| 1. |
- Enmark, Martin, et al.
(författare)
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A Deeper Investigation of Strange Preparative Band Shapes of a Simple Racemic Solute on tris-(3, 5- dimethylphenyl)carbamoyl Cellulose as Chiral Stationary Phase
- 2011
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Konferensbidrag (refereegranskat)abstract
- The adsorption equilibria of racemic methyl mandelate on a tris-(3, 5-dimethylphenyl)carbamoyl cellulose chiral stationary phase (CSP) has a peculiar behavior. The preparative band shape of the more retained enantiomer was very unusual with an inflection point at low concentrations whereas the less retained enantiomer shows normal type I adsorption behavior. For a deeper understanding of this separation adsorption isotherms were determined and further analyzed with Scatchard plots combined with adsorption energy distribution calculations. The less retained enantiomer was best described by Tóth adsorption isotherm while the second enantiomer was best described with a bi-Moreau adsorption isotherm. The Moreau model is an extension of the Langmuir model including non-ideal adsorbate-adsorbate interactions; here the unusual model provided an explanation to the non-ideal adsorption of the more retained enantiomer. Furthermore, the possibility of using the Moreau model as a local model for adsorption in AED calculations was evaluated by synthetically generated raw adsorption slope data. It was demonstrated that the AED accurately could predict the number of adsorption sites for the generated data. The adsorption behavior of both enantiomers was also studied at several different temperatures and it was found to be exothermic; in addition, the non-ideal adsorbate-adsorbate interaction strength decreases with increasing temperature. Stochastic analysis of the adsorption process could identify a single kinetic site for each enantiomer. The average amount of adsorption/desorption events increases and the sojourn time decreases with increasing temperature
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| 2. |
- Enmark, Martin, et al.
(författare)
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Characterization of an unusual adsorption behavior of racemic methyl-mandelate on a tris-(3,5-dimethylphenyl) carbamoyl cellulose chiral stationary phase
- 2011
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1218:38, s. 6688-6696
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Tidskriftsartikel (refereegranskat)abstract
- An interesting adsorption behavior of racemic methyl mandelate on a tris-(3,5-dimethylphenyl)carbamoyl cellulose chiral stationary phase was theoretically and experimentally investigated. The overloaded band of the more retained enantiomer had a peculiar shape indicating a type V adsorption isotherm whereas the overloaded band of the less retained enantiomer had a normal shape indicating a type I adsorption behavior. For a closer characterization of this separation, adsorption isotherms were determined and analyzed using an approach were Scatchard plots and adsorption energy distribution (AED) calculations are combined for a deeper analysis. It was found that the less retained enantiomer was best described by a Tóth adsorption isotherm while the second one was best described with a bi-Moreau adsorption isotherm. The latter model comprises non-ideal adsorbate–adsorbate interactions, providing an explanation to the non-ideal adsorption of the more retained enantiomer. Furthermore, the possibility of using the Moreau model as a local model for adsorption in AED calculations was evaluated using synthetically generated raw adsorption slope data. It was found that the AED accurately could predict the number of adsorption sites for the generated data. The adsorption behavior of both enantiomers was also studied at several different temperatures and found to be exothermic; i.e. the adsorbate–adsorbate interaction strength decreases with increasing temperature. Stochastic analysis of the adsorption process revealed that the average amount of adsorption/desorption events increases and the sojourn time decreases with increasing temperature.
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| 3. |
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| 4. |
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| 5. |
- Samuelsson, Jörgen, et al.
(författare)
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Expanding the elution by characteristic point method for determination of various types of adsorption isotherms
- 2011
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1218:24, s. 3737-3742
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Tidskriftsartikel (refereegranskat)abstract
- Important improvements have recently been made on the elution by characteristic point (ECP) method to increase the accuracy of the determined adsorption isotherms. However, the method has so far been limited/used for only type I adsorption isotherms (e.g. Langmuir, Toth, bi-Langmuir). In this study, general strategies are developed to expand the ECP method for the determination of more complex adsorption isotherms including such containing inflection points. We will exemplify the methodology with type II, type III and type V isotherms. Guidelines are given for how to determine such isotherms using the ECP method and for the experimental considerations that must be taken into account or that may be eliminated in the particular case.
