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• Simultaneous gradient elution separation of siRNA duplexes and lipids via IP-RPLC. • Investigated 5 UHPLC columns and 4 ion-pairing agents. • Best retention conditions achieved with phenyl column and DBAA. • Optimal temperature depends on melting characteristics of siRNA duplex. • Forced-stress testing and analysis of lipid nanoparticle formulation of siRNA. RNA interference offers a novel approach for the development of new therapeutics for targets that are otherwise "undruggable" using traditional modalities. The safety and efficacy of siRNA-based therapy mainly rely on lipid or polymer-based nanocarriers to overcome inherent barriers to a systemic delivery of siRNA. A multicomponent lipid nanoparticle (LNP) system is a promising delivery platform, typically consisting of a cationic lipid, phospholipid, PEG-containing short-chain lipid, and cholesterol. Characterization and chemical analysis of the LNP formulation is important to assure drug product stability, a key consideration for chemistry, manufacturing and control strategy. Here we report an ion-pair reversed phase UHPLC method capable of simultaneously separating both siRNA and functional lipids in LNPs with a minimal retention gap for two classes of biologically essential yet chemically distinct molecules. Key chromatographic parameters critical to the separation are discussed, including the structure of the ion-pair agent, stationary phase chemistry, column temperature and an organic additive. The results showed that the retention time of siRNA is tunable by using various ion-pair reagents. The retention factor of the siRNA exhibited a first order relationship with the number of carbons in the alkyl chain of the ion-pair reagents. In contrast, the type of ion-pair reagent has no significant impact on the separation of phospholipids. Separations using a BEH phenyl column and dibutylammonium acetate as the ion-pair reagent showed satisfactory selectivity for a range of double-stranded siRNAs and phospholipids, key components for lipid nanoparticle formulations. Furthermore, the method was applied to the separation of an experimental LNP formulation, demonstrating good selectivity for siRNA, functional lipids and their potential degradation products. [ABSTRACT FROM AUTHOR]

The size-exclusion separation of a water-soluble polyelectrolyte polymer, sodium polystyrene sulfonate (NaPSS), was demonstrated on common reversed-phase (C 18, C 4 , phenyl, and cyano) and hydrophilic interaction chromatography (HILIC) columns. The effect of common solvents – acetonitrile (ACN), tetrahydrofuran (THF), and methanol (MeOH), used as mobile phase modifiers – on the elution of NaPSS and the effect of column temperature (within a relatively narrow range corresponding to typical chromatographic conditions, i.e. , 10 °C–60 °C) on the partition coefficient, K S E C , were also investigated. Non-size-exclusion chromatography (non-SEC) effects can be minimized by the addition of an electrolyte and an organic modifier to the mobile phase, and by increasing the column temperature ( e.g., to 50 °C or 60 °C). Strong solvents such as THF and ACN are more successful in the reduction of such effects than is the weaker solvent MeOH. The best performance is seen on medium polarity and polar stationary phases, such as cyanopropyl- and diol-modified silica (HILIC), where the elution of the NaPSS polyelectrolyte is by a near-ideal SEC mechanism. Hydrophobic stationary phases, such as C 18 , C 4 , and phenyl, require a higher concentration of a strong solvent modifier (THF) in the mobile phase to reduce non-SEC interactions of the solute with the stationary phase. [ABSTRACT FROM AUTHOR]

Common reversed-phase columns (C 18 , C 4 , phenyl, and cyano) offer inert surfaces suitable for the analysis of polymers by size-exclusion chromatography (SEC). The effect of tetrahydrofuran (THF) solvent and the mixtures of THF with a variety of common solvents used in high performance liquid chromatography (acetonitrile, methanol, dimethylformamide, 2-propanol, ethanol, acetone and chloroform) on reversed-phase stationary phase characteristics relevant to size exclusion were studied. The effect of solvent on the elution of polystyrene (PS) and poly(methyl methacrylate) (PMMA) and the effect of column temperature (within a relatively narrow range corresponding to typical chromatographic conditions, i.e., 10 °C–60 °C) on the SEC partition coefficients K SEC of PS and PMMA polymers, were also investigated. The bonded phases show remarkable differences in size separations when binary mixtures of THF with other solvents are used as the mobile phase. The solvent impact can be two-fold: ( i ) change of the polymeric coil size, and possible shape, and ( ii ) change of the stationary phase pore volume. If the effect of this impact is properly moderated, then the greatest benefit of optimized solute resolution can be achieved. Additionally, this work provides an insight on solvent-stationary phase interactions and their effects on column pore volume. The only effect of temperature observed in our studies was a decreased elution volume of the polymers with increasing temperature. SEC partition coefficients were temperature-independent in the range of 10 °C–60 °C and therefore, over this temperature range elution of PS and PMMA polymers is by near-ideal SEC on reversed-phase columns. Non-ideal SEC appears to occur for high molar mass PMMA polymers on a cyano column when alcohols are used as mobile phase modifiers. [ABSTRACT FROM AUTHOR]

