Modelling the pH dependent retention and competitive adsorption of charged and ionizable solutes in mixed-mode and reversed-phase liquid chromatography.

This study investigated the influence of pH on the retention of solutes using a mixed-mode column with carboxyl (-COOH) groups acting as weak cation exchanger bonded to the terminal of C18 ligands (C18-WCX column) and a traditional reversed-phase C18 column. First, a model based on electrostatic theory was derived and successfully used to predict the retention of charged solutes (charged, and ionizable) as a function of mobile phase pH on a C18-WCX column. While the Horváth model predicts the pH-dependent retention of ionizable solutes in reversed-phase liquid chromatography (RPLC) solely based on solute ionization, the developed model incorporates the concept of surface potential generated on the surface of the stationary phase and its variation with pH. To comprehensively understand the adsorption process, adsorption isotherms for these solutes were individually acquired on the C18-WCX and reversed-phase C18 columns. The adsorption isotherms followed the Langmuir model for the uncharged solute and the electrostatically modified Langmuir model for charged solutes. The elution profiles for the single components were calculated from these isotherms using the equilibrium dispersion column model and were found to be in close agreement with the experimental elution profiles. To enable modelling of two-component cases involving charged solute(s), a competitive adsorption isotherm model based on electrostatic theory was derived. This model was later successfully used to calculate the elution profiles of two components for scenarios involving (a) a C18 Column: two charged solutes, (b) a C18 Column: one charged and one uncharged solute, and (c) a C18-WCX Column: two charged solutes. The strong alignment between the experimental and calculated elution profiles in all three scenarios validated the developed competitive adsorption model.

Prediction of overloaded concentration profiles under ultra-high-pressure liquid chromatographic conditions.

In this study, overloaded elution profiles under ultra-high-pressure liquid chromatographic (UHPLC) conditions and accounting for the severe pressure and temperature gradients generated, are compared with experimental data. The model system consisted of an C18 column packed with 1.7-µm particles (i.e., a UHPLC column) and the solute was 1,3,5-tri­tert-butylbenzene eluted with a mobile phase composed of 85/15 (v/v) acetonitrile/water. Two thermal modes were considered, and the solute was eluted at the very high inlet pressures necessary to achieve a highly efficient and rapid chromatographic process, as provided by using columns packed with small particles. However, the high inlet pressure and high linear velocity of the mobile phase caused the production of a significant amount of heat, and consequently, the formation of axial and radial temperature gradients. Due to these gradients, the retention and the mobile phase velocity were no longer constant. Thus, simple mathematical models consisting only of the mass balance equations are unsuitable to properly model the elution profiles. Here, the elution concentration profiles were predicted using a combined two-dimensional heat and mass transfer model, also including the calculation of the mobile phase velocity distribution. The isotherm adsorption model was the bi-Langmuir isotherm model with Henry constants that depended on the local temperature and pressure in the column. These adjustments allowed us to precisely account for changes in the shape and retention of the overloaded concentration profiles in the mobile phase. The proposed model provided accurate predictions of the overloaded concentration profiles, demonstrating good agreement with experimental profiles eluted under severe pressure and temperature gradients in the column even in the most extreme cases where the pressure drops reached 846 bar and the temperature gradients equaled 0.15 K mm-1 and 0.95 K mm-1 in the axial and the radial directions, respectively. In such cases 36 % decrease of the retention factor was observed along the column and 2 % increase in radial direction. These changes, combined with the velocity distribution, shifted the overloaded elution profile's shock towards the center of the column, advancing approximately 3 mm from its initial position close to the column wall. Ultimately, this resulted in the broadening of the elution band.

Strategies for predictive modeling of overloaded oligonucleotide elution profiles in ion-pair chromatography.