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| 6. |
- Samuelsson, Jörgen, et al.
(författare)
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Improvement in the generation of adsorption isotherm data in the elution by characteristic points method- : the ECP-slope approach
- 2010
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Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1217:46, s. 7215-7221
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Tidskriftsartikel (refereegranskat)abstract
- The elution by characteristic points (ECP) method is a very rapid and precise method for determination of the phase system equilibrium of phase systems in broad solute concentration ranges. Thus, the method is especially suitable for rapid characterization of high efficient separation systems. One important source of error, the effects by the post-loop dispersion, was eliminated in a recent investigation. In this study, the systematic error caused by the selection of the integration starting point at concentration equal to 0 is eliminated. This is done by developing and validating a new procedure for isotherm data generation; the ECP-slope method. The method generates raw slope data of the adsorption isotherm instead of raw adsorption data by integrations as the classical ECP does. Both numerical and experimental data were used for the comparison of the classical ECP approach with the slope-ECP method
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| 7. |
- Undin, Torgny, 1975-
(författare)
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Development and improvement of methods for characterization of HPLC stationary phases
- 2011
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- High Performance Liquid Chromatography (HPLC) is a widely used tech-nique both for detecting and purifying substances in academy and in the industry. In order to facilitate the use of, and knowledge in HPLC, character-ization of stationary phases is of utmost importance. Tailor made characteri-zation methods and workflows are steadily increasing the speed and accura-cy in which new separation systems and methods are developed. In the field fundamental separation science and of preparative chromatography there is always the need for faster and more accurate methods of adsorption isotherm determination. Some of that demand are met with the steadily increase of computational power, but the practical aspects on models and methods must also be further developed. These nonlinear characterization methods will not only give models capable of describing the adsorption isotherm but also actual values of local adsorption energies and monolayer saturation capacity of an individual interaction sites etc.The studies presented in this thesis use modern alkali stable stationary phas-es as a model phase, which will give an insight in hybrid materials and their separation mechanism. This thesis will include an update and expansion in using the Elution by Characteristic Points (ECP) method for determination of adsorption isotherms. The precision is even further increased due to the ability to use slope data as well as an increase in usability by assigning a set of guidance rules to be applied when determine adsorption isotherms having inflection points. This thesis will further provide the reader with information about stationary phase characterization and the power of using existing tech-niques; combine them with each other, and also what the expansion of meth-ods can revile in terms of precision and increased usability. A more holistic view of what benefits that comes with combining a non-linear characteriza-tion of a stationary phase with more common linear characterization meth-ods are presented.
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| 8. |
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| 9. |
- Undin, Torgny, et al.
(författare)
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Evaluation of a combined linear–nonlinear approach for column characterization using modern alkaline-stable columns as model
- 2013
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Ingår i: Journal of Separation Science. - : Wiley. - 1615-9306 .- 1615-9314. ; 36:11, s. 1753-1761
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Tidskriftsartikel (refereegranskat)abstract
- This study investigates if deeper understanding is achieved when combining nonlinear and linear chromatographic column characterization methods. As test systems, two hybrid columns (Phenomenex Gemini-NX C18 and Kromasil Eternity C18) and one classic one (Kromasil-C18) were selected. The nonlinear methods were based on firm adsorption theory and involved determination of adsorption isotherms followed by calculations with a new numerical tool, adsorption energy distribution, on probe components at different pH values. The linear methods involved the hydrophobic subtraction model and selected probe components retention factors as a function of pH. The combined analysis indicated that both complementary and confirmative information can be achieved regarding the actual model systems.
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| 10. |
- Agmo Hernández, Víctor, et al.
