Influence Of Mobile Phase Composition Research Articles

Optimizing Messenger RNA Analysis Using Ultra-Wide Pore Size Exclusion Chromatography Columns.

Biopharmaceutical products, in particular messenger ribonucleic acid (mRNA), have the potential to dramatically improve the quality of life for patients suffering from respiratory and infectious diseases, rare genetic disorders, and cancer. However, the quality and safety of such products are particularly critical for patients and require close scrutiny. Key product-related impurities, such as fragments and aggregates, among others, can significantly reduce the efficacy of mRNA therapies. In the present work, the possibilities offered by size exclusion chromatography (SEC) for the characterization of mRNA samples were explored using state-of-the-art ultra-wide pore columns with average pore diameters of 1000 and 2500 Å. Our investigation shows that a column with 1000 Å pores proved to be optimal for the analysis of mRNA products, whatever the size between 500 and 5000 nucleotides (nt). We also studied the influence of mobile phase composition and found that the addition of 10 mM magnesium chloride (MgCl2) can be beneficial in improving the resolution and recovery of large size variants for some mRNA samples. We demonstrate that caution should be exercised when increasing column length or decreasing the flow rate. While these adjustments slightly improve resolution, they also lead to an apparent increase in the amount of low-molecular-weight species (LMWS) and monomer peak tailing, which can be attributed to the prolonged residence time inside the column. Finally, our optimal SEC method has been successfully applied to a wide range of mRNA products, ranging from 1000 to 4500 nt in length, as well as mRNA from different suppliers and stressed/unstressed samples.

Altering the mobile phase composition to enhance self-disproportionation of enantiomers in achiral chromatography

The influence of mobile phase composition on the efficiency of enantiomer separation by achiral chromatography (ACh) was investigated. The separation was induced by the phenomenon of self-disproportionation of enantiomers (SDE) triggered by their homo and hetero-chiral interactions in an achiral environment. Typically, SDE occurs in apolar mobile phases of weak elution strength, which causes the separation time to extend and the process productivity to deteriorate. To mitigate that effect, we altered the content of a strong solvent (modifier) in the mobile phase by use of a solvent gradient in which the target enantiomer was separated in the presence of the weak solvent, whereas the unresolved mixture of enantiomers was eluted by increasing the modifier content in the mobile phase. This enabled accelerating the solute elution while preserving the separation selectivity. The approach was examined for the separation of nonracemic mixtures of two structurally different compounds that exhibited the SDE effect in ACh, i.e., metalaxyl (MX) and methyl p-tolyl sulfoxide (MTSO). The target compound of the separation was the more abundant enantiomer in the enantiomeric mixture. The process realization was preceded by the determination of the effect of the modifier content on the separation yield for enantiomeric mixtures of MX and MTSO of different enantiomeric excess (ee). In the case of MX, yield of the pure target enantiomer varied from 2 %, for the maximum concentration of the modifier, to 45 % for the minimum modifier concentration and the largest ee used in the experiments. In the case of MTSO, the yield varied from minimum 40 % to maximum 66 %. To predict the process, we employed a dynamic model, in which underlying thermodynamic dependencies were implemented.

Influence of mobile phase composition on the analytical sensitivity of LC–ESI–MS/MS for the concurrent analysis of bisphenols, parabens, chlorophenols, benzophenones, and alkylphenols

Phenols are significant environmental endocrine disruptors that can have adverse health effects on exposed individuals. Correlating phenol exposure to potential health implications requires the development of a comprehensive and sensitive analytical method capable of analyzing multiple phenols in a single sample preparation and analytical run. Currently, no such method is available for multiple classes of phenols due to electrospray ionization (ESI) limitations in concurrent ionization and lack of sensitivity to certain phenols, particularly alkylphenols. In this study, we investigated the influence of mobile phase compositions in ESI on concurrent ionization and analytical sensitivity of liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) during the analysis of multiple classes of phenols, and we propose a comprehensive and sensitive analytical method for various classes of phenols (i.e., bisphenols, parabens, benzophenones, chlorophenols, and alkylphenols). The proposed method was affected by 0.5 mM ammonium fluoride under methanol conditions. It enabled the concurrent ionization of all the phenols and significantly improved the analytical sensitivity for bisphenols and alkylphenols, which typically have poor ionization efficiency. This method, combined with a “dilute and shoot” approach, allowed us to simultaneously quantify 38 phenols with good chromatographic behavior and sensitivity. Furthermore, the method was successfully applied to the analysis of 61 urine samples collected from aquatic (swimming) and land (indoor volleyball and outdoor football) athletes.