The origins of the peak capacity concept are described and the important contributions to the development of that concept in chromatography and electrophoresis are reviewed. Whereas numerous quantitative expressions have been reported for one- and two-dimensional separations, most are focused on chromatographic separations and few, if any, quantitative unbiased expressions have been developed for capillary or microchip zone electrophoresis. Making the common assumption that longitudinal diffusion is the predominant source of zone broadening in capillary electrophoresis, analytical expressions for the peak capacity are derived, first in terms of migration time, diffusion coefficient, migration distance, and desired resolution, and then in terms of the remaining underlying fundamental parameters (electric field, electroosmotic and electrophoretic mobilities) that determine the migration time. The latter expressions clearly illustrate the direct square root dependence of peak capacity on electric field and migration distance and the inverse square root dependence on solute diffusion coefficient. Conditions that result in a high peak capacity will result in a low peak capacity per unit time and vice-versa. For a given symmetrical range of relative electrophoretic mobilities for co- and counter-electroosmotic species (cations and anions), the peak capacity increases with the square root of the electric field even as the temporal window narrows considerably, resulting in a significant reduction in analysis time. Over a broad relative electrophoretic mobility interval [-0.9, 0.9], an approximately two-fold greater amount of peak capacity can be generated for counter-electroosmotic species although it takes about five-fold longer to do so, consistent with the well-known bias in migration time and resolving power for co- and counter-electroosmotic species. The optimum lower bound of the relative electrophoretic mobility interval [μ r,Z , μ r,A ] that provides the maximum peak capacity per unit time is a simple function of the upper bound, but its direct application is limited to samples with analytes whose electrophoretic mobilities can be varied independently of electroosmotic flow. For samples containing both co- and counter-electroosmotic ions whose electrophoretic mobilities cannot be easily manipulated, comparable levels of peak capacity and peak capacity per unit time for all ions can be obtained by adjusting the EOF to devote the same amount of time to the separation of each class of ions; this corresponds to μ r,Z = −0.5. [ABSTRACT FROM AUTHOR]

Described as intrinsically powerful building blocks for two-dimensional separations by Giddings [Anal. Chem., 56 (1984) 1258A–1270A], the coupling of chromatography and electrophoresis has been proven to enhance the resolution of a wide array of molecules in complex biological, environmental and food samples. This review provides a comprehensive overview of multidimensional chromato-electrophoretic (LC - E) and electrophero-chromatographic (E - LC) separation systems from inception to the most recent published examples. LC separation modes include reversed phase, ion exchange, and size exclusion. Electromigration separation modes include capillary, microchip or free flow electrophoresis; micellar electrokinetic chromatography; electrochromatography; and isoelectric focusing. The advantages and disadvantages of various non-gel based off-line and on-line hyphenation technologies of LC - E and E - LC are discussed, with conditions and system characteristics also provided. [ABSTRACT FROM AUTHOR]

A stochastic approach was utilized to estimate the probability of a successful isocratic or gradient separation in conventional chromatography for numbers of sample components, peak capacities, and saturation factors ranging from 2 to 30, 20–300, and 0.017–1, respectively. The stochastic probabilities were obtained under conditions of (i) constant peak width (“gradient” conditions) and (ii) peak width increasing linearly with time (“isocratic/constant N” conditions). The isocratic and gradient probabilities obtained stochastically were compared with the probabilities predicted by Martin et al. [Anal. Chem., 58 (1986) 2200–2207] and Davis and Stoll [J. Chromatogr. A, (2014) 128–142]; for a given number of components and peak capacity the same trend is always observed: probability obtained with the isocratic stochastic approach < probability obtained with the gradient stochastic approach ≤ probability predicted by Davis and Stoll << probability predicted by Martin et al. The differences are explained by the positive bias of the Martin equation and the lower average resolution observed for the isocratic simulations compared to the gradient simulations with the same peak capacity. When the stochastic results are applied to conventional HPLC and sequential elution liquid chromatography (SE-LC), the latter is shown to provide much greater probabilities of success for moderately complex samples (e.g., P HPLC = 31.2% versus P SE-LC = 69.1% for 12 components and the same analysis time). For a given number of components, the density of probability data provided over the range of peak capacities is sufficient to allow accurate interpolation of probabilities for peak capacities not reported, <1.5% error for saturation factors <0.20. Additional applications for the stochastic approach include isothermal and programmed-temperature gas chromatography. [ABSTRACT FROM AUTHOR]

