Properties Of Mobile Phase Research Articles

Tuning mobile phase properties to improve empty and full particle separation in adeno-associated virus productions by anion exchange chromatography.

In the past decade, recombinant adeno-associated virus (rAAV) has gained increased attention as a prominent gene therapy technology to treat monogenetic diseases. One of the challenges in rAAV production is the enrichment of full rAAV particles containing the gene of interest (GOI) payload. By adjusting the mobile phase properties of anion-exchange chromatography (AEX), it was demonstrated that empty and full separation of rAAV was improved in monolith based preparative AEX chromatography. When compared to the baseline method using NaCl, the use of tetraethylammonium acetate (TEA-Ac) in the AEX mobile phase resulted in enhanced resolution from 0.75 to 1.23 between "Empty" and "Full" peaks by salt linear gradient elution, as well as increased the percentage of full rAAV particles from 20% to 36% and genome recovery from 59% to 62%. Furthermore, a dual wash plus step elution AEX method was developed. Wherein, the first wash step harnesses TEA-Ac to separate empty and full capsids, which is followed by a second wash step that ensures no TEA-Ac salt is carried over into AEX eluate. The resulting optimized AEX purification method has the potential to be adapted for manufacturing and purification processes involving various rAAV production platforms that experience empty and full rAAV separation challenges.

Green method development approach of superheated water liquid chromatography for separation and trace determination of non-steroidal anti-inflammatory compounds in pharmaceutical and water samples and their extraction

Non-steroidal anti-inflammatory drugs (NSAIDs) are pharmaceutical compounds with anti-inflammatory, analgesic, and antipyretic effects. Herein, a simple and rapid high-temperature liquid chromatography and superheated water chromatography method was developed and validated for the trace determination of NSAID residues of ketoprofen, naproxen, sodium diclofenac, and ibuprofen in water samples. The NSAIDs were separated in less than five minutes using buffered distilled water as the mobile phase and ODS Zirconia RP-C18 column as the stationary phase. Linearity was observed in the van’t Hoff plots of the tested drugs by employing a low acetonitrile percentage (20% ACN) in the mobile phase, without any significant changes in their retention mechanisms. However, nonlinear van’t Hoff plots were obtained for the superheated water chromatography data of the tested drugs because of significant changes in their retention factors, transition stage of the stationary phase, or the mobile phase properties. The limits of detection for ketoprofen, naproxen, sodium diclofenac, and ibuprofen were 14, 2, 4.2, and 32 µg L −1 , respectively, and their limits of quantification were 44, 8, 12, and 98 µg L −1 , respectively. The accuracy and precision parameters were determined for selected drugs, where the relative standard deviations were in the range of ± 0.2179–2.6741%. In addition, these conditions were employed for the removal of NSAIDs from the water samples using carbon nanotubes. The proposed system was applied for the separation and analyses of drugs in water and pharmaceutical samples, and acceptable recoveries of 90.48–98.15% for the water samples and 99.9–100.08% for the pharmaceutical samples were obtained.

Unified chromatography – Mass spectrometry as a versatile tool for determination of food dyes

Unified chromatography with mass spectrometric detection was assessed for determination of food dyes. Nineteen substances representing azo, triphenylmethane, xanthone, indigoid, quinoline and polyene classes covering an unprecedented range from nonpolar β-Carotene (logD 13.6) to ionic Tartrazine (logD −7.5) were analyzed simultaneously. The dyes were separated in a single experimental run by an 18-min gradient elution from 98% CO2 to 100% aqueous-methanolic modifier on a diol column. Isomeric substances were resolved, and Isatin Sulfonic acid was detected as a degradation product of Indigo Carmine. Mobile phase properties reproducibly changed from supercritical to liquid state ensuring stable retention times (inter-day RSD<0.5%). Quantitative analysis of sports drinks after straightforward 10- or 25-fold dilution with dimethyl sulfoxide confirmed the method applicability to real-life samples. Sufficient limits of detection (typically 0.025 mg L−1 in processed samples, equivalent to 0.25 mg L−1 in drink) and a wide linear range (typically 0.5–50 mg L−1 or 1.3–125 mg L−1 in drink for 10× or 25× dilution, respectively) were demonstrated during validation. A comparison of method performance with competitive liquid chromatography procedures is also provided. Unified chromatography is a promising tool for comprehensive multiclass analysis of dyes in the context of food safety.