Due to their potential for gene regulation, oligonucleotides have moved into focus as one of the preferred modalities modulating currently undruggable disease-associated targets. In the course of synthesis and storage of oligonucleotides a significant number of compound-related impurities can be generated. Purification protocols and analytical methods have become crucial for the therapeutic application of any oligonucleotides, be they antisense oligonucleotides (ASOs), small interfering ribonucleic acids (siRNAs) or conjugates. Ion-pair chromatography is currently the standard method for separating and analyzing therapeutic oligonucleotides. Although mathematical modeling can improve the accuracy and efficiency of ion-pair chromatography, its application remains challenging. Simple models may not be suitable to treat advanced single molecules, while complex models are still inefficient for industrial oligonucleotide optimization processes. Therefore, fundamental research to improve the accuracy and simplicity of mathematical models in ion-pair chromatography is still a necessity. In this study, we predict overloaded concentration profiles of oligonucleotides in ion-pair chromatography and compare relatively simple and more advanced predictive models. The experimental system consists of a traditional C18 column using (dibutyl)amine as the ion-pair reagent and acetonitrile as organic modifier. The models were built and tested based on three crude 16-mer oligonucleotides with varying degrees of phosphorothioation, as well as their respective n - 1 and (P = O)1 impurities. In short, the proposed models were suitable to predict the overloaded concentration profiles for different slopes of the organic modifier gradient and column load.

Capillary electrokinetic chromatography for studying interactions between ß-blockers and Intralipid emulsion.

Toxicity of ß-blockers is one of the most common causes of poison-induced cardiogenic shock throughout the world. Therefore, methodologies for in vivo removal of the drugs from the body have been under investigation. Intralipid emulsion (ILE) is a common commercial lipid emulsion used for parenteral nutrition, but it has also been administered to patients suffering from drug toxicities. In this work, a set of ß-blockers of different hydrophobicity's (log KD values ranging from 0.16 to 3.8) were investigated. The relative strength of the interactions between these compounds and the ILE was quantitatively assessed by means of binding constants and adsorption constants of the formed ß-blocker-ILE complexes. The binding constants were determined by capillary electrokinetic chromatography and the adsorption constants were calculated based on different adsorption isotherms. Expectedly, the binding constants were strongly related to the log KD values of the ß-blockers. The binding and adsorption constants also show that less hydrophobic ß-blockers interact with ILE, suggesting that this emulsion could be useful for capturing such compounds in cases of their overdoses. Thus, the use of ILE for treatment of toxicities caused by a larger range of ß-blockers is worth further investigation.

Assessment of physicochemical properties of sorbent materials in passive and active sampling systems towards gaseous nitrogen-containing compounds.

The adsorption and desorption behavior of volatile nitrogen-containing compounds in vapor phase by solid-phase microextraction Arrow (SPME-Arrow) and in-tube extraction (ITEX) sampling systems, were investigated experimentally using gas chromatography-mass spectrometry. Three different SPME-Arrow coating materials, DVB/PDMS, MCM-41, and MCM-41-TP and two ITEX adsorbents, TENAX-GR and MCM-41-TP were compared to clarify the selectivity of the sorbents towards nitrogen-containing compounds. In addition, saturated vapor pressures for these compounds were estimated, both experimentally and theoretically. In this study, the adsorption of nitrogen-containing compounds on various adsorbents followed the Elovich model well, while a pseudo-first-order kinetics model best described the desorption kinetics. Pore volume and pore sizes of the coating sorbents were essential parameters for the determination of the adsorption performance for the SPME-Arrow sampling system. MCM-41-TP coating with the smallest pore size gave the slowest adsorption rate compared to that of DVB/PDMS and MCM-41 in the SPME-Arrow sampling system. Both adsorbent and adsorbate properties, such as hydrophobicity and basicity, affected the adsorption and desorption kinetics in SPME-Arrow system. The adsorption and desorption rates of studied C6H15N isomers in the MCM-41 and MCM-41-TP sorbent materials of SPME-Arrow system were higher for dipropylamine and triethylamine (branched amines) than for hexylamine (linear chain amines). DVB/PDMS-SPME-Arrow gave fast adsorption rates for the aromatic-ringed pyridine and o-toluidine. All studied nitrogen-containing compounds demonstrated high desorption rates with DVB/PDMS-SPME-Arrow. Chemisorption and physisorption were the sorption mechanisms in MCM-41- and MCM-41-TP- SPME-Arrow, but additional experiments are needed to confirm this. An active sampling technique ITEX gave comparable adsorption and desorption rates on the selective MCM-41-TP and universal TENAX-GR sorbent materials for all the compounds studied. Vapor pressures of nitrogen-containing compounds were experimentally estimated by using retention index approach and these values were compared with the theoretical ones, calculated using the COnductor-like Screening MOdel for Real Solvent (COSMO-RS) model. Both values agreed well with those found in the literature proving that these methods can be successfully used in predicting VOC's vapor pressures, e.g. for the formation of secondary organic aerosols.