A chromatographic analytical method for the direct determination of amino acids by hydrophilic interaction liquid chromatography (HILIC) was developed. A dual gradient simultaneously varying the pH 3.2 ammonium formate buffer concentration and level of acetonitrile (ACN) in the mobile phase was employed. Using a charged aerosol detector (CAD) and a 2 nd order regression analysis, the fit of the calibration curve showed R 2 values between 0.9997 and 0.9985 from 1.5 mg/mL to 50 μg/mL (600 ng to 20 ng on column). Analyte chromatographic parameters such as the sensitivity of retention to the water fraction in the mobile phase values (m HILIC ) were determined as part of method development. A degradation product of glutamine (5-pyrrolidone-2-carboxylic acid; pGlu) was observed and resolved chromatographically with no method modifications. The separation was used to quantitate amino acid content in acid hydrolysates of various protein samples. [ABSTRACT FROM AUTHOR]

This work describes the applicability of common reversed phase and HILIC columns for size exclusion chromatography of synthetic and natural polymers. Depending on the nature of the solute and column stationary phase, a “non-retention” condition must be created with the aid of the mobile phase to achieve a unique size-based separation in isocratic mode. The various bonded phases show remarkable differences in size separations that are controlled by mobile phase conditions. Polymer-mobile phase and column-mobile phase solvation interactions determine polymer hydrodynamic volume (or solute bulkiness) and polymer-column steric interaction. Solvation interactions in turn depend on polymer, mobile phase and stationary phase polarities. Column-mobile phase solvation interactions determine the structural order of the bonded ligands that can vary from ordered (extended, aligned away from the silica substrate) to disordered (folded, pointing toward the silica substrate). Chain order increases with increased solvent penetration into the bonded phase. Increased chain order reduces pore volume, and therefore decreases the size-separation efficiency of a column. Conversely, decreased chain order increases pore volume and therefore increases the size-separation efficiency. The thermodynamic quality of the mobile phase also plays a significant role in the separation of polymers. “Poor” solvents can significantly reduce the hydrodynamic diameter of a solute and thus change their retention behavior. Medium polarity stationary phases, such as fluoro-phenyl and cyano, exhibit a unique retention behavior. With an appropriate polarity mobile phase, polar and non-polar synthetic polymers of the same molecular masses can be eluted at the same retention volumes. [ABSTRACT FROM AUTHOR]

Dual-opposite-injection capillary electrophoresis (DOI-CE) is a separation technique that utilizes both ends of the capillary for sample introduction. Depending on the individual electrophoretic mobilities of the analytes of interest and the migration distance that each analyte travels to the detection window, the elution order of analytes in a DOI-CE separation can vary widely. One consequence of a variable elution order is that co-detection of oppositely charged analytes becomes possible under certain experimental conditions. While several approaches currently exist to avoid co-detection, they are often time consuming and inefficient. This paper describes a systematic approach to easily avoid co-detection by analyzing the net mobilities of the analytes of interest to aid in choosing appropriate capillary lengths for a particular separation. Simulated electropherograms that highlight various aspects of this new approach are shown. An electropherogram from the literature is replicated and the derived relationship is applied to show the potential advantages of this new approach. [ABSTRACT FROM AUTHOR]

• Investigation via > 109 simulations of the potential intrinsic advantage of tandem-column liquid chromatography (TC-LC) in terms of selectivity and resolution. • Simulated separations of 16 test solutes extracted from the hydrophobic subtraction model (HSM) database. • Simulated separations of > 104 synthetic samples containing 5–32 compounds per sample. • Amount of increased resolution (up to 0.75 units) depends on sample complexity and number of tandem-column combinations investigated. An approach is described for determining if there is an intrinsic advantage, from a selectivity and resolution perspective, of using two different UHPLC/HPLC reversed-phase columns in tandem for a separation of a given sample compared to a single U/HPLC reversed-phase column that provides the same plate number. Retention data for 16 compounds extracted directly from the hydrophobic subtraction model (HSM) database at HPLCColumns.org are used to simulate and then compare the critical resolution of those compounds obtained using HSM conditions (isocratic elution at 35°C using 50% acetonitrile, 50% aqueous phosphate buffer at pH 2.8 or 7) for each of 662 U/HPLC single columns or 218,791 combinations of tandem columns and assuming a modest plate number of 8000. The critical resolution obtained for 16 additional "n-1" samples created by the systematic removal of one of the original 16 compounds was also compared using single- and tandem-column LC, as was the critical resolution obtained for thousands of synthetic samples generated by randomly varying HSM solute descriptors for each synthetic compound. When all possible single-column or tandem-column results were compared, a significant advantage was observed with tandem-column liquid chromatography (TC-LC), with an average increase in critical resolution of 0.63 (pH 2.8) or 0.75 (pH 7) units observed for the synthetic samples with the smallest number of components (m = 5). As the number of components in a sample increased, the average improvement in critical resolution (∆R s,crit) using TC-LC gradually decreased from about 0.70 for m = 5 to 0.18 for m = 32 components. The average improvement in critical resolution achieved by switching from SC-LC to TC-LC was also lower when a smaller number of columns and column combinations were available to explore, as would be the case for a finite column inventory in a real laboratory. Nevertheless, on average there does appear to be an intrinsic advantage of tandem-column liquid chromatography, however small, which can be amplified by using high efficiency columns. [ABSTRACT FROM AUTHOR]

