between the two respective column ends. Furthermore, we can conclude that the shorter columns are axially more homogeneous than the longer ones, thus the column length is an influencing factor on the column packing procedure.

The use of alteration analysis in supercritical fluid chromatography to monitor changes in a series of chromatograms.

Two-dimensional correlation analysis (2DCOR) is a unique chemometric method introduced in spectroscopy which became successful in a wide range of analytical fields. It was applied in chromatography as well but has not gained wide-spread popularity. In our previous work, we introduced an alternative method, Alteration Analysis (ALA), which is built upon the basic properties of 2DCOR, but it is fine-tuned for chromatographic applications and can be used on higher dimensional data sets as well. We explored its merits through computer generated examples. In this study, we present the application of ALA to two various data-sets in chromatography. First, we used a series of samples where the concentrations of the compounds were adjusted according to the changes we studied in our previous in-silico experiments. We compared the alteration maps from the computer generated and measured sources. The results demonstrated that ALA can provide the same properties from measured data as laid down in theory. The second one is a test concerning the effect of sample solvent composition in supercritical fluid chromatography (SFC). ALA maps show the influence of increasing methanol concentration on the peak location and shape of compounds in the chromatogram. With these two examples, we demonstrate that ALA can be used not only in theory, but it has also practical potentials and importance.

Subantimicrobial Dose Doxycycline Worsens Chronic Arthritis-Induced Bone Microarchitectural Alterations in a Mouse Model: Role of Matrix Metalloproteinases?

Background: Rheumatoid arthritis (RA) is a chronic inflammatory joint disease hallmarked by irreversible damage of cartilage and bone. Matrix metalloproteinases (MMPs) involved in connective tissue remodeling play an important role in this process. Numerous MMPs have been examined in humans and animals, but their functions are still not fully understood. Therefore, we investigated the role of MMPs in the K/BxN serum-transfer model of RA with the broad-spectrum MMP inhibitor subantimicrobial dose doxycycline (SDD) using complex in vivo and in vitro methodolgy. Methods: Chronic arthritis was induced by repetitive i.p. injections of K/BxN serum in C57BL/6J mice. SDD was administered daily in acidified drinking water (0.5 mg/mL, 80 mg/kg) during the 30 days experimental period. Mechanonociceptive threshold of the paw was evaluated by aesthesiometry, grasping ability by grid test, arthritis severity by scoring, neutrophil myeloperoxidase activity by luminescence, vascular hyperpermeability and MMP activity by fluorescence in vivo imaging and the latter also by gelatin zymography, bone structure by micro-computed tomography (micro-CT). Plasma concentrations of doxycycline were determined by liquid chromatography-mass spectrometry analysis. Results: K/BxN serum induced significant inflammatory signs, mechanical hyperalgesia, joint function impairment, increased myeloperoxidase activity and vascular hyperpermeability. Significant increase of MMP activity was also observed both in vivo and ex vivo with elevation of the 57-60, 75, and 92 kDa gelatinolytic isoforms in the arthritic ankle joints, but neither MMP activity nor any above described functional parameters were influenced by SDD. Most importantly, SDD significantly reduced bone mineral density in the distal tibia and enhanced the Euler number in the ankle. Arthritis-induced microarchitectural alterations demonstrating increased irregularity and cancellous bone remodeling, such as increased Euler number was significantly elevated by SDD in both regions. Conclusion: We showed increase of various MMP activities in the joints by in vivo fluorescence imaging together with ex vivo zymography, and investigated their functional significance using the broad-spectrum MMP inhibitor SDD in the translational RA model. This is the first demonstration that SDD worsens arthritis-induced bone microarchitectural alterations, but it appears to be independent of MMP inhibition.

Characterization of radial and axial heterogeneities of chromatographic columns by flow reversal.

The impact of the column length (5, 10, and 15 cm) and packing mode (constant pressure and constant flow rate up to 15,000 psi) on the radial and axial heterogeneities of 3.0 mm i.d. research prototype columns packed with the same batch of 2.4 µm BEH-C18 particles was investigated by the flow reversal technique. The data were gathered for a non-retained marker (uracil, acetonitrile/water eluent mixture, 80/20, v/v, flow rate 0.5 mL/min, T = 297 K) and revealed that the radial heterogeneity of the packed bed, characterized by the center-to-wall relative velocity bias (ωß) and its length scale, is nearly independent on the packing mode: the velocity biases extend over a same length scale estimated at 154 µm while ωß is in between 4% and 6% for all columns. Secondly, the data revealed that the column length has a slight impact on ωß: assuming a two-region (wall and center regions) stochastic model of transcolumn eddy dispersion, ωß increases from 4.6% to 5.1% and to 6.1% for 5, 10, and 15 cm long columns, respectively, when packed at constant flow rate. For columns packed at constant pressure, ωß increases from 5.0% to 5.2% and to 5.6%, respectively. Finally, it is found that all columns are axially heterogeneous: the bottom half, which is packed first (column inlet), is slightly more uniform than the top half (column outlet) which is packed last. Overall, the results of the flow reversal experiments corroborate recent observations (130 µm thick wall region and ωß = 5.0%) based on flow simulations in a focused-ion-beam scanning electron microscopy (FIB-SEM) based 3D reconstruction from a 2.1 mm × 50 mm column packed with 2 µm BEH-C18 particles.

The effect of the frictional heat on retention and efficiency in thermostated or insulated chromatographic columns packed with sub-2-µm particles.