Simultaneous separation of insulin and six therapeutic analogues on a mixed mode column: HPLC-UV method development and application

Human insulin and six most used therapeutic analogues are very similar in terms of retention on a reversed-phase column. Thus, the LC methods prescribed in the European Pharmacopoeia monographs for insulin and insulin analogues include many similar separation methods, which tend to be time consuming when separating individual products of insulins or are inadequate when handling a mixture. In this study, we present a simple, robust, versatile and accessible HPLC-UV separation method for identification and quantification of human insulin and its analogues in one run. The simultaneous separation and detection is possible by fine-tuning the mobile phase properties that affect the separation mechanism on a mixed mode column combining anion exchange and reversed-phase characteristics. Also developed was a simple and effective SPE sample cleaning procedure with insulin recoveries ranging from 80 to 100% for all analogues. On the other hand, the concentration of major excipients such as phenol and m-cresol fall below 1%. The two developed and validated separation methods differ in their compatibility with the use of a quaternary or binary pump, thus enabling sample characterisation independent of the HPLC solvent delivery system. The methods are compatible with the use of a mass spectrometric detector for an indisputable identification.

Cross-scale quality assessment of a mechanistic cation exchange chromatography model.

Cation exchange chromatography (CEX) is an essential part of most monoclonal antibody (mAb) purification platforms. Process characterization and root cause investigation of chromatographic unit operations are performed using scale down models (SDM). SDM chromatography columns typically have the identical bed height as the respective manufacturing-scale, but a significantly reduced inner diameter. While SDMs enable process development demanding less material and time, their comparability to manufacturing-scale can be affected by variability in feed composition, mobile phase and resin properties, or dispersion effects depending on the chromatography system at hand. Mechanistic models can help to close gaps between scales and reduce experimental efforts compared to experimental SDM applications. In this study, a multicomponent steric mass-action (SMA) adsorption model was applied to the scale-up of a CEX polishing step. Based on chromatograms and elution pool data ranging from laboratory- to manufacturing-scale, the proposed modeling workflow enabled early identification of differences between scales, for example, system dispersion effects or ionic capacity variability. A multistage model qualification approach was introduced to measure the model quality and to understand the model's limitations across scales. The experimental SDM and the in silico model were qualified against large-scale data using the identical state of the art equivalence testing procedure. The mechanistic chromatography model avoided limitations of the SDM by capturing effects of bed height, loading density, feed composition, and mobile phase properties. The results demonstrate the applicability of mechanistic chromatography models as a possible alternative to conventional SDM approaches.

Determination of physicochemical properties of small molecules by reversed-phase liquid chromatography.

The physicochemical properties of small molecules that can be determined by retention measurements in reversed-phase liquid chromatography include solvent-based properties inferred from equilibrium processes occurring predominantly in the mobile phase and sorption properties for materials which can be used as stationary phases inferred from solute-stationary phase interactions. In addition, physicochemical properties can be estimated from correlation models based on surrogate chromatographic systems with a similar capability for intermolecular interactions to the chemical or biological system. Examples of properties determined by direct methods include molecular descriptors (solvation parameter model), acid dissociation constants, formation constants, and surface properties of solids determined by inverse liquid chromatography. Examples of properties estimated by indirect methods include hydrophobicity, lipophilicity, n-octanol-water partition constant, soil-water sorption constant, non-specific toxicity to fish and microorganisms, and permeation coefficients for the blood-brain and skin-water barriers. Since all approaches depend on an accurate measurement of chromatographic retention parameters typical operational and mechanistic problems are discussed from the perspective of data quality. Fundamentally the accuracy of direct methods is limited by stationary phase heterogeneity and indirect methods by the limited number of suitable surrogate chromatographic models.