Highlights: [•] A theory for sequential, selective mobile phases is significantly extended. [•] The separation disorder is significantly reduced. [•] The increase in peak capacity is proportional to the number of sequential elution modes. [•] The probability of resolving all sample components is markedly increased. [ABSTRACT FROM AUTHOR]

Abstract: Numerous combinations of one-, two-, and three-chiral-component microemulsions have been previously prepared in our group, using N-dodecoxycarbonylvaline (DDCV), 2-hexanol, and ethyl acetate, dibutyl tartrate, or diethyl tartrate. A few results of the various formulations investigated suggested the possible presence of minor impurities in one or more components of the microemulsion. In this study, the purity of the current lots of R- and S-surfactant were measured, as was the subsequent effect of minor impurities on the relevant chromatographic figures of merit (CFOMs) that describe a chiral separation, i.e., efficiency, enantioselectivity, retention, migration window (elution range), and resolution. Two related methods are proposed for correcting enantioselectivities measured in the presence of chiral impurities in the chiral microemulsion. [Copyright &y& Elsevier]

Abstract: The effect of triethylamine as a mobile phase modifier on chromatographic efficiency in micellar liquid chromatography (MLC) is reported for nine different columns with various bonded stationary phases and silica pore sizes, including large-pore short alkyl chain, non-porous, and perfluorinated. Reduced plate height (h) versus reduced velocity (ν) plots were constructed for each column and the A′ and C′ terms calculated using a simplified Van Deemter equation introduced in our previous work. To further explore the practicality of using triethylamine in the micellar mobile phase, the efficiency of nine polar and non-polar substituted benzenes was studied on seven columns. Surfactant adsorption isotherms were measured for five columns with three micellar mobile phases to understand the relationship between adsorbed surfactant, mobile phase additive, and column efficiency. Clear improvements in efficiency were observed with the addition of 2% (v/v) triethylamine to a 1-butanol modified aqueous micellar mobile phase. This finding is supported by the lower amount of surfactant adsorbed onto the stationary phase when TEA is present in the mobile phase compared to an SDS only or a 1-butanol modified SDS mobile phase. [Copyright &y& Elsevier]

Abstract: Micellar liquid chromatography (MLC) remains hindered by reduced chromatographic efficiency compared to reversed phase liquid chromatography (RPLC) using hydro-organic mobile phases. The reduced efficiency has been partially explained by the adsorption of surfactant monomers onto the stationary phase, resulting in a slow mass transfer of the analyte within the interfacial region of the mobile phase and stationary phase. Using an array of 12 columns, the effects of various bonded stationary phases and silica pore sizes, including large-pore short alkyl chain, non-porous, superficially porous and perfluorinated, were evaluated to determine their impact on efficiency in MLC. Additionally, each stationary phase was evaluated using 1-propanol and 1-butanol as separate micellar mobile phase alcohol additives, with several columns also evaluated using 1-pentanol. A simplified equation for calculation of A′ and C′ terms from reduced plate height (h) versus reduced velocity (ν) plots was used to compare the efficiency data obtained with the different columns and mobile phases. Analyte diffusion coefficients needed for the h versus ν plots were determined by the Taylor-Aris dispersion technique. The use of a short alkyl chain, wide-pore silica column, specifically, Nucleosil C4, 1000Å, was shown to have the most improved efficiency when using a micellar mobile phase compared to a hydro-organic mobile phase for all columns evaluated. The use of 1-propanol was also shown to provide improved efficiency over 1-butanol or 1-pentanol in most cases. In a second series of experiments, column temperatures were varied from 40 to 70°C to determine the effect of temperature on efficiency for a subset of the stationary phases. Efficiency improvements ranging from 9% for a Chromegabond C8 column to 58% for a Zorbax ODS column were observed over the temperature range. Based on these observed improvements, higher column temperatures may often yield significant gains in column efficiency, assuming the column is thermally stable. [Copyright &y& Elsevier]