Bisphenol A and its structural analogues in infant formulas available in the Brazilian market: Optimisation of a UPLC-MS/MS method, occurrence, and dietary exposure assessment

For the first time, structural analogues to bisphenol A were investigated in infant formulas marketed in Brazil. A fast and high throughput UPLC-MS/MS method was established for simultaneous analysis of bisphenol A, B, E, F, and S in complex infant formula matrices. The influence of mobile phase composition on electrospray ionization response in negative mode was studied to improve the detectability of the method. As also, the main sample preparation variables that could affect the extraction and cleanup were screened by the Plackett-Burman design. The method performance characteristics were adequate, including reliable limits of detection (5–10 µg kg−1) and quantification (10–20 µg kg−1) with suitable recoveries (84.2–108.9 %) and precision (≤18 %). Sixty-one infant formulas were analyzed, and 36 % of total samples contained at least one bisphenol analogue, whose levels ranged between 10.9 and 198.9 µg kg−1. Based on a deterministic approach, the estimated daily intakes for babies up to 6 months old, fed exclusively with infant formula, were below the temporary tolerable daily intake of 4 µg kg−1 body weight set for bisphenol A by the European Food Safety Authority.

Chiral phenethylamine synergistic tricarboxylic acid modified β-cyclodextrin immobilized on porous silica for enantioseparation

In this work, a series of chiral phenethylamine synergistic tricarboxylic acid modified β-cyclodextrin bonded stationary phase for high performance liquid chromatography (HPLC) were synthesized via a simple one-pot synthesis approach. Various racemates (aryl alcohols, flavanones, triazoles, benzoin, etc.) were well separated on the tricarboxylic acid modified chiral stationary phases in both normal and reversed modes with good reproducibility and stability, and the influence of mobile phase composition on resolution (Rs) were deeply investigated. The RSD values of Rs for repeatability and column-to-column were below 1.28% and 3.05%, respectively. Hence, the fabrication of tricarboxylic acid modified chiral stationary phase (CSPs) is a new efficient strategy to improve the application of β-cyclodextrin as CSPs in the field of chromatography.

Studies on the retention behavior of quercetin, phenol, and caffeine as test substances on selected neutral and charged hydrophilic interaction liquid chromatography stationary phases

AbstractThe influence of mobile phase composition and column temperature on the retention behavior of quercetin, phenol, and caffeine as test analytes was analyzed. The experiments were conducted in columns filled with the charged amine phase and the neutral amide phase. As mobile phases, the varying fractions of methanol or acetonitrile in water solutions were applied. The most likely retention mechanisms have been discussed. The results of investigations confirm the importance of a mixed adsorption‐partition retention mechanism in the hydrophilic interaction liquid chromatography process, regardless of the mobile phase composition and type of stationary phase. Investigations have also shown that in the tested systems the retention mechanism is also strongly column temperature‐dependent. Additionally, the efficiency of the tested columns was determined and analyzed as a function of the linear flow velocity. The obtained literature known as van Deemter plots confirms generally higher column efficiencies in the systems with acetonitrile as an organic component of the mobile phase. However, in some cases, the application of methanol allows obtaining similar column efficiencies. Furthermore, the results show that both columns tested exhibit comparable efficiency in the most tested systems and provide very good efficiency preservation over the wide range of eluent velocities.

Influence of Mobile Phase Composition on Analytical Sensitivity of Lc–Esi–Ms/Ms for the Concurrent Analysis of Bisphenols, Parabens, Chlorophenols, Benzophenones, and Alkylphenols

Influence of Mobile Phase Composition on Analytical Sensitivity of Lc–Esi–Ms/Ms for the Concurrent Analysis of Bisphenols, Parabens, Chlorophenols, Benzophenones, and Alkylphenols

Enantioselective separation of nonsteroidal anti-inflammatory drugs with amylose tris(3-chloro-5-methylphenylcarbamate) stationary phase in HPLC with a focus on enantiomeric quality control in six pharmaceutical formulations containing racemic mixtures or single stereoisomers.