Systematic evaluation of selected supercritical fluid chromatography diol‐ and diethylamine‐based columns for application in hydrophilic interaction liquid chromatography

AbstractHydrophilic interaction liquid chromatography has become a useful method for separation of polar and hydrophilic analytes, i.e. compounds that are difficult to separate in reversed‐phase chromatography. The retention mechanism in hydrophilic interaction liquid chromatography is rather complex. Different types of interactions contribute to retention, depending on the properties of stationary phase, mobile phase and nature of analytes. Many columns for hydrophilic interaction liquid chromatography are available and the new stationary phases are still under development. In our study systematic examination to describe two novel columns, intended for supercritical fluid chromatography, as potential stationary phases for hydrophilic interaction liquid chromatography was performed. The supercritical fluid chromatography columns contained polar diethylamine ligand and diol ligand bonded to ethylene bridged hybrid particles. The retentivity and selectivity of the stationary phases were evaluated using a variety of test compounds. The impacts of acetonitrile content, buffer pH and concentration on the retention were investigated. Distinct mixed‐mode behavior of the diethylamine column was observed; significant contribution of anion‐exchange interaction to retention was confirmed. The DIOL phase has neutral character and retained analytes predominantly by hydrophilic interaction liquid chromatography mechanism.

A method for designing flow-through chromatography processes

Continuous manufacturing is expected to increase the productivity of the production of biologics such as protein drugs. However, it is not easy to build the continuous process especially for downstream processing as many unit operations (chromatography and membrane filtration) are involved. An operation method known as flow-through chromatography (FTC) is considered to be an efficient method for separating two components as the flow is continuous. In FTC, a target protein is eluted from the chromatography column without adsorption whereas contaminants are strongly bound. Since at least two different modes of chromatography are needed in order to remove contaminants, two FTC columns have to be connected in order to build the continuous process. This is not an easy task since the mobile phase properties (pH, salt, buffer ions) are different for the two columns. In this paper, we developed a method for designing FTC processes based on the mechanistic models that we have developed for linear gradient elution (LGE) of proteins. The distribution coefficient K as a function of salt concentration, I determined from LGE data were used for the simulation. It was found that the process is quite sensitive to a small change in I. This indicates that FTC processes are carefully monitored and controlled for the stable operation.

Connected flow-through chromatography processes as continuous downstream processing of proteins

Continuous manufacturing is expected to increase the productivity of protein drug production. However, it is not easy to build the continuous process especially for downstream processing as many unit operations (chromatography and membrane filtration) are involved. An operation method known as flow-through chromatography (FTC) is considered to be an efficient method for separating two components as the flow is continuous. In FTC, a target protein is eluted from the chromatography column without adsorption whereas contaminants are strongly bound. Since at least two different modes of chromatography are needed in order to remove contaminants, two FTC columns have to be connected in order to build the continuous process. This is not an easy task since the mobile phase properties (pH, salt, buffer ions) are different for the two columns. In this paper, we investigated how connected FTC columns can remove impurities efficiently from the cell culture broth containing monoclonal antibody. It was found that the sequence (activated carbon - anion exchange chromatography - cation exchange chromatography) is most efficient when the mobile phase pH and conductivity were properly chosen.

The Evolution of Electrospray Generated Droplets is Not Affected by Ionization Mode.

Ionization efficiency and mechanism in ESI is strongly affected by the properties of mobile phase. The use of mobile-phase properties to accurately describe droplets in ESI source is convenient but may be inadequate as the composition of the droplets is changing in the plume due to electrochemical reactions occurring in the needle tip as well as continuous drying and fission of droplets. Presently, there is paucity of research on the effect of the polarity of the ESI mode on mobile phase composition in the droplets. In this paper, the change in the organic solvent content, pH, and droplet size are studied in the ESI plume in both ESI+ and ESI- ionization mode. We introduce a rigorous way - the absolute pH (pHabsH2O) - to describe pH change in the plume that takes into account organic solvent content in the mobile phase. pHabsH2O enables comparing acidities of ESI droplets with different organic solvent contents. The results are surprisingly similar for both ionization modes, indicating that the dynamics of the change of mobile-phase properties is independent from the ESI mode used. This allows us to conclude that the evolution of ESI droplets first of all proceeds via the evaporation of the organic modifier and to a lesser extent via fission of smaller droplets from parent droplets. Secondly, our study shows that qualitative findings related to the ESI process obtained on the ESI+ mode can almost directly be applied also in the ESI- mode. Graphical Abstract ᅟ.