To optimize separations in capillary electrophoresis, it is important to control the electroosmotic mobility of the running buffer and the factors that affect it. Through the application of a site-dissociation-site-binding model, we demonstrated that the electroosmotic mobility could be controlled qualitatively and quantitatively by the parameters related to the physical and chemical properties of the running buffer: pH, cation valence, ionic strength, viscosity, activity, and dissociation constant. Our study illustrated that the logarithm of the number of apparent silanol sites on a fused-silica surface has a linear relationship with the pH of a buffer solution. The extension of the chemical kinetics approach allowed us to obtain the thickness of the electrical double layer when multivalent inorganic cations are present with monovalent cations in a buffer solution, and we found that the thickness of the electrical double layer does not depend on the charge of anions. The general equation to predict the electroosmotic mobility suggested here also indicates the increase of electroosmotic mobility with temperature. The general equation was experimentally verified by three buffer scenarios: (i) buffers containing only monovalent cations; (ii) buffers containing multivalent inorganic cations; and (iii) buffers containing cations and neutral additives. The general equation can explain the experimental observations of (i) a maximum electroosmotic mobility for the first scenario as the pH was varied at constant ionic strength and (ii) the inversion and maximum value of the electroosmotic mobility for the second scenario when the concentration of divalent cations was varied at constant pH. A good agreement between theory and experiment was obtained for each scenario. [ABSTRACT FROM AUTHOR]

Abstract: Dodecoxycarbonylvaline (DDCV) microemulsions (1% and 4%, w/v) were employed to evaluate the retention mechanism of a series of enantiomers over a temperature range of 15–35°C. From the acquired retention data, van’t Hoff plots were constructed and enthalpy and entropy of transfer were calculated from the slope and intercept, respectively. Resolution, enantioselectivity, distribution coefficients and Gibb''s free energy were also calculated, as well as between enantiomer differences in enthalpy, entropy and Gibb''s free energy. Finally, comparisons were made between the microemulsion thermodynamic data and a corresponding set of micellar data. While the 4% DDCV microemulsion did not provide a linear van’t Hoff relationship, the 1% DDCV microemulsion was linear over a temperature range of 15–30°C. For the 1% DDCV microemulsion, the enthalpic contribution to retention was consistently favorable (ΔH &#60;0), whereas the entropic contribution varied from compound to compound. Finally, while the achiral attraction of the analytes was greater for the micellar phase, the microemulsion seemed to provide a suitable difference in entropy (and Gibb''s free energy) between enantiomers to achieve chiral discrimination. [Copyright &y& Elsevier]

Abstract: One of the main limitations of micellar liquid chromatography (MLC) is the lower efficiency compared to reversed-phase liquid chromatography (RPLC) with hydro-organic mobile phases. The main contribution to the reduced efficiency has been shown to be due to the slow mass transfer between micelles, the aqueous phase, and the stationary phase mainly due to surfactant adsorption onto the stationary phase. The use of a variety of stationary phases, including large-pore short alkyl chain, non-porous, superficially porous, and perfluorinated, is shown to have differing effects on remediation of the reduced efficiency. Diffusion coefficients were determined by the Taylor–Aris dispersion technique for the construction of Knox plots. The Knox plots are used to compare the efficiency data obtained with the different columns using several alkylphenones in both micellar mobile phase and hydro-organic mobile phase. [Copyright &y& Elsevier]

Sertraline is a basic compound and of pharmaceutical application for antidepressant treatment. The compound has two chiral centers. Separation of the three enantiomeric impurities from the parent compound is challenging. In this study, we successfully separated all four stereoisomers by electrokinetic chromatography using highly sulfated γ-cyclodextrin and highly sulfated α-cyclodextrin as the chiral selectors. The two chiral selectors provided different selectivity and therefore affected the overall separation profiles. This may be due to the size difference between the dichlorophenyl moiety end and naphthalenamine moiety end, resulting in two different types of inclusion complexes with the different cyclodextrins. For routine analysis, highly sulfated γ-cyclodextrin was better than highly sulfated α-cyclodextrin. For each stereoisomeric impurity, the method using sulfated γ-cyclodextrin provided a limit of quantitation at or lower than 0.1% of the drug substance with adequate resolution. The critical resolution at this concentration level was not less than 4.0. Experimental data suggested that an internal standard was necessary for the purpose of quantitation, and the practical linearity range for analysis of sertraline stereoisomeric impurities was of about two orders of magnitude. [Copyright &y& Elsevier]

Micellar liquid chromatography (MLC) is useful in bioanalysis because proteinaceous biofluids can be directly injected onto the column. The technique has been limited in part because of the apparently weak eluting power of micellar mobile phases. It has recently been shown [Anal. Chem. 72 (2000) 294] that this may be overcome by the use of large pore size stationary phases. In this work, large-pore (1000 A˚) C18 stationary phases were evaluated relative to conventional small-pore (100 A˚) C18 stationary phases for the direct sample injection of drugs in plasma. Furthermore, the difference between the large and small pore phases in gradient elution separations of mixtures of widely varying hydrophobicities was investigated. Large-pore stationary phases were found to be very effective for eluting moderately to highly hydrophobic compounds such as ibuprofen, crotamiton, propranolol, and dodecanophenone, which were highly retained on the small-pore stationary phases typically used in MLC. The advantages of direct introduction of biological samples (drugs in plasma) and rapid column re-equilibration after gradient elution in MLC were maintained with large-pore phases. Finally, recoveries, precision, linearity, and detection limits for the determination of quinidine and DPC 961 in spiked bovine plasma were somewhat better using MLC with wide pore phases. [Copyright &y& Elsevier]