Development of a unified gradient theory for ion-pair chromatography using oligonucleotide separations as a model case.

Ion-pair chromatography is the de facto standard for separating oligonucleotides and related impurities, particularly for analysis but also often for small-scale purification. Currently, there is limited understanding of the quantitative modeling of both analytical and overloaded elution profiles obtained during gradient elution in ion-pair chromatography. Here we will investigate a recently introduced gradient mode, the so-called ion-pairing reagent gradient mode, for both analytical and overloaded separations of oligonucleotides. The first part of the study demonstrates how the electrostatic theory of ion-pair chromatography can be applied for modeling gradient elution of oligonucleotides. When the ion-pair gradient mode is used in a region where the electrostatic surface potential can be linearized, a closed-form expression of retention time can be derived. A unified retention model was then derived, applicable for both ion-pair reagent gradient mode as well as co-solvent gradient mode. The model was verified for two different experimental systems and homo- and heteromeric oligonucleotides of different lengths. Quantitative modeling of overloaded chromatography using the ion-pairing reagent gradient mode was also investigated. Firstly, a unified adsorption isotherm model was developed for both gradient modes. Then, adsorption isotherms parameter of a model oligonucleotide and two major synthetic impurities were estimated using the inverse method. Secondly, the parameters of the adsorption isotherm were then used to investigate how the productivity of oligonucleotide varies with injection volume, gradient slope, and initial retention factor. Here, the productivity increased when using a shallow gradient slope combined with a low initial retention factor. Finally, experiments were conducted to confirming some of the model predictions. Comparison with the conventional co-solvent gradient mode showed that the ion-pairing reagent gradient leads to both higher yield and productivity while consuming less co-solvent.

Method development for the acquisition of adsorption isotherm of ion pair reagents Tributylamine and Triethylamine in ion pair chromatography.

Tributylamine (TBuA) and triethylamine (TEtA) are the most commonly used ion pair reagents in ion pair chromatography especially for the analysis of oligonucleotides. In order to improve the understanding of the retention and separation mechanism of oligonucleotides in ion pair chromatography, it is important to understand the retention mechanism and the nature of interaction of these ion pair reagents with the stationary phase in the chromatographic column. Adsorption isotherm is helpful in evaluating such interactions, and subsequently predicting the retention mechanism. Alkylamines are very polar molecules which lack suitable chromophore and are commonly present in charged forms. Therefore, their determination and the subsequent acquisition of their adsorption isotherms using traditional liquid chromatography is very difficult. In this study, we first developed an analytical method for the determination of TBuA and TEtA in a typical chromatographic mobile phase (acetonitrile-water) and then used the same method to acquire the adsorption isotherms for tributylammonium acetate (TBuAA) and triethylammonium acetate (TEtAA). This method started with the conversion of the alkylammonium ions to free neutral forms by treating the sample with a strong base, followed by pentane-mediated extraction and finally the analysis of the extracts using gas chromatography-flame ionization detector (GC-FID). This three-step method was validated for parameters like range, linearity, intra-day and inter-day precision and accuracy, limit of detection and limit of quantitation. For the adsorption isotherms, the C18 column was first equilibrated with the solutions having different concentrations of alkylammonium ions and then stripped with eluent devoid of alkylammonium ions. Several stripping eluents were investigated and it was discovered that the eluent requirement could be decreased by the addition of sodium chloride. The effluents from the stripping phase were collected and analyzed using the developed analytical method to acquire the adsorption data. Under the investigated conditions, adsorption of TBuAA and TEtAA showed type III and type I isotherm behavior respectively.

A closer study of overloaded elution bands and their perturbation peaks in ion-pair chromatography.

