| 1. |
- Bagge, Joakim, et al.
(författare)
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Impact of stationary-phase pore size on chromatographic performance using oligonucleotide separation as a model
- 2020
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Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1634, s. 1-10
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Tidskriftsartikel (refereegranskat)abstract
- A combined experimental and theoretical study was performed to understand how the pore size of packing materials with pores 60-300 angstrom 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 angstrom rather than 100 angstrom 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 angstrom-pore-size packing material provided a 10% increase in productivity as compared with a 300 A packing material, although the surface area of the 60 angstrom packing is as much as five time larger.
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| 2. |
- Enmark, Martin, 1984-, et al.
(författare)
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Investigation of robustness for supercritical fluid chromatography separation of peptides : Isocratic vs gradient mode
- 2018
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Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1568, s. 177-187
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Tidskriftsartikel (refereegranskat)abstract
- We investigated and compared the robustness of supercritical fluid chromatography (SFC) separations of the peptide gramicidin, using either isocratic or gradient elution. This was done using design of experiments in a design space of co-solvent fraction, water mass fraction in co-solvent, pressure, and temperature. The density of the eluent (CO2-MeOH-H2O) was experimentally determined using a Coriolis mass flow meter to calculate the volumetric flow rate required by the design. For both retention models, the most important factor was the total co-solvent fraction and water mass fraction in co-solvent. Comparing the elution modes, we found that gradient elution was more than three times more robust than isocratic elution. We also observed a relationship between the sensitivity to changes and the gradient steepness and used this to draw general conclusions beyond the studied experimental system. To test the robustness in a practical context, both the isocratic and gradient separations were transferred to another laboratory. The gradient elution was highly reproducible between laboratories, whereas the isocratic system was not. Using measurements of the actual operational conditions (not the set system conditions), the isocratic deviation was quantitatively explained using the retention model. The findings indicate the benefits of using gradient elution in SFC as well as the importance of measuring the actual operational conditions to be able to explain observed differences between laboratories when conducting method transfer.
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| 3. |
- Lesko, Marek, et al.
(författare)
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A closer study of overloaded elution bands and their perturbation peaks in ion-pair chromatography
- 2022
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Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1682
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Tidskriftsartikel (refereegranskat)abstract
- 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, Leśko 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.
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| 4. |
- Lesko, Marek, et al.
(författare)
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Evaluating the advantages of higher heat conductivity in a recently developed type of core-shell diamond stationary phase particle in UHPLC
- 2020
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Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1625
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Lesko, Marek, et al.
(författare)
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Experimental and theoretical investigation of high-concentration elution bands in ion-pair chromatography
- 2021
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Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1656
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Lesko, Marek, et al.
(författare)
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Prediction of overloaded concentration profiles under ultra-high-pressure liquid chromatographic conditions
- 2024
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Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1718
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Tidskriftsartikel (refereegranskat)abstract
- 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-tri‑tert-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.
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| 7. |
- Lesko, Marek, et al.
(författare)
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Predictions of overloaded concentration profiles in supercritical fluid chromatography
- 2021
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Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1639
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Lesko, Marek, et al.
(författare)
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Strategies for predictive modeling of overloaded oligonucleotide elution profiles in ion-pair chromatography
- 2023
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Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1711
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Samuelsson, Jörgen, 1971-, et al.
(författare)
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Fundamental investigation of impact of water and TFA additions in peptide sub/supercritical fluid separations
- 2024
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Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1732
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Tidskriftsartikel (refereegranskat)abstract
- The retention of three peptides was studied under analytical and overloaded conditions at different concentrations of trifluoroacetic acid (TFA) and water added to the co-solvent methanol (MeOH). Four columns with different stationary phase properties, i.e., silica, diol, 2-ethylpyridine and cyanopropyl (CN) columns, were evaluated in this investigation. The overall aim was to get a deeper understanding on how column chemistry as well as water and TFA in the co-solvent affect the analytical and overloaded elution profiles using multivariate design of experiments and adsorption measurements of co-solvent components. Multivariate experimental design modeling indicated that water had on average around five times higher effect on the retention than the addition of TFA. The results also showed that the retention increases with the addition of TFA and water to the co-solvent on all columns except the CN column, on which the retention decreased. When examining the effect of adding water to the co-solvent, evidence of a hydrophilic interaction liquid chromatography (HILIC)-like retention mechanism was found on the three other columns with more polar stationary phases. However, on the CN column water acted as an additive, decreasing the retention due to competition with the peptide for available adsorption surface. Adsorption isotherm measurements of the polar solvent MeOH showed that MeOH adsorbs much weaker on the CN column than on the other columns. Addition of TFA and water to the co-solvent substantially sharpened the elution profiles under both overloaded and analytical conditions. Adding a small amount of TFA (from 0 % to 0.05 %) to the co-solvent substantially improved the peak shape of the elution profiles, while further addition (from 0.05 % to 0.15 %) had only a minor effect on the elution profile shape. The reduced retention on the CN column could not be explained by TFA adsorption, which was very weak on all studied columns (retention factor, 0.05–0.15). One could therefore speculate that the ion-pairing complex formed between the peptide and TFA in the mobile phase, reduce the retention due to its reduced polarity. On the other columns displaying HILIC-like properties, the TFA probably just decreased the pH of the mobile phase, thereby promoting the partitioning of the peptide into the water-rich layer. Finally, peak deformation due to diluent–eluent mismatch was observed under overloaded conditions. This was most severe in the cases where MeOH adsorption to the stationary phase was strong and the peptides were only mildly retained. Adding 1,4-dioxan to the diluent resolved this issue.
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| 10. |
- Åsberg, Dennis, 1988-, et al.
(författare)
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A practical approach for predicting retention time shifts due to pressure and temperature gradients in ultra-high-pressure liquid chromatography
- 2017
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Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1479, s. 107-120
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Tidskriftsartikel (refereegranskat)abstract
- Large pressure gradients are generated in ultra-high-pressure liquid chromatography (UHPLC) using sub–2 μm particles causing significant temperature gradients over the column due to viscous heating. These pressure and temperature gradients affect retention and ultimately result in important selectivity shifts. In this study, we developed an approach for predicting the retention time shifts due to these gradients. The approach is presented as a step-by-step procedure and it is based on empirical linear relationships describing how retention varies as a function of temperature and pressure and how the average column temperature increases with the flow rate. It requires only four experiments on standard equipment, is based on straightforward calculations, and is therefore easy to use in method development. The approach was rigorously validated against experimental data obtained with a quality control method for the active pharmaceutical ingredient omeprazole. The accuracy of retention time predictions was very good with relative errors always less than 1% and in many cases around 0.5% (n = 32). Selectivity shifts observed between omeprazole and the related impurities when changing the flow rate could also be accurately predicted resulting in good estimates of the resolution between critical peak pairs. The approximations which the presented approach are based on were all justified. The retention factor as a function of pressure and temperature was studied in an experimental design while the temperature distribution in the column was obtained by solving the fundamental heat and mass balance equations for the different experimental conditions. We strongly believe that this approach is sufficiently accurate and experimentally feasible for this separation to be a valuable tool when developing a UHPLC method. After further validation with other separation systems, it could become a useful approach in UHPLC method development, especially in the pharmaceutical industry where demands are high for robustness and regulatory oversight.
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