Analyzes the effect of liquid chromatographic separation on matrix-related signal suppression in electrospray ionization mass spectrometry. Induction of matrix-related signal suppression by column overload and matrix component-analyte celation; Effectiveness of on-line orthogonal liquid chromatography separations.

Studies the effect of stationary-phase pore size on retention behavior in micellar liquid chromatography. Differences in retention behavior between various solute types and stationary phases; Partitioning behavior of representative solutes; Differences in the stationary-phase characteristics between the small- and large-pore size C18 columns.

Presents the results of a study which investigated the influence of dodecyl sulfate micelles on efficiency, selectivity, retention, elution range and resolution in micellar electrokinetic chromatography. Effects of using the counterions lithium and potassium; Effect of acetonitrile; Comparisons between the counterions; Operating currents.

Small interfering RNA (siRNA) therapeutics have received significant interest in recent years owing to their ability to elicit sequence-specific gene knockdown and subsequent suppression of protein expression. Chemical modifications can improve the hydrolytic stability and susceptibility of siRNAs to enzymatic degradation. One commonly used modification to improve hydrolytic stability, the replacement of the native phosphodiester linkage with a phosphorothioate moiety, introduces an additional phosphorous stereocenter into the molecule, resulting in the formation of diastereomers. The chromatographic separation of stereoisomeric siRNAs containing such phosphothioates is a challenging problem, especially when multiple phosphothioates are present within a modified siRNA duplex giving rise to multiple stereoisomers. In this study, we report an investigation into the use of an ion-pairing reversed phase UHPLC (or IP-RP UHPLC) method for the baseline separation of closely related diastereomers of an Apo-B gene targeting siRNA duplex under denaturing conditions. The related siRNA species consist of two diastereomers from the sense strand and the two pairs of diastereomers from the antisense strand. Key chromatographic parameters critical to stereoisomer separation are highlighted, including the structure of the ion-pairing agent, chemical composition of the stationary phase, and organic modifier. The method was applied to the separation of an siRNA stressed with iodine and demonstrated satisfactory selectivity for the parent siRNA and the desulfurization product. [ABSTRACT FROM AUTHOR]

Longitudinal on-column thermal modulation for comprehensive two-dimensional liquid chromatography is introduced. Modulation optimization involved a systematic investigation of heat transfer, analyte retention, and migration velocity at a range of temperatures. Longitudinal on-column thermal modulation was realized using a set of alkylphenones and compared to a conventional valve-modulator employing sample loops. The thermal modulator showed a reduced modulation-induced pressure impact than valve modulation, resulting in reduced baseline perturbation by a factor of 6; yielding a 6-14-fold improvement in signal-to-noise. A red wine sample was analyzed to demonstrate the potential of the longitudinal on-column thermal modulator for separation of a complex sample. Discrete peaks in the second dimension using the thermal modulator were 30-55% narrower than with the valve modulator. The results shown herein demonstrate the benefits of an active focusing modulator, such as reduced detection limits and increased total peak capacity. [ABSTRACT FROM AUTHOR]

Highlights: [•] Oligonucleotide analysis by ion-pair reversed-phase liquid chromatography. [•] Evaluation of a core–shell C18 column for improved separation of oligonucleotides. [•] Long-term column stability test at pH 7 mobile phase and high column temperature. [•] Evaluation of SunShell C18 column showed the column remained stable after more than 300 injections. [ABSTRACT FROM AUTHOR]

Abstract: An investigation into the use of core–shell particle columns for separation of short (∼21 base pairs) RNA oligonucleotides by ion-pair reversed-phase liquid chromatography (IP-RPLC) showed improved resolution for a number of test analytes relative to conventional (fully-porous) reversed-phase columns. The best resolutions were obtained using columns packed with smaller sub-2μm core–shell particles. [Copyright &y& Elsevier]

Abstract: The effect of deuterated solvents in size-exclusion chromatography (SEC) was studied by comparing intrinsic viscosity measurements, SEC calibration curves, and column efficiency using water-soluble polymers. For aqueous SEC, the use of deuterium oxide slightly increases the SEC elution volume. To verify that adsorption onto the packing was absent, data from exclusion experiments were compared at 35 and 50°C. Our results indicate that adsorption is not occurring for pullulan or polyethylene glycol (PEG)/poly(ethylene oxide) (PEO); for the latter, however, the elution volume increased using both D2O and H2O, indicative of slight hydrodynamic volume contraction of PEG/PEO at higher temperatures. A moderate increase in band broadening (moderate decrease in column efficiency) was observed using D2O. Finally, the effects of chloroform versus deuterated chloroform were evaluated, but no hydrodynamic volume changes were observed. [Copyright &y& Elsevier]

Studies the experimental aspects of the sampling process in relation to comprehensive two-dimensional liquid chromatography. How is the total analysis time determined; Optimization of multidimensional separation systems; Discussion of the sensitivity of the resolution with respect to the sampling phase; Indications of the findings.