(författare)
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Enhanced interpretation of adsorption data generated by liquid chromatography and by modern biosensors
- 2013
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1317, s. 22-31
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Tidskriftsartikel (refereegranskat)abstract
- In this study we demonstrate the importance of proper data processing in adsorption isotherm estimations. This was done by investigating and reprocessing data from five cases on two closely related platforms: liquid chromatography (LC) and biosensors. The previously acquired adsorption data were reevaluated and reprocessed using a three-step numerical procedure: (i) preprocessing of adsorption data, (ii) adsorption data analysis and (iii) final rival model fit. For each case, we will discuss what we really measure and what additional information can be obtained by numerical processing of the data. These cases clearly demonstrate that numerical processing of LC and biosensor data can be used to gain deeper understanding of molecular interactions with adsorption media. This is important because adsorption data, especially from biosensors, is often processed using old and simplified methods. (C) 2013 Elsevier B.V. All rights reserved.
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| 11. |
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| 12. |
- Arnell, Robert, et al.
(författare)
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Adsorption behaviour of a quinidine carbamate-based chiral stationary phase : Role of the additive
- 2009
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1216:16, s. 3480-3487
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Tidskriftsartikel (refereegranskat)abstract
- In this study, we incorporate the additive properties into the theoretical model of a general preparative chromatographic system; this is normally not done and this limits a proper process optimization. As a model phase system, we used the adsorption of 9H-fluoren-9-ylmethoxycarbonyl-allylglycine (Fmoc-allylglycine) enantiomers on a quinidine carbamate-based chiral stationary phase (anion exchanger) together with a methanol-glacial acetic acid-ammonium acetate eluent. The inverse method was used to measure the competitive adsorption isotherms of both the Fmoc-allylglycine enantiomers as well as the non-detectable additive acetic acid. It was concluded that this enantioselective preparative system is well described by a non-heterogeneous adsorption model and that the loading capacity is very high. The proposed model is valid over a wide range of additive concentrations, which is important for process optimization.
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| 13. |
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| 14. |
- Arnell, Robert, et al.
(författare)
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Analytical Characterization of Chiral Drug-Protein Interactions: Comparison between the Optical Biosensor (Surface Plasmon Resonance) Assay and the HPLC Perturbation Method
- 2006
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Ingår i: Analytical Chemistry. ; 78:5, s. 1682-1689
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Tidskriftsartikel (refereegranskat)abstract
- Two modern, fundamentally different methods were used for a detailed investigation of enantioselective drug-protein interactions, a surface plasmon resonance (SPR)-based Biacore 2000 biosensor assay and the previously validated HPLC perturbation method (HPLC-PM). This is the first time SPR has been used for this purpose. The fundamental features of the two methods were investigated, and the consequences for operation and data evaluation were addressed. With HPLC-PM, chiral data could be obtained directly from the racemic mixture, whereas a separate analysis of each pure enantiomer was required to obtain chiral data with SPR. It was shown that if chirality is not attributed in the SPR analysis, misleading average racemic binding constants will be obtained. Both drug and protein consumption were considerably higher with HPLC-PM. HPLC-PM was found to be best suited for measurements of weak affinity interactions, whereas the SPR method was best for strong interactions. With both methods, the presence of DMSO in the samples severely affected the interactions, introducing errors. The binding of the -blockers alprenolol and propranolol to Cel7a cellulase was used as a model system. These methods gave results that agreed quite well qualitatively, but considerable quantitative deviations were sometimes obtained.
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| 15. |
- Arnell, Robert, et al.