In the present study, an accurate, rapid, and simple chiral HPLC-UV method with amylose tris(3-chloro-5-methylphenylcarbamate) as stationary phase was developed and applied for enantiomeric determination of six nonsteroidal anti-inflammatory drugs (NSAIDs) in the commercial pharmaceutical formulations, including (R,S)-ibuprofen, S-ibuprofen, (R,S)-ketoprofen, S-ketoprofen, S-naproxen, and (R,S)-loxoprofen sodium. Experiments on the influence of mobile phase composition, proportion of organic modifier, percentage of acid additives, and column temperature on enantioseparation were conducted to obtain the best separation condition. It was indicated that one mobile phase simply composed of acetonitrile-water (0.1% formic acid, v/v) at the proportion of 50:50 (v/v) with a flow rate of 0.6ml/min at 22°C could simultaneously provide the excellent enantiomeric resolutions for all selected NSAIDs, which made the enantioseparation process more applicable and operable. The newly developed method was then applied for determination of NSAID enantiomers in pharmaceutical formulations containing racemic mixtures or single stereoisomers. Calibration curve of each enantiomer at the concentration of 5.0-100 ug/ml showed good linearity with the correlation coefficient above 0.9996. Satisfactory recovery (96.54-101.54%), good intra-day precision (RSD 0.52-1.46%), and inter-day precision (RSD 0.13-1.09%) were also obtained. The newly developed method was then applied for determination of NSAID enantiomers in pharmaceutical formulations containing racemic mixtures or single stereoisomers. Quantitative results of the commercial capsules and tablets demonstrated that the difference between the declared and measured values did not exceed 1.52%.

Enantioselective Separation of Nonsteroidal Anti-Inflammatory Drugs with Amylose Tris (3-Chloro-5-Methylphenylcarbamate) Stationary Phase in HPLC with a Focus on Enantiomeric Quality Control in Six Pharmaceutical Formulations Containing Racemic Mixtures or Single Stereoisomers

In the present study, an accurate, rapid and simple chiral HPLC-UV method with amylose tris (3-chloro-5-methylphenylcarbamate) as stationary phase was developed and applied for enantiomeric determination of six nonsteroidal anti-inflammatory drugs (NSAIDs) in the commercial pharmaceutical formulations, including Rac-ibuprofen, S-ibuprofen, Rac-ketoprofen, S-ketoprofen, S-naproxen and Rac-loxoprofen sodium. Experiments on the influence of mobile phase composition, proportion of organic modifier, percentage of acid additives as well as column temperature on enantioseparation were conducted to obtain the best separation condition. It was indicated that one mobile phase simply composed of acetonitrile-water (0.1% formic acid) at the proportion of 50:50 (v/v) with a flow rate of 0.6 mL/min at 22℃ could simultaneously provid the excellent enantiomeric resolutions for all selected NSAIDs, which made the enantioseparation process more applicable and operable. The newly developed method was then applied for determination of NSAID enantiomers in pharmaceutical formulations containing racemic mixtures or single stereoisomers. Calibration curve of each enantiomer at the concentration of 5.0－100 ug/ml showed good linearity with the correlation coefficient above 0.9996. Satisfactory recovery (96.54－101.54%), good intra-day precision (RSD 0.52－1.46%) and inter-day precision (RSD 0.13－1.09%) were also obtained. Quantitative results of the commercial capsules and tablets demonstrated that the difference between the declared and measured values did not exceed 1.52%.

Influence of mobile phase composition and temperature on the retention behavior of selected test substances in diol-type column

In this work the effect of mobile phase composition and temperature on the retention behavior of several test compounds (including non-polar and polar substances) on diol-Acclaim Mixed Mode HILIC-1 column in methanol-water systems with high (RPLC mode) and low (HILIC mode) water content was analyzed. In the studied systems both linear, slightly curved and curved van’t Hoff plots were observed. This indicates, that depending on solute physicochemical properties and mobile phase composition, both single and more complex retention mechanism(s) may influence the retention behavior of tested substances. The possibilities of different retention mechanisms existence (e.g. connected with adsorbent surface heterogeneity, hydrophobic and hydrogen bond interactions) in the analyzed systems have been also discussed.