There is strong renewed interest in ion-pair chromatography (IPC) because of its great importance for separating new-generation biosimilar pharmaceuticals such as oligonucleotides. Due to the complexity of the IPC process, its mathematical modeling is challenging, especially in preparative mode. In a recent study, Lesko et al. (2021) developed a mathematical model for predicting, with good accuracy, overloaded concentration profiles for sodium benzenesulfonate, describing how the overloaded solute concentration profiles change from Langmuirian to complicated U-shaped, and then back again to Langmuirian profiles, with increasing concentration of the ion-pair reagent in the mobile phase. This study identifies and explains the underlying mechanism generating these complex peak shapes and band-shape transformations; this was only possible by visualizing and modeling the underlying equilibrium perturbations that occur upon injection in preparative IPC. In the 2021 study, the model was derived based on the concentration profiles obtained using a conventional UV detector principle, so the concentration gradients and perturbation zones of the mobile-phase components were not visualized. In this study, the necessary mechanistic information was obtained via complementary experiments combining two detection principles, i.e., refractive index detection and UV detection, with modeling efforts. The models correctly described the invisible equilibrium perturbations and how these formed internal gradients of the mobile-phase components. The models also explained the complex overloaded solute-band deformations reported in the recent study. In addition, a rule of thumb was developed for predicting experimental conditions that could result in deformed solute elution profiles and/or for avoiding these deformations. The latter is crucial for the practical chromatographer, since such U-shaped solute-band profiles are undesirable in preparative separation due to the broader elution zones, resulting in lower productivity than that of normal band shapes.

Building machine-learning-based models for retention time and resolution predictions in ion pair chromatography of oligonucleotides.

Support vector regression models are created and used to predict the retention times of oligonucleotides separated using gradient ion-pair chromatography with high accuracy. The experimental dataset consisted of fully phosphorothioated oligonucleotides. Two models were trained and validated using two pseudo-orthogonal gradient modes and three gradient slopes. The results show that the spread in retention time differs between the two gradient modes, which indicated varying degree of sequence dependent separation. Peak widths from the experimental dataset were calculated and correlated with the guanine-cytosine content and retention time of the sequence for each gradient slope. This data was used to predict the resolution of the n - 1 impurity among 250 000 random 12- and 16-mer sequences; showing one of the investigated gradient modes has a much higher probability of exceeding a resolution of 1.5, particularly for the 16-mer sequences. Sequences having a high guanine-cytosine content and a terminal C are more likely to not reach critical resolution. The trained SVR models can both be used to identify characteristics of different separation methods and to assist in the choice of method conditions, i.e. to optimize resolution for arbitrary sequences. The methodology presented in this study can be expected to be applicable to predict retention times of other oligonucleotide synthesis and degradation impurities if provided enough training data.

Experimental and theoretical investigation of high- concentration elution bands in ion-pair chromatography.

The effective separation of many solutes, including pharmaceuticals, can be performed using an ion-pair reagent (IPR) in the mobile phase. However, chromatographic separation and mathematical modelling are a challenge in ionpair chromatography (IPC), especially in preparative mode, due to the complicated chromatographic process. In this study, we present a retention mechanism and a mathematical model that predict overloaded concentration profiles in IPC using a system with X-Bridge C18 as stationary phase and tetrabutylammonium bromide in the 0 - 15 mM concentration range as the IPR. Two different mobile phases were used: (i) 15/85 [v/v] acetonitrile/water, (ii) 25/75 methanol/water. The model compounds were sodium salts of organic compounds with sulfonic acid functions. The analytical and preparative elution profiles were obtained for specified conditions. The analytical data were utilized to calculate the difference in electrical potential between the surface and bulk solution using firm electrostatic theory. In the preparative mode in a certain range of IPR concentrations, complicated U-shaped overloaded profiles were observed. In the other considered cases, Langmuir overloaded elution profiles were recorded. A multilayer adsorption model was derived, which is consistent with the dynamic ion exchange models. The model assumes that lipophilic IPR adsorbs on the stationary phase, creating charged active sites that serve as exchange sites for the solutes. The molecules of the solute can adsorb on the already formed IPR layer. It was also assumed that a subsequent layer of solute can form on the formed layer of complexes due to interactions between the solute molecules. The model takes into account the electrostatic attraction and repulsion of the molecules, depending on the considered situation. The proposed model allowed prediction of the overloaded concentration profiles with very good agreement for the model solute and followed the progression from Langmuirian, through U-shaped, to again Langmuirian profiles.

Selectivity limits of and opportunities for ion pair chromatographic separation of oligonucleotides.