(författare)
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Biotechnological Approach to the Synthesis of 9α-Hydroxylated Steroids
- 2007
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Ingår i: Preparative Biochemistry & Biotechnology. - : Informa UK Limited. - 1082-6068 .- 1532-2297. ; 37:4, s. 309-321
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Tidskriftsartikel (refereegranskat)abstract
- The steroid 9α-hydroxylase gene has been cloned from Mycobacterium smegmatis into Escherichia coli BL21. Progesterone added to bioreactors was subjected to in vivo transformation into 9α-hydroxyprogesterone. In 7 days, 43.6 mg9α-hydroxyprogesterone was formed from 53.8 mg/L progesterone. The enzyme also has shown evidence of processing 4-androstene-3,17-dione in vivo. An extensive analytical method development, including LLE, HPLC-DAD, MS, andNMR was performed to verify the product and to enable a quantitative analysis. Protocols for analytical and preparative separation have been developed, using binaphtol as internal standard. Both the growth pattern and the bioconversion ratewere unaffected by the presence of binaphtol in the bioreactor. The enzyme was purified by immobilised metal affinity and ion exchange chromatography, resulting in low in vitro activity.
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| 16. |
- Arnell, Robert, 1976-
(författare)
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Development and Validation of Methods for Characterization of Multi-Component Systems in Preparative LC
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis concerns the development and validation of methods for characterization of multi-component preparative LC systems. Measurements of competitive adsorption isotherms are performed to gain detailed information about the interactions inside the chromatography column. This information increases our understanding of the separation process and makes it possible to perform computer simulations and numerical optimizations to find optimal operating conditions.The methods under focus are called “the tracer-pulse method”, “the inverse method”, and “the inverse method on plateaus”. They are extensions of existing methods, with new experimental and numerical procedures to enable rapid and accurate multi-component adsorption isotherm determination. In the validation it was shown that they can produce results agreeing with traditional methods and that the acquired adsorption isotherm parameters can be used in simulations to accurately predict the outcome of preparative LC separations.The methods were used to characterize several complex LC systems and two phenomena were discovered and theoretically treated: 1) The presence of invisible deformed peaks in single-component systems. 2) Peak deformations encountered with modern chiral stationary phases, caused by strongly adsorbed eluent additives. The latter type of deformation was highly tuneable and it was possible to adjust the enantiomer peak shapes so that the peaks tailed in opposite directions with the sharp sides in between, yielding baseline resolution at remarkably high sample loads.In a final applied study both the LC-based perturbation peak method and a biosensor method based on surface plasmon resonance (SPR) were used for the first time for detailed characterization of chiral drug-protein interactions. The fundamental properties of the two very different methods were compared and it was found that the LC method is more suitable for multi-component analysis and that the SPR method is more suitable for stronger interactions.
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| 17. |
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| 18. |
- Arnell, Robert, et al.
(författare)
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Validation of the Tracer-Pulse Method for Multi-Component Liquid Chromatography. A Classical Paradox Revisited
- 2006
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 78:13, s. 4615-4623
-
Tidskriftsartikel (refereegranskat)abstract
- The tracer-pulse method was extended and validated for the determination of multicomponent adsorption isotherms in liquid chromatography. Competitive adsorption isotherms can be determined for any number of solutes, up to the column resolution limit. The basic principle is to equilibrate the column with an eluent containing a mixture of the solutes and then measure the migration velocity of each of them through the column. It is easy to calculate the stationary phase concentrations from these velocities, given the eluent composition. As in frontal analysis, real competitive isotherm data are measured using this method, unlike other methods, which only produce parametric estimates. The method was used to measure the binary isotherms of beta-blockers on a Kromasil C8 column. The data were fitted to competitive bi-Langmuir adsorption isotherm functions and was found to agree well with the results of frontal analysis and the perturbation method. Computer simulations based on the isotherm parameters were performed and displayed very good agreement with the experimental chromatograms. An intriguing and seemingly paradoxical property is visualized and discussed: the fact that the injected molecules are not found in the detected peaks.
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| 19. |
- 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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| 20. |
- Cilpa-Karhu, Geraldine, et al.