A prerequisite for ordered two-dimensional (2D) separations and full utilization of the enhanced 2D peak capacity is selective exploitation of the sample attributes, described as sample dimensionality. In order to take sample dimensionality into account prior to optimization of a 2D separation, a new concept based on construction of 2D separation selectivity maps is proposed and demonstrated for ion chromatography x capillary electrophoresis (ICxCE) separation of low-molecular-mass organic acids as test analytes. For this purpose, 1D separation selectivity maps were constructed based on calculation of pairwise separation factors and identification of critical pairs for four IC stationary phases and 28 levels of background electrolyte pH in CE. The derived IC and CE maps were then superimposed and the effectiveness of the respective 2D separations assessed using an in silico approach, followed by testing examples of one successful and one unsuccessful 2D combination experimentally. The results confirmed the efficacy of the predictions, which require a minimal number of experiments compared to the traditional one-at-a-time approach. Following the same principles, the proposed framework can also be adapted for optimization of separation selectivity in various 2D combinations and for other applications. [ABSTRACT FROM AUTHOR]

• Developed a workflow to prioritize single and tandem-column pairs for RPLC separations. • Separation predictions based upon hydrophobic subtraction model (HSM) parameters. • Methodology applied to separation of Linrodostat and 10 related impurities. • Prediction of potentially successful columns greatly simplifies method development. • For the Linrodostat study, tandem-column LC outperformed single column LC under HSM conditions. The liquid chromatography (LC) analysis of small molecule pharmaceutical compounds and related impurities is crucial in the development of new drug substances, but developing these separations is usually challenging due to analyte structural similarities. Tandem-column LC (TC-LC) has emerged as a powerful approach to achieve alternative separation selectivity compared to conventional single column separations. However, one of the bottlenecks associated with use of tandem column approaches is time-consuming column pair screening and selection. Herein, we compared critical resolution (R c) in single column vs. TC-LC separations for a given set of small molecule pharmaceutical compounds and developed a column selection workflow that uses separation simulations based on parameters from the hydrophobic subtraction model (HSM) of reversed-phase selectivity. In this study, HSM solute parameters were experimentally determined for a small molecule pharmaceutical (Linrodostat) and ten of its related impurities using multiple linear regression of their retentions on 16 selected RPLC columns against in-house determined HSM column parameters. R c values were calculated based on HSM database column parameters for a pool of about 200 available stationary phases in both single-phase column (2.1 mm i.d. × 100 mm) or tandem column paired (two 2.1 mm i.d. × 50 mm) formats. Four column configurations (two single and two tandem) were predicted to achieve successful separations under isocratic HSM separation conditions, with a fifth tandem pair predicted to have a single co-elution. Of these five potential candidates, one tandem pair yielded compete baseline resolution of the 11-component mixture in an experimental separation. In this specific case, the tandem column pairs outperformed single-phase columns, with better predicted and experimental R c values for the Linrodostat mixture under the HSM separation conditions. The results reported in this study demonstrated the enormous selectivity potential of TC-LC in pharmaceutical compound separations and are consistent with our previous study that examined the potential of tandem column approaches using purely computational means, though there is room for substantial improvement in the prediction accuracy. The proposed workflow can be used to prioritize a small number of column combinations by computational means before any experiments are conducted. This is highly attractive from the point of view of time and resource savings considering over 200,000 different tandem column pairings are possible using columns for which there are data in the HSM database. [Display omitted] [ABSTRACT FROM AUTHOR]

Synthetic oligonucleotides have become increasingly popular as a result of the recent discovery of ribonucleic acid interference (RNAi), a natural process for silencing gene expression. As biomedical researchers evaluate the use of antisense and small interfering RNAs (siRNAs) as potential therapies for the treatment of disease, the need for improved methods for the chromatographic separation and analysis of oligonucleotides has become apparent. This article presents a review of different liquid chromatography (LC) methods and strategies for the chromatographic separation of short RNA oligonucleotides. [ABSTRACT FROM AUTHOR]