Synthesis and evaluation of novel chiral stationary phases based on N‑cyclobutylcarbonyl chitosan derivatives

Five halo-containing chitosan 3,6‑bis(phenylcarbamate)‑2‑(cyclobutylformamide) derivatives were synthesized with well-deacetylated chitosan and halo-substituted phenyl isocyanates. The structures of the derivatives were characterized by IR, elemental analysis, and 1H NMR spectrum. After being coated on silica gel, five new CSPs were obtained and their enantioseparation abilities were evaluated, and the enantioseparation abilities were compared with that of commercial CSP of Chiralcel OD-H. The results demonstrated that all the prepared CSPs showed satisfactory enantioseparation ability. The performance of CSP 2 with 3,4‑dichloro substituent on the phenyl group was the best with 11 analytes being baseline separated and 2 analytes being partially separated among 15 tested analytes. Besides, the influence of mobile phase composition, substituent on the phenyl moieties of the chitosan derivatives, as well as structure of analytes on enantioseparation ability was also discussed. In this study, the prepared CSPs exhibited excellent tolerance to “unusual solvents”, and some of them even could work in pure tetrahydrofuran. Therefore, the CSPs bear a universal solvent compatibility in comparison with coated-type CSPs of cellulose/amylose derivatives.

Preparation of a poly(1, 6-hexylene dimethacrylate) conventional size high performance liquid chromatographic monolithic column for separation of small molecules

A porous monolithic column was prepared by in situ free-radical polymerization in a stainless steel column (4.6 mm i.d. × 100 mm). The polymerization reacted using 1,6-hexylene dimethacrylate as single crosslinking monomer, methanol and 1-dodecanol as the binary porogens and 2,2-azobisisobutyronitrile asinitiator. During the column preparation process, the ratio of monomer to porogen was optimized to obtain the best column. The chemical groups and morphology of monolithic column were characterized by the Fourier transform infrared spectroscopy and scanning electron microscopy, respectively. Furthermore, the influence of mobile phase composition and flow rate on the separation of aromatic compounds was investigated.The results showed that the poly(1,6-hexylene dimethacrylate) monolithic column had high mechanical stability at flow rate of 4 mL/min. Phenol, 1-naphthol, biphenyl and phenanthrene were easily separated on the prepared monolithic column using acetonitrile/water (58/42, v/v) as mobile phase at flow rate of 2 mL/min. The relative standard deviation of run-to-run and column-to-column of the retention times for the four standard compounds were <0.3% and 3.3%, respectively. Three kinds of small molecules (phenols, aromatic hydrocarbons, aromatic acids) were successfully separated on the poly(1,6-hexylene dimethacrylate) monolithic column. The possible chromatogramphic retention mechanism on poly(1,6-hexylene dimethacrylate) monolithic column was determined as hydrophobic interaction and dipole-dipole interaction. The conventional size poly(1,6-hexylene dimethacrylate) monolithic column has potential to separate small molecule compounds rapidly by high performance liquid chromatography.

Characterization of Klason lignin samples isolated from beech and aspen using microbore column size-exclusion chromatography.

The present study is focused on the separation and characterization of lignin samples isolated by Klason method from European beech (Fagus sylvatica) broadleaf hardwood and European aspen (Populus tremula) broadleaf softwood by size-exclusion chromatography. The separation was carried out using dimethylformamide as major component of the mobile phase and a 3mm id microbore column packed with hydroxyethyl methacrylate gel, calibrated with polystyrene standards. The influence of mobile phase composition and sample solvent composition on the chromatographic behavior and molar mass distributions was investigated.