(författare)
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Three complementary techniques for the clarification of temperature effect on low-density lipoprotein–chondroitin-6-sulfate interaction
- 2013
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 443:2, s. 139-147
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Tidskriftsartikel (refereegranskat)abstract
- Abstract A rigorous processing of adsorption data from quartz crystal microbalance technology was successfully combined with the data obtained by partial filling affinity capillary electrophoresis and molecular dynamics for the clarification of the temperature effect on the interaction of a major glycosaminoglycan chain chondroitin-6-sulfate (C6S) of proteoglycans with low-density lipoprotein (LDL) and with a peptide fragment of apolipoprotein B-100 (residues 3359–3377 of LDL, PPBS). Two experimental techniques and computational atomistic methods demonstrated a nonlinear pattern of the affinity of C6S at temperatures above 38.0 °C to both LDL and PPBS. The temperature affects the interaction of C6S with LDL and PPBS by influencing the structural behavior of glycosaminoglycan C6S and/or that of LDL.
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| 21. |
- Encarnacao, Joao Crispim, et al.
(författare)
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Impact of assay temperature on antibody binding characteristics in living cells : A case study
- 2017
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Ingår i: BIOMEDICAL REPORTS. - : Spandidos Publications. - 2049-9434 .- 2049-9442. ; 7:5, s. 400-406
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Tidskriftsartikel (refereegranskat)abstract
- Kinetic and thermodynamic studies of ligand-receptor interactions are essential for increasing the understanding of receptor activation mechanisms and drug behavior. The characterization of molecular interactions on living cells in real-time goes beyond most current binding assays, and provides valuable information about the dynamics and underlying mechanism of the molecules in a living system. The effect of temperature on interactions in cell-based assays is, however, rarely discussed. In the present study, the effect of temperature on binding of monoclonal antibodies, cetuximab and pertuzumab to specific receptors on living cancer cells was evaluated, and the affinity and kinetics of the interactions were estimated at selected key temperatures. Changes in the behavior of the interactions, particularly in the on- and off-rates were observed, leading to greatly extended time to reach the equilibrium at 21 degrees C compared with at 37 degrees C. However, the observed changes in kinetic characteristics were less than a factor of 10. It was concluded that it is possible to conduct real-time measurements with living cells at different temperatures, and demonstrated that influences of the ambient temperature on the interaction behavior are likely to be less than one order of magnitude.
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| 22. |
- Enmark, Martin, 1984-, et al.
(författare)
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A closer study of peak distortions in supercritical fluid chromatography as generated by the injection
- 2015
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Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1400, s. 131-139
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Tidskriftsartikel (refereegranskat)abstract
- Abstract In SFC the sample cannot be dissolved in the mobile phase, so it is often dissolved in pure modifier, or another liquid, sometimes resulting in serious distortions of the eluted peak profiles already at moderately high injection volumes. It is suspected the reasons for these effects are solvent strength mismatch and/or viscosity mismatch. This study presents a systematic and fundamental investigation of the origin of these peak deformations due to the injection solvent effects in SFC, using both systematic experiments and numerical modeling. The first set of experiments proved that the injection volume and the elution strength of the sample solution had a major impact of the shapes of the eluted peaks. Secondly, the sample band elution profile was numerically modeled on a theoretical basis assuming both un-retained and retained co-solvent injection plugs, respectively. These calculations quantitatively confirmed our first set of experiments but also pointed out that there is also an additional significant effect. Third, viscous fingering experiments were performed using viscosity contrast conditions imitating those encountered in SFC. These experiments clearly proved that viscous fingering effects play a significant role. A new method for determination of adsorption isotherms of solvents was also developed, called the “Retention Time Peak Method” (RTPM). The RTPM was used for fast estimation of the adsorption isotherms of the modifier and required using only two experiments.
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| 23. |
- Enmark, Martin, et al.