Here it was investigated how oligonucleotide retention and selectivity factors are affected by electrostatic and non-electrostatic interactions in ion pair chromatography. A framework was derived describing how selectivity depends on the electrostatic potential generated by the ion-pair reagent concentration, co-solvent volume fraction, charge difference between the analytes, and temperature. Isocratic experiments verified that, in separation problems concerning oligonucleotides of different charges, selectivity increases with increasing surface potential and analyte charge difference and with decreasing co-solvent volume fraction and temperature. For analytes of the same charge, for example, diastereomers of phosphorothioated oligonucleotides, selectivity can be increased by decreasing the co-solvent volume fraction or the temperature and has only a minor dependency on the ion-pairing reagent concentration. An important observation is that oligonucleotide retention is driven predominantly by electrostatic interaction generated by the adsorption of the ion-pairing reagent. We therefore compared classical gradient elution in which the co-solvent volume fraction increases over time versus gradient elution with a constant co-solvent volume fraction but with decreasing ion-pair reagent concentration over time. Both modes decrease the electrostatic potential. Oligonucleotide selectivity was found to increase with decreasing ion-pairing reagent concentration. The two elution modes were finally applied to two different model antisense oligonucleotide separation problems, and it was shown that the ion-pair reagent gradient increases the selectivity of non-charge-based separation problems while maintaining charge-difference-based selectivity.

A Retention-Matching Strategy for Method Transfer in Supercritical Fluid Chromatography: Introducing the Isomolar Plot Approach.

A strategy to match any retention shifts due to increased or decreased pressure drop during supercritical fluid chromatography (SFC) method transfer is presented. The strategy relies on adjusting the co-solvent molarity without the need to adjust the back-pressure regulator. Exact matching can be obtained with minimal changes in separation selectivity. To accomplish this, we introduce the isomolar plot approach, which shows the variation in molar co-solvent concentration depending on the mass fraction of co-solvent, pressure, and temperature, here exemplified by CO2-methanol. This plot allowed us to unify the effects of the co-solvent mass fraction and density on retention in SFC. The approach, which was verified on 12 known empirical retention models for each enantiomer of six basic pharmaceuticals, allowed us to numerically calculate the apparent retention factor for any column pressure drop. The strategy can be implemented either using a mechanistic approach if retention models are known or empirically by iteratively adjusting the co-solvent mass fraction. As a rule of thumb for the empirical approach, we found that the relative mass fraction adjustment needed is proportional to the relative change in the retention factor caused by a change in the pressure drop. Different proportionality constants were required to match retention in the case of increasing or decreasing pressure drops.

Predictions of overloaded concentration profiles in supercritical fluid chromatography.

Here, overloaded concentration profiles were predicted in supercritical fluid chromatography using a combined two-dimensional heat and mass transfer model. The heat balance equation provided the temperature and pressure profiles inside the column. From this the density, viscosity, and mobile phase velocity profiles in the column were calculated. The adsorption model is here expressed as a function of the density and temperature of the mobile phase. The model system consisted of a Kromasil Diol column packed with 2.2-µm particles (i.e., a UHPSFC column) and the solute was phenol eluted with neat carbon dioxide at three different outlet pressures and five different mobile phase flow rates. The proposed model successfully predicted the eluted concentration profiles in all experimental runs with good agreement even with high-density drops along the column. It could be concluded that the radial temperature and density gradients did not significantly influence the overloaded concentration elution profiles.

Impact of Methanol Adsorption on the Robustness of Analytical Supercritical Fluid Chromatography in Transfer from SFC to UHPSFC.

In supercritical fluid chromatography (SFC), the retention of a solute depends on the temperature, density, pressure, and cosolvent fraction. Here, we investigate how the adsorption of the cosolvent MeOH changes with pressure and temperature and how this affects the retention of several solutes. The lower the pressure, the stronger the MeOH adsorption to the stationary phase; in addition, at low pressure, perturbing the pressure results in significant changes in the amounts of MeOH adsorbed to the stationary phase. The robustness of the solute retention was lowest when operating the systems at low pressures, high temperatures, and low cosolvent fractions in the eluent. Here, we found a clear relationship between the sensitivity of MeOH adsorption to the stationary phase and the robustness of the separation system. Finally, we show that going from classical SFC to ultrahigh-performance SFC (UHPSFC), that is, separations conducted with much smaller packing diameters, results in retention factors that are more sensitive to fluctuations in the flow rate than with traditional SFC. The calculated density profiles indicate only a slight density drop over the traditional SFC column (3%, visualized as lighter → darker blue in the TOC), whereas the drop for the UHPSFC one was considerably larger (20%, visualized as dark red → light green in the TOC). The corresponding temperature drops were calculated to be 0.8 and 6.5 °C for the SFC and UHPSFC systems, respectively. These increased density and temperature drops are the underlying reasons for the decreased robustness of UHPSFC.

Impact of stationary-phase pore size on chromatographic performance using oligonucleotide separation as a model.