Influence of selected mobile phase properties on the TLC retention behavior of ziprasidone and its impurities

ABSTRACTInfluence of nine different solvents, either alone or in a mixture, on the retention behavior of ziprasidone and its five impurities were examined by normal-phase thin-layer chromatography. Migration distances of the examined compounds obtained under the examined chromatographic conditions were correlated with calculated mobile phase properties, such as Snyder polarity and Hansen solubility. Linear or second-order polynomial relationships with high correlation coefficients were established between investigated variables. The obtained mathematical functions and statistical results indicated that selected mobile phase properties can be used for the prediction of the retention behavior of ziprasidone and its five impurities.

Adduct Formation in ESI/MS by Mobile Phase Additives.

Adduct formation is a common ionization method in electrospray ionization mass spectrometry (ESI/MS). However, this process is poorly understood and complicated to control. We demonstrate possibilities to control adduct formation via mobile phase additives in ESI positive mode for 17 oxygen and nitrogen bases. Mobile phase additives were found to be a very effective measure for manipulating the formation efficiencies of adducts. An appropriate choice of additive may increase sensitivity by up to three orders of magnitude. In general, sodium adduct [M + Na]+ and protonated molecule [M + H]+ formation efficiencies were found to be in good correlation; however, the former were significantly more influenced by mobile phase properties. Although the highest formation efficiencies for both species were observed in water/acetonitrile mixtures not containing additives, the repeatability of the formation efficiencies was found to be improved by additives. It is concluded that mobile phase additives are powerful, yet not limiting factors, for altering adduct formation. Graphical Abstract ᅟ.

Structural features and thermal degradation behaviors of extracts obtained by heat reflux extraction of low rank coals with cyclohexanone

Investigation on the properties of mobile phase in low rank coals and its pyrolysis characteristics is an alternative approach for understanding the pyrolysis mechanism and structure features of these coals. In this work, heat reflux extraction with cyclohexanone (CYC) was developed to dissolve the mobile phases in two low rank coals and afford one extract from Chifeng coal (ECF) and another extract from Hami coal (EHM). The structural features of the extracts were investigated using multiple analytical techniques first. Then, thermal reactivity and gaseous product evolution of the extracts, residues, and raw coals were evaluated by TG–FTIR. Detailed volatile specie composition during fast pyrolysis of extracts were further detected on the pyrolysis gas chromatography/time of flight mass spectrometer (Py–GC/TOF–MS). The results suggest that heat reflux extraction with CYC is an effective method for dissolving a fairly high fraction of the organic matters in low rank coals. Two low rank coals mainly showed one mass loss stage of devolatilization of the organic matters near 430°C and large numbers of gaseous products were released such as hydrocarbons (CH4, C2H4, C2+ aliphatics, and arenes), CO2, CO and CO bonds containing species, which were the result of the cracking and cleavage of the alkyl sides, strongly bonded functional groups, and bridged bonds, respectively. Forming of CO during pyrolysis of low rank coals was mainly derived from the cracking of the O-containing primary tar fragment and the condensation reactions of the aromatic clusters above 600°C rather than from the initial cleavage of the organic structures in low rank coals. Thermal dissolution effect of CYC can depolymerize the macromolecular structure of raw coals and thereby the dissociated coal structures (residues) are beneficial to crack the alkyl side chains and break the O-containing structures at lower temperatures. Two extracts can be pyrolyzed at temperatures lower than that of their parent coals and large quantities of O-containing gaseous products and C2+ aliphatics release during pyrolysis process. Furthermore, aliphatic hydrocarbons, arenes, ketones, phenols, and benzofurans are the most abundant species in the products during fast pyrolysis of both two extracts. Notably, the cleavage of CC bonds near the aromatic rings in longer-chain alkylbenzenes is a crucial reaction during fast pyrolysis of ECF and EHM.