Highlights: [•] Mixed-mode stationary phases were evaluated for the analysis of oligonucleotides. [•] A single column and method can be used for complete oligonucleotide analysis. [•] Mixed-mode columns with SAX-LC conditions provided the best separation. [•] SM-C18 column with NaCl salt gradient provided the best peak shape and separation. [ABSTRACT FROM AUTHOR]

Abstract: The S enantiomers of ester functionalized NHC ligand precursors, 1-[(2S)-1-ethoxy-3-methyl-1-oxobutan-2-yl]-3-ethyl-1H-imidazol-3-ium bromide (3a) and the methyl ester analog 3b, were synthesized from l-valine and fully characterized. The absolute stereochemistry of the chiral center was confirmed by single crystal X-ray diffraction (for 3a) and enantiopurity (of 3a and 3b) was determined by micellar electrokinetic chromatography. Deprotonation of this NHC ligand precursor (3a or 3b) and coordination to [RuCl2 (p-cymene)]2 resulted in the isolation of a monodentate complex (with only NHC coordination) (4a or 4b) and a bidentate complex (5). In the bidentate complex, in addition to NHC coordination, a carboxylate donor group (from in situ hydrolysis of the ester) coordinates to Ru. All ruthenium complexes have been characterized by spectroscopic and analytical methods, plus single crystal X-ray diffraction for 4a and 5. The evidence shows that both Ru complexes are racemic, but with alternate synthetic methods perhaps racemization during complexation of 3a or 3b to metals could be avoided. The ruthenium complexes described herein serve as catalysts for ketone transfer hydrogenation in basic isopropanol. [Copyright &y& Elsevier]

Abstract: A capillary sodium dodecyl sulfate gel electrophoresis (cSDS) method has been developed and qualified for purity and impurity analysis of monoclonal antibodies. This method was optimized and qualified for the analysis of monoclonal antibody (mAb1) under reduced and non-reduced conditions. Some of the sample preparation parameters including sample buffer pH, incubation temperature and duration, alkylation conditions with iodoacetamide (IAM), and reduction conditions with 2-mercaptoethanol (2-ME) were optimized. It was observed that under slightly acidic conditions (pH 5.5–6.5) the thermally induced fragmentation of non-reduced mAb1 was greatly decreased. As such, a citrate–phosphate buffer at pH 6.5 was used for sample preparation to replace the original Beckman sample buffer (pH 9.0). The optimal sample preparation conditions were found to be as follows: (1) incubation temperature and duration (reduced and non-reduced), 65°C for 5min; (2) alkylation condition, 10μL of 0.25M IAM; (3) reduction condition, 10μL of 5-fold diluted 2-ME. The method was qualified by evaluating specificity, accuracy, precision, limit of quantitation (LOQ), and linearity. The method exhibited no interference from sample buffer matrix. The method was found to be linear, accurate, and precise in the range of 0.25–3.0mg/mL protein concentration. The LOQ of the method was determined to be 0.02mg/mL for reduced and non-reduced mAb1. In addition, some aspects of sample stability were examined during qualification. [ABSTRACT FROM AUTHOR]

Abstract: The European Pharmacopoeia (Ph. Eur.) monograph for Erythropoietin Concentrated Solution describes a capillary zone electrophoresis method for identification of recombinant human erythropoietin. However, this method has shown poor reproducibility due to inadequate capillary conditioning. We have modified the Ph. Eur. method to make it more robust and suitable for the quality control laboratory for the analysis of epoetin alfa and epoetin alfa after formulation with polysorbate 80. This study qualified the modified method by showing improved robustness and reproducibility. The study also characterized and qualified a secondary standard of epoetin alfa as a substitute for the primary standard, Ph. Eur. erythropoietin Biological Reference Preparation, which is available in limited supply. Four sets of analyses were performed to assess repeatability, intermediate precision, and the secondary standard. The results showed that the modified method is suitable for its intended purpose to test epoetin alfa and formulated epoetin alfa samples. The epoetin alfa secondary standard is a suitable substitute for the primary standard. Further, we developed a procedure for the removal of polysorbate 80 from formulated epoetin alfa, allowing the material to be analyzed by the modified Ph. Eur. method. [Copyright &y& Elsevier]

Looks at fundamental developments in liquid chromatography during the period October 1995 to October 1997. Information on the theory and optimization of liquid chromatography; Analysis of the normal phase of liquid chromatography; Questions raised with regards to reversed-phase liquid chromatography; Discussion on bipolymer separations; Details on affinity and ion chromatography; Investigation and utilization of secondary chemical equilibria.

Presents information on a study which examined the electrokinetic chromatography of a novel mixed surfactant system using thermodynamically stable vesicles and mixed micelles formed from oppositely charged surfactants. What the surfactant system consisted of; Comparison of polar group selectivity, retention, and efficiency between vesicles, mixed micelles, and sodium dodecyl sulfate micelles.