(författare)
-
A Deeper Understanding of a Complex Adsorption Behavior in a Common Chiral, Separation System
- 2011
-
Konferensbidrag (refereegranskat)abstract
- The adsorption equilibria of racemic methyl mandelate on a tris-(3,5-dimethylphenyl)carbamoyl cellulose chiral stationary phase (CSP) was investigated. This separation has previously been performed and analyzed on a methylcellulose tribenzoate coated CSP. In that case, both enantiomers could be described with a bi-Langmuir adsorption isotherm, convex upwards (type I). In our case, two fundamentally different properties were observed. First, the elution order was reversed. Furthermore, only the less retained enantiomer shows type I adsorption behavior, while the adsorption isotherm of the more retained compound contained an inflection point at low concentration. To analyze these differences, adsorption isotherms were determined and further analyzed using Scatchard plots and adsorption energy distribution calculations. The less retained enantiomer was best described by Tóth adsorption isotherm while the second enantiomer was best described with a bi-Moreau adsorption isotherm. The Moreau model is an extension of the Langmuir model including non-ideal adsorbate-adsorbate interactions; here the unusual model provided an explanation to the non-ideal adsorption of the more retained enantiomer. Furthermore, the possibility of using the Moreau model as a local model for adsorption in AED calculations was evaluated by synthetically generated raw adsorption slope data. It was demonstrated that the AED accurately could predict the number of adsorption sites for the generated data. The adsorption behavior of both enantiomers was also studied at several different temperatures and it was found to be exothermic; in addition, the non-idealadsorbate-adsorbate interaction strength decreases with increasing temperature. Stochastic analysis of the adsorption process could identify a single kinetic site for each enantiomer. The average amount of adsorption/desorption events increases and the sojourn time decreases with increasing temperature.
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| 24. |
- Enmark, Martin, 1984-, et al.
(författare)
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A Retention-Matching Strategy for Method Transfer in Supercritical Fluid Chromatography : Introducing the Isomolar Plot Approach
- 2021
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 93:16, s. 6385-6393
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 25. |
- Enmark, Martin, et al.
(författare)
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A systematic investigation of algorithm impact in preparative chromatography with experimental verifications
- 2011
-
Ingår i: Journal of Chromatography A. - Amsterdam : Elsevier. - 0021-9673 .- 1873-3778. ; 1218:5, s. 662-672
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Tidskriftsartikel (refereegranskat)abstract
- Computer-assisted optimization of chromatographic separations requires finding the numerical solution of the Equilibrium-Dispersive (ED) mass balance equation. Furthermore, the competitive adsorption isotherms needed for optimization are often estimated numerically using the inverse method that also solves the ED equations. This means that the accuracy of the estimated adsorption isotherm parameters explicitly depends on the numerical accuracy of the algorithm that is used to solve the ED equations. The fast and commonly used algorithm for this purpose, the Rouchon Finite Difference (RFD) algorithm, has often been reported not to be able to accurately solve the ED equations for all practical preparative experimental conditions, but its limitations has never been completely and systematically investigated. In this study, we thoroughly investigate three different algorithms used to solve the ED equations: the RFD algorithm, the Orthogonal Collocation on Finite Elements (OCFE) method and a Central Difference Method (CDM) algorithm, both for increased theoretical understanding and for real cases of industrial interest. We identified discrepancies between the conventional RFD algorithm and the more accurate OCFE and CDM algorithms for several conditions, such as low efficiency, increasing number of simulated components and components present at different concentrations. Given high enough efficiency, we experimentally demonstrate good prediction of experimental data of a quaternary separation problem using either algorithm, but better prediction using OCFE/CDM for a binary low efficiency separation problem or separations when the compounds have different efficiency. Our conclusion is to use the RFD algorithm with caution when such conditions are present and that the rule of thumb that the number of theoretical plates should be greater than 1000 for application of the RFD algorithm is underestimated in many cases
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| 26. |
- Enmark, Martin, et al.