Optimization of normal phase chromatographic conditions for lipid analysis and comparison of associated detection techniques

One important challenge in lipid class analysis is to develop a method suitable or, at least adaptable, for a vast diversity of samples. In the current study, an improved normal-phase liquid chromatography (NPLC) method allowed analyzing the lipid classes present in mammalian, vegetable as well as microorganism (yeast and bacteria) lipid samples. The method effectively separated 30 lipid classes or subclasses with a special focus on medium polarity lipids. The separation was carried out with bare silica stationary phase and was coupled to evaporative light scattering detection (ELSD), charged aerosol detection (Corona-CAD®) and mass spectrometry. Solutions are provided to circumvent technical issues (such as pumping solvents of low viscosity, solvent purity, rinsing step). The influence of mobile phase composition and addition of ionic modifiers on the chromatographic behavior of particular lipid classes is documented. A comparison between ELSD and Corona-CAD® confirmed the interest of this later detector for samples with a wide range of concentration of different lipids. Three common atmospheric pressure ionization interfaces were used for coupling the NPLC separation to a LTQ Velos Pro® mass spectrometer. The comparison of the chromatographic profiles showed that atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) are both suitable to detect the different lipid classes whereas APPI allows a better sensitivity for lipids at low-concentration.

Chiral separation of aryloxyphenoxy-propionate herbicides in a permethyl-β-cyclodextrin based column. Influence of temperature and mobile phase composition on enantioselectivity.

We used a permethyl-β-cyclodextrin chiral stationary phase under reversed-phase conditions for the chiral separation of four aryloxyphenoxy-propionate herbicides (fenoxaprop-p-ethyl, quizalofop-p-ethyl and tefuryl, and haloxyfop-p-methyl) with mixtures of methanol, ethanol, 2-propanol, n-propanol, tert-butanol, or acetonitrile and water as mobile phases and investigated the influence of mobile phase composition and column temperature (from 0 to 50°C) on the separation. The retention factors (k) and selectivity factors (α) of all the herbicides investigated decreased with increasing temperature. The lnα versus 1/T and lnk versus 1/T plots for the enantiomers of the chiral pesticides were linear within the range of 0-50°C with all alcohol/water mixtures constituting the mobile phase, but the lnk versus 1/T plots were nonlinear for all the enantiomers chromatographed in acetonitrile/water mixtures. The thermodynamic parameters based on linear van't Hoff plots were calculated. The influence of temperature and mobile phase composition on the enantioseparation of the solutes has rarely been considered simultaneously. The temperature and the solvents used in the mobile phase, however, were found to have a profound effect on the enantioseparation of these herbicides.

Research Article. The Influence of Some Parameters on Chiral Separation of Ibuprofen by High-Performance Liquid Chromatography and Capillary Electrophoresis

Abstract Objective: The aim of the study was to compare the influence of mobile phase composition and temperature on chiral separation of racemic ibuprofen by capillary electrophoresis and high performance liquid chromatography with UV detection. Materials and methods: Racemic ibuprofen was analysed on a chiral OVM column with an HPLC system 1100 Agilent Technologies, under isocratic elution, by using potassium dihydrogen phosphate 20 mM and ethanol in mobile phase. The flow rate was set at 1 mL/min, UV detector at 220 nm and different column temperatures were tested. For electrophoresis separation an Agilent CE G1600AX Capillary Electrophoresis System system, with UV detection, was used. The electrophoresis analysis was performed at different pH values and temperatures, with phosphate buffer 25 mM and methyl-β-cyclodextrin as chiral selector. Results: The chromatograhic analysis reveals a high influence of mobile phase pH on ibuprofen enantiomers separation. An elution with a mixture of potassium dihydrogen phosphate 20 mM pH=3 and ethanol, at 25°C, allowed enantiomers separation with good resolution in less than 8 min. Conclusions: The proposed HPLC method proved suitable for the separation of ibuprofen enantiomers with a good resolution, but the capillary electrophoresis tested parameters did not allow chiral discrimination.