The mass-transfer properties of a core-shell packing material with 1.6 µm particle diameter, and that of a fully porous packing material with 1.7 µm particle diameter were investigated and compared. The first absolute and the second central moments of the peaks of the homologous series of alkylbenzenes, over a wide range of mobile phase velocities were measured and used for the calculation of the mass-transfer coefficients. For the evaluation of the band broadening caused by the thermal dissimilarities, the measurements were carried out under thermostated conditions and also at near adiabatic insulation of the columns.

Rate constant determination of interconverting enantiomers by chiral chromatography using a stochastic model.

It may happen under the conditions employed that enantiomers interconvert to each other. In this case, obviously, the kinetics of the process is to be examined. When enantiomers dynamically interconvert to each other during the separation process, a plateau is observed between the adjacent peaks (so-called Batman peak appears). The peak shape depends on the rate constant of this dynamic reaction. A novel stochastic model was derived which takes both the separation and the interconversion into account at the molecular level - thus the effects of the parameters affecting the separation can be investigated. The novel model was used for the study of quetiapine, a drug molecule that interconverts during the separation to evaluate the rate constant based on the enantiomerization. Various flow rates and temperatures were used, and good agreement was obtained with the rate constant obtained from optical rotation experiments and with the software written by Trapp [1]. The most important result we concluded is the need of mild conditions during the separation to ascertain the rate constant the most accurately (low flow rates and temperatures where the enantiomerization process is limited to a few interconversions). The comparison of the rate constants of the on-column and the off-column experiments should be done by considering the stationary phase effects that are absent in the off-column experiments.

Correction to: Use of non-living lyophilized Phanerochaete chrysosporium cultivated in various media for phenol removal.

In the original publication of this paper, the Acknowledgements section is missing the statement below.

Use of non-living lyophilized Phanerochaete chrysosporium cultivated in various media for phenol removal.

The biosorption of phenol on non-living lyophilized mycelial pellets of Phanerochaete chrysosporium cultivated in liquid medium of various compositions was studied in batch biosorption system. The fungal cell surfaces were characterized by FTIR spectroscopy and specific surface charge determination. The sorption kinetics and equilibrium were evaluated using linear and non-linear regression. For adsorption equilibrium, a comparative evaluation is also presented using non-linear least-square estimation and linearization of the Langmuir and anti-Langmuir equations. The presence of mineral and vitamin materials in the liquid medium enhanced the adsorption capacity of fungal biomass for phenol. At optimum pH 5-6, the values of specific surface charge were 0.023 and 0.069 meq g-1 for various cultivations, and the maximum amounts of phenol can be adsorbed at these pH values. The maximum adsorbed phenol amounts by cells cultivated in simple and complex media were 4.53 and 13.48 mg g-1, respectively, at an initial phenol concentration of 100 mg l-1. Graphical abstract ᅟ.

Influence of pressure on the retention of resorcinarene-based cavitands.

The thermodynamics of the retention mechanism of resorcinarene-based cavitands in RPLC as well as the nature of the binding sites have been studied recently. In the present study, the influence of pressure on the retention of the cyclic tetramers on alkylsilyl and polar-embedded C8 and C18 stationary phases is investigated using aqueous methanol mobile phase. The pressure effect for cavity-shaped molecules has been scarcely studied so far. We observed that the retention factors of the analytes increased with the increase of the average column pressure (1-400 bar) when using restricting capillary tubes. The calculated molar volume changes were negative, between -ΔVm = 5-19 mL/mol on all types of stationary phases. Comparing the different stationary phases, we found that the molar volume changes for both the apolar and more polar analytes were twice larger on the Hypersil BDS (base deactivated silica) than on the XTerra columns and they were independent of the length of the alkyl chains of the stationary phases.

Multilayer adsorption in liquid chromatography - The surface heterogeneity below an adsorbed multilayer.

A numerical method was introduced for the estimation of the surface heterogeneity below an adsorbed multilayer of the analyte. The calculation procedure is based on the raw adsorption isotherm data points obtained by frontal analysis experiments. To permit the mapping of the nature of the analyte-surface interaction, a numerical procedure was used to pre-estimate the adsorbate-adsorbate interactions occurring during the adsorption process. The surface heterogeneity estimation was carried out using the affinity-energy distribution calculations with assuming local BET isotherm. In the local BET isotherm the pre-estimated adsorbate-adsorbate interaction constant was used, and the surface heterogeneity was described. After the test of the numerical method with benchmark isotherms, the algorithm was tested on several experimental isotherms. The isotherms were measured using phenol as test molecule on reversed phase adsorbents, with different surface coverage of the octadecyl ligands. The surface of the non-end-capped stationary phases showed detectable heterogeneity, while the surface end-capped phases were found to be homogeneous.

Investigation of the temperature dependence of water adsorption on silica-based stationary phases in hydrophilic interaction liquid chromatography.

In the present work, the adsorption of water was investigated in aqueous normal-phase liquid chromatography on Cogent Silica C and Cogent Phenyl hydride stationary phases at different temperatures by frontal analysis - using coulometric Karl Fischer titration - to compare the temperature dependence of adsorption of water from aqueous acetonitrile. The Cogent Silica-C and Cogent Phenyl Hydride columns have a silicon hydride surface (silica hydride) with less than 2% free silanol group; therefore, they do not have a strong association with water. The adsorption behavior of water on the mentioned stationary phases was modeled by Langmuir isotherm. The preferentially adsorbed water was expressed in terms of a hypothetical monomolecular water layer equivalent in the inner pores. The uptake of water slightly depends on the temperature. The adsorbed water may fill four to eight percent of the pore volume over the studied temperature range, which approximately corresponds to the equivalent of 0.24-0.68 water layer coverage of the adsorbent surface. The phenyl hydride stationary phase shows decreased water uptake in comparison to the Silica C stationary phase.