Molecular structure characterization of the tetrahydrofuran-microwave-extracted portions from three Chinese low-rank coals

The existence form and structure properties of mobile phase (MP) in low-rank coals (LRCs) can significantly influence the initial stage of thermal conversion. In the present work, three Chinese LRCs, namely, Shenfu, Zhundong and Hongshaquan, were extracted with tetrahydrofuran using the microwave-assisted heating. The tetrahydrofuran-microwave-extracted (TME) portion as the representative of MP was further separated to four fractions defined as oil, resin, asphaltene and preasphaltene, respectively. Diffuse reflectance Fourier transform-infrared spectroscopy (DRIFT), gas chromatography/mass spectrometer (GC/MS), matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) and X-ray photoelectron spectroscopy (XPS) were used to comprehensively investigate the molecular characteristics of the derived materials. The results indicated that the studied TME portions were mainly consisted of asphaltenes and rich in highly branched aliphatic hydrocarbons due to the relatively low CH2/CH3 and Har/Hal ratios. Para-alkyl substituted aromatic structures with 1–2 rings were the main aromatic structures in the TME portions. CO bonds were the main oxygen-containing structures in the TME portions and could be more likely seen in aliphatic compounds. Combining the MALDI-TOF-MS and DRIFT analyses, the ratio of aliphatic side chains and aromatic hydrogens (3000–2800cm−1/900–700cm−1, I2) derived from IR spectra seemed to be a suitable parameter for assessing the average molecular weight (AMW) of the specific fraction in TME portion of LRCs when the ratio of CO/CO was at very low level. The results made a further explanation for the detailed chemical structure of mobile phase in coal and could be helpful for studying the formation mechanism of volatiles during pyrolysis process.

Application of the Solvatic Model for Prediction of Retention in RP-LC for Multi-Step Gradient Profiles

Application of the solvatic retention model of reversed-phase liquid chromatography was studied to predict retention of phenylisothiocyanate derivatives of amino acids from structural formulae and stationary and mobile phase properties. The gradient elution mode with methanol and acetonitrile aqueous mobile phases was used. It was shown that practically acceptable prediction or retention time values can be achieved after the first approximation step when experimental data of one run are used. The zero approximation level predictions—from structural formulae, column and mobile phase properties can be used as a “first guess” method from which further optimization can begin.

Design of monoliths through their mechanical properties

Chromatographic monoliths have several interesting properties making them attractive supports for analytics but also for purification, especially of large biomolecules and bioassemblies. Although many of monolith features were thoroughly investigated, there is no data available to predict how monolith mechanical properties affect its chromatographic performance. In this work, we investigated the effect of porosity, pore size and chemical modification on methacrylate monolith compression modulus. While a linear correlation between pore size and compression modulus was found, the effect of porosity was highly exponential. Through these correlations it was concluded that chemical modification affects monolith porosity without changing the monolith skeleton integrity. Mathematical model to describe the change of monolith permeability as a function of monolith compression modulus was derived and successfully validated for monoliths of different geometries and pore sizes. It enables the prediction of pressure drop increase due to monolith compressibility for any monolith structural characteristics, such as geometry, porosity, pore size or mobile phase properties like viscosity or flow rate, based solely on the data of compression modulus and structural data of non-compressed monolith. Furthermore, it enables simple determination of monolith pore size at which monolith compressibility is the smallest and the most robust performance is expected. Data of monolith compression modulus in combination with developed mathematical model can therefore be used for the prediction of monolith permeability during its implementation but also to accelerate the design of novel chromatographic monoliths with desired hydrodynamic properties for particular application.

Using linear solvation energy relationship model to study the retention factor of solute in liquid chromatography

This review highlights the use of linear solvation energy relationships (LSERs) to evaluate liquid chromatography systems. From the systems investigated, this paper discusses the descriptors of solutes and the coefficients of various descriptors in detail. Descriptors, such as the solute size, polarity/polarizability, hydrogen bond acidity, and hydrogen bond basicity, in the LSER were examined. The effects of the stationary phase and mobile phase properties on the coefficients of these descriptors were reviewed. The LSER coefficients were evaluated in terms of the corresponding properties of the stationary phase and mobile phase. Copyright © 2012 John Wiley & Sons, Ltd.