A combined experimental and theoretical study was performed to understand how the pore size of packing materials with pores 60-300 Å in size affects the separation of 5-50-mer oligonucleotides. For this purpose, we developed a model in which the solutes were described as thin rods to estimate the accessible surface area of the solute as a function of the pore size and solute size. First, an analytical investigation was conducted in which we found that the selectivity increased by a factor of 2.5 when separating 5- and 15-mer oligonucleotides using packing with 300 Å rather than 100 Å pores. We complemented the analytical investigation by theoretically demonstrating how the selectivity is dependent on the column's accessible surface area as a function of solute size. In the preparative investigation, we determined adsorption isotherms for oligonucleotides using the inverse method for separations of a 9- and a 10-mer. We found that preparative columns with a 60 Å-pore-size packing material provided a 10% increase in productivity as compared with a 300 Å packing material, although the surface area of the 60 Å packing is as much as five time larger.

Advanced Analysis of Biosensor Data for SARS-CoV-2 RBD and ACE2 Interactions.

The traditional approach for analyzing interaction data from biosensors instruments is based on the simplified assumption that also larger biomolecules interactions are homogeneous. It was recently reported that the human receptor angiotensin-converting enzyme 2 (ACE2) plays a key role for capturing SARS-CoV-2 into the human target body, and binding studies were performed using biosensors techniques based on surface plasmon resonance and bio-layer interferometry. The published affinity constants for the interactions, derived using the traditional approach, described a single interaction between ACE2 and the SARS-CoV-2 receptor binding domain (RBD). We reanalyzed these data sets using our advanced four-step approach based on an adaptive interaction distribution algorithm (AIDA) that accounts for the great complexity of larger biomolecules and gives a two-dimensional distribution of association and dissociation rate constants. Our results showed that in both cases the standard assumption about a single interaction was erroneous, and in one of the cases, the value of the affinity constant KD differed more than 300% between the reported value and our calculation. This information can prove very useful in providing mechanistic information and insights about the mechanism of interactions between ACE2 and SARS-CoV-2 RBD or similar systems.

Evaluating the advantages of higher heat conductivity in a recently developed type of core-shell diamond stationary phase particle in UHPLC.

In recent studies, the nature and magnitude of the temperature gradients developed in ultra-high pressure liquid chromatography (UHPLC), were found to be dependent on the heat conductivity properties of the column matrices, but also, on the principle used for controlling the temperature over the column. Here, we investigated the potential of using highly heat conductive diamond-based stationary phases (85 times higher than silica), for reducing the temperature gradients. The stationary phases investigated were a (i) Diamond Analytics FLARE column, based on particles comprised of a graphite core surrounded by a very thin diamond shell, and two silica hybrid columns: (ii) a core-shell silica Kromasil Eternity Shell column and (iii) a fully porous silica Kromasil Eternity XT column. Models were developed based on two-dimensional heat transfer theory and mass transfer theory, which were used to model the temperature profiles and the migration of an analyte band accounting for column efficiencies at different flow rates. For the silica-based columns, using water-controlled temperature mode, the temperature gradients along the column axes are suppressed whereas temperature gradients in the radial direction prevails resulting in decreased column efficiencies. Using these columns with air-controlled temperature mode, the radial temperature gradients are reduced whereas temperature gradients along the column prevails resulting in decreased retention times. With the Diamond FLARE column, there was no loss in column efficiency using the water-controlled temperature mode and the van Deemter curves are almost identical using both temperature control modes. Thus, for the Diamond FLARE column, in contrast to the silica-based columns, there are almost no losses of column efficiencies due to reduced radial temperature gradients independent on how the column temperature was controlled.

Systematic investigations of peak distortions due to additives in supercritical fluid chromatography.

The impact of eluent components added to improve separation performance in supercritical fluid chromatography was systematically, and fundamentally, investigated. The model system comprised basic pharmaceuticals as solutes and eluents containing an amine (i.e., triethylamine, diethylamine, or isopropylamine) as additive with MeOH as the co-solvent. First, an analytical-scale study was performed, systematically investigating the impact of the additives/co-solvent on solute peak shapes and retentions, using a design of experiments approach; here, the total additive concentration in the eluent ranged between 0.021 and 0.105 % (v/v) and the MeOH fraction in the eluent between 16 and 26 % (v/v). The co-solvent fraction was found to be the most efficient tool for adjusting retentions, whereas the additive fraction was the prime tool for improving column efficiency and peak analytical performance. Next, the impacts of the amine additives on the shapes of the so-called overloaded solute elution profiles were investigated. Two principal types of preparative peak deformations appeared and were investigated in depth, analyzed using computer simulation with mechanistic modeling. The first type of deformation was due to the solute eluting too close to the additive perturbation peak, resulting in severe peak deformation caused by co-elution. The second type of deformation was also due to additive-solute interactions, but here the amine additives acted as kosmotropic agents, promoting the multilayer adsorption to the stationary phase of solutes with bulkier aryl groups.