Simultaneous Determination of Nonylphenol and Nonylphenol Ethoxylates in Wastewater Samples from Biodegradation Process by High Performance Liquid Chromatography Method

The degradation products of nonylphenol ethoxylates (NPnEO), nonylphenol (NP) and short-chain NPnEO are representative of environmental endocrine disruptors. They possess strong lipophilicity, toxicity, cumulative property and estrogenic effect. They can pollute the environment, cause body precocious, and induce the body's estrogen-sensitive cancer cell proliferation. A fast method using high performance liquid chromatography (HPLC) was developed to simultaneously quantify NP and 11 kinds of NPnEO in wastewater samples. The influence of mobile phase composition, mobile phase ratio, mobile phase flow rate, column temperature and sample injection volume on the separation effect was studied. Under the optimized conditions, NP and 11 kinds of NPnEO were separated successfully within 35 min. The method showed good linearity for NP and 11 kinds of NPnEO (n = 1-11). The linear correlation coefficients for the standard curves were 0.9720- 0.9999. The precision degree of the method was reliable and all the relative standard deviation (RSD) values (n = 0-11) obtained were less than 5.0%.

Development and Validation of Chiral HPLC Method for Quantification of 3-(S)-Quinuclidinol in Pharmaceutically Important Precursor, 3-(R)-Quinuclidinol, by Precolumn Derivatization.

A sensitive and specific high-performance liquid chromatography (HPLC) method for the separation and quantification of 3-(S)-quinuclidinol in 3-(R)-quinuclidinol, precursor for active pharmaceutical ingredients (solifenacin, revatropate and talsaclidine), has been developed and validated by precolumn derivatization. Several chiral columns were tested in a normal phase system. Excellent enantioseparation with the resolution more than 11.4 was achieved on Chiralpak IC column using isocratic mobile phase consisting of n-hexane, ethanol, 2-propanol and diethylamine (80:8:12:0.4, v/v). The detection was carried out using UV detector at 230 nm. The influence of mobile phase composition, namely organic modifiers, additives, aliphatic alkanes and water content in mobile phase, on retention and enantioseparation was studied. Validation of the developed method including specificity, system suitability, limit of detection, limit of quantification, linearity, repeatability, intermediate precision, accuracy and solution stability was performed according to the International Conference on Harmonization guidelines. The advantage of the method is a good HPLC enantioseparation by precolumn derivatization using reaction mixture as sample, simpler UV detection, short analysis time (<30 min), and therefore this method is suitable option for routine quantification of 3-(S)-quinuclidinol in 3-(R)-quinuclidinol.

Effect of Chromatographic Conditions on Enantioseparation of Bedaquiline Using Polysaccharide-based Chiral Stationary Phases in RP-HPLC.

A sensitive and specific high-performance liquid chromatography method for the separation and determination of bedaquiline stereoisomers has been developed and validated. Ten different chiral columns were tested in a reversed-phase system. Excellent enantioseparation with a resolution of all isomers >2.0 was achieved for all stereoisomers on a Chiralcel OJ-3R column, using a mixture of 10 mM buffer of triethylamine/phosphoric acid pH 7.0 and acetonitrile (40 : 60; v/v). Bedaquiline (BDQ) stereoisomers were detected using UV-vis detector at a wavelength of 227 nm. The influence of mobile phase composition, namely buffer type, mobile phase pH and acetonitrile content in mobile phase, on retention and enantioseparation was studied. Validation of the developed method including linearity, limit of detection, limit of quantification, precision, accuracy and selectivity was performed according to the International Conference on Harmonisation guidelines. The advantages of the method are good enantioseparation and excellent selectivity; therefore, this is suitable for routine determination of chiral purity of (1R,2S)-BDQ in enantiopure active pharmaceutical ingredient.

Studies on the sorption behavior of quercetin, phenol, and caffeine as test substances on diol mixed-mode HILIC column

ABSTRACTThe influence of mobile phase composition and column temperature on the retention behavior of quercetin (Q), phenol (PH), and caffeine (CAF) as test analytes on diol- Acclaim Mixed Mode HILIC-1 column in mobile phases containing varying fractions of methanol in water and acetonitrile in water was analyzed. Brief analysis of the column efficiency was also presented. Using the frontal analysis (FA) method the adsorption studies at the different mobile phase compositions were undertaken and the adsorption energy distributions (AED) have been calculated prior to selection of proper adsorption model. The calculations revealed heterogeneous interactions for Q and PH in the analyzed systems while homogeneous for CAF in systems with low water content in eluent and heterogeneous in systems with high water content. The Langmuir and bi-Langmuir isotherm turned out to be excellent in description of these adsorption trends.