The mobile phase motion in ascending Micellar thin-layer chromatography with normal-phase plates

The physical chemical characteristics (surface tension and viscosity) of micellar mobile phases based on the cationic surfactant cetylpiridinium chloride and additives of alcohols (ethanol, 1-propanol, 1-butanol, 1-pentanol) have been obtained in this work. The effect of mobile phase properties on the motion of mobile phase in the ascending mode of thin-layer chromatography (TLC) was investigated. The applicability of a quadratic relationship between the time of mobile phase motion and the distance from the solvent entry position of mobile phase and front of the mobile phase was validated for micellar TLC. It was observed that the retardation factors of hydrophilic test compounds change significantly only at low contents of the modifier in micellar mobile phase.

Successful application of monolithic innovative technology using a carbonyldiimidazole disk to purify supercoiled plasmid DNA suitable for pharmaceutical applications

The growing demand on plasmid DNA (pDNA) manufacture for therapeutic applications requires a final product with higher quality and quantity, spending the least time. Most of the current processes for pDNA production use at least one chromatographic step, which often constitutes a key-step in the purification sequence. Monolithic stationary phases are new alternatives to the conventional matrices, which offer fast separation of pDNA due to their excellent mass transfer properties and their high binding capacity for large molecules, as pDNA. However, the efficient recovery of pure pDNA focuses on a suitable balance of the feedstock, adsorbent and mobile phase properties. To satisfy the increasing demand for pharmaceutical grade plasmids, we developed a novel downstream process which overcomes the bottlenecks of common lab-scale techniques while complying with all regulatory requirements. This work reports an integrative approach using the carbonyldiimidazole monolith to efficiently purify the supercoiled (sc) pDNA active conformation from other plasmid topologies and Escherichia coli impurities present in a clarified lysate. The monolith specificity and selectivity was also assessed by performing experiments with plasmids of several sizes of 2.7, 6.05 and 7.4 kilo base pairs (kbp), verifying the applicability to purify different plasmids. Hence, the process yield of the pDNA purification step using the CDI monolith was 89%, with an extremely reduced level of impurities (endotoxins and gDNA), which was reflected in good transfection experiments of the sc plasmid DNA sample. Overall, the analytical results and transfection studies performed with the pDNA sample purified with this monolithic enabling technology, confirmed the suitability of this pDNA to be used in pharmaceutical applications.

Reversed-phase high performance liquid chromatography (RP-HPLC) characteristics of some 9,10-anthraquinone derivatives using binary acetonitrile–water mixtures as mobile phase

The retention behavior of 28 synthesized 9,10-anthraquinone derivatives in a reversed-phase (RP) high performance liquid chromatography (HPLC) system has been studied on a C18-RP column using acetonitrile–water mixtures as mobile phase. The influences of the composition of mobile phase and the solute structure on the retention times of 9,10-anthraquinone derivatives were investigated by linear solvation free energy relationship (LSFER) and quantitative structure-retention relationship (QSRR) analyses. Among different solvatochromic parameters of solvent systems, their polarity/polarizability parameter ( π*) was identified as the controlling factor affecting retention behavior of these compounds. A four-parametric QSRR model was obtained between solute structures and retention indices. Finally, a unified model containing both the molecular structure parameters and mobile phase properties was developed to describe the chromatographic behavior of the systems studied. The resulted QSRR models could explain and predict higher than 90% of variances in the retention indices. Among the solvent properties, polarity/polarizability parameter ( π*), and among the solute properties, HATS5v (leverage-weighted autocorrelation of lag 5/weighted by atomic van der Waals volumes, GETAWAY descriptors), Mor14p (3D-MoRSE-signal 14/weighted by atomic polarizabilities, 3D-MoRSE descriptors), GATS5p (Geary autocorrelation-lag 5/weighted by atomic polarizabilities, 2D autocorrelations) and R6u+(R maximal autocorrelation of lag 6/unweighted, GETAWAY descriptors) were identified as controlling factors in the RP-HPLC behavior of 9,10-anthraquinone derivatives in actonitrile–water binary solvents.