Analytical and preparative separation of phosphorothioated oligonucleotides: columns and ion-pair reagents.

Oligonucleotide drugs represent an emerging area in the pharmaceutical industry. Solid-phase synthesis generates many structurally closely related impurities, making efficient separation systems for purification and analysis a key challenge during pharmaceutical drug development. To increase the fundamental understanding of the important preparative separation step, mass-overloaded injections of a fully phosphorothioated 16mer, i.e., deoxythymidine oligonucleotide, were performed on a C18 and a phenyl column. The narrowest elution profiles were obtained using the phenyl column, and the 16mer could be collected with high purity and yield on both columns. The most likely contribution to the successful purification was the quantifiable displacement of the early-eluting shortmers on both columns. In addition, the phenyl column displayed better separation of later-eluting impurities, such as the 17mer impurity. The mass-overloaded injections resulted in classical Langmuirian elution profiles on all columns, provided the concentration of the ion-pairing reagent in the eluent was sufficiently high. Two additional column chemistries, C4 and C8, were also investigated in terms of their selectivity and elution profile characteristics for the separation of 5-20mers fully phosphorothioated deoxythymidine oligonucleotides. When using triethylamine as ion-pairing reagent to separate phosphorothioated oligonucleotides, we observed peak broadening caused by the partial separation of diastereomers, predominantly seen on the C4 and C18 columns. When using the ion-pair reagent tributylamine, to suppress diastereomer separation, the greatest selectivity was found using the phenyl column followed by C18. The present results will be useful when designing and optimizing efficient preparative separations of synthetic oligonucleotides.

Determining gradient conditions for peptide purification in RPLC with machine-learning-based retention time predictions.

A strategy for determining a suitable solvent gradient in silico in preparative peptide separations is presented. The strategy utilizes a machine-learning-based method, called ELUDE, for peptide retention time predictions based on the amino acid sequences of the peptides. A suitable gradient is calculated according to linear solvent strength theory by predicting the retention times of the peptides being purified at three different gradient slopes. The advantage of this strategy is that fewer experiments are needed to develop a purification method, making it useful for labs conducting many separations but with limited resources for method development. The preparative separation of met-enkephalin and leu-enkephalin was used as model solutes on two stationary phases: XBridge C18 and CSH C18. The ELUDE algorithm contains a support vector regression and is pre-trained, meaning that only 10-50 peptides are needed to calibrate a model for a certain stationary phase and gradient. The calibration is done once and the model can then be used for new peptides similar in size to those in the calibration set. We found that the accuracy of the retention time predictions is good enough to usefully estimate a suitable gradient and that it was possible to compare the selectivity on different stationary phases in silico. The absolute relative errors in retention time for the predicted gradients were 4.2% and 3.7% for met-enkephalin and leu-enkephalin, respectively, on the XBridge C18 column and 2.0% and 2.8% on the CSH C18 column. The predicted retention times were also used as initial values for adsorption isotherm parameter determination, facilitating the numerical calculation of overloaded elution profiles. Changing the trifluoroacetic acid (TFA) concentration from 0.05% to 0.15% in the eluent did not seriously affect the error in the retention time predictions for the XBridge C18 column, an increase of 1.0 min (in retention factor, 1.3). For the CSH C18 column the error was, on average, 2.6 times larger. This indicates that the model needs to be recalibrated when changing the TFA concentration for the CSH column. Studying possible scale-up complications from UHPLC to HPLC such as pressure, viscous heating (i.e., temperature gradients), and stationary-phase properties (e.g., packing heterogeneity and surface chemistry) revealed that all these factors were minor to negligible. The pressure effect had the largest effect on the retention, but increased retention by only 3%. In the presented case, method development can therefore proceed using UHPLC and then be robustly transferred to HPLC.