(författare)
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A systematic investigation on the accuracy of computer simulations for optical isomers in industrial settings
- 2010
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Konferensbidrag (refereegranskat)abstract
- Predicting the band profiles of typical chromatographic separations using the approach of the inverse method (IM) [2] requires finding the numerical solution of the Equilibrium-Dispersive (ED) mass balance equation [1]. The accuracy of the determination of the competitive adsorption isotherms is therefore explicitly dependent on the choice of algorithm to solve the ED model. Earlier studies have shown that the well known RFD algorithm [3] has limitations in its accuracy for certain simulated conditions such as column efficiency and adsorption isotherm type [5]. In this study, we thoroughly investigate three different algorithms, the rapid and well known Rouchon Finite Difference (RFD) algorithm [3], the Orthogonal Collocation on Finite Elements (OCFE) method [4] and the Central Difference Method (CDM) algorithm developed by us. Firstly, a systematic investigation is made, comparing the different algorithms under a broad range of different synthetic conditions. Secondly, we apply all three algorithms using the IM on real experimental systems; (I) one high efficiency quaternary separation and (II) one pharmaceutical industry application with a chiral intermediate separation. We have found discrepancies between the conventional (RFD) and the more accurate (OCFE and CDM) algorithms for several synthetic conditions such as low efficiency, increasing number of simulated components and components present at different concentrations. Given high enough efficiency, we experimentally demonstrate good prediction of experimental data of a quaternary separation problem using either algorithm but better prediction of OCFE/CDM for a binary low efficiency separation problem. Our conclusion is to use the RFD algorithm with caution when any of the particular conditions we have investigated is valid. When the highest accuracy is sought, there is no doubt that OCFE or CDM should be the algorithms of choice. However, given the computational speed of RFD, we also recommend it for preliminary parameter fitting. For conditions where we have shown the algorithms to produce practically identical solutions, RFD can be applied with greater certainty. It remains to be thoroughly investigated the effect on the particular shape of the adsorption isotherm on the obtained solution, for example type III having inflection points.
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| 27. |
- Enmark, Martin, et al.
(författare)
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A thermodynamic and kinetic study of an unusual adsorption behavior-Methyl Mandelate on commercially available Tris-(3,5- dimethylphenyl)carbamoyl Cellulose Chiral Stationary Phase
- 2012
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The adsorption equilibria of racemic methyl mandelate on a tris-(3,5- dimethylphenyl)carbamoyl cellulose chiral stationary phase (CSP) was investigated. The following were observed, the less retained enantiomer shows “Langmuirian†(type I) adsorption behavior, while the adsorption isotherm of the more retained compound contained an inflection point at low concentration. To analyze these differences, adsorption isotherms were determined and further analyzed using Scatchard plots and adsorption energy distribution (AED) calculations. The less retained enantiomer was best described by heterogeneous unimodal adsorption model (Tóth) while the second enantiomer was best described with a heterogeneous adsorption model with adsorbate-adsorbate interactions (bi-Moreau). The adsorption behavior of both enantiomers was also studied at several different temperatures and it was found to be exothermic; in addition, the non-idealadsorbate-adsorbate interaction strength decreases with increasing temperature. Stochastic analysis of the adsorption process could identify a single kinetic site for each enantiomer. The average amount of adsorption/desorption events increases and the sojourn time decreases with increasing temperature. This is an industrial – academic cooperation in the Fundamental Separation Science Group www.separationscience.se
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| 28. |
- Enmark, Martin, et al.
(författare)
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Adsorption Isotherm Determination for Reliable Phase System Characterization in SFC : Challenges and Pitfalls
- 2012
-
Konferensbidrag (refereegranskat)abstract
- Recently the pharmaceutical industry has started to replace HPLC with SFC because of incentives to lower the environmental impact and as well as increasing performance. Reliable characterization of the adsorption processes in SFC, is therefore of utmost importance. The key thermodynamic phase system information is obtained by rigorous determination of adsorption isotherms over a broad concentration range. If properly processed, this data gives not only correct information about the degree of heterogeneity but also the values of the energy of interactions and monolayer capacities of each individual type of adsorption site in the phase system; ultimately, this can result in identification of the types of interactions (dipole-dipole, van der Waals interactions etc.). In this study, we will present the transfer of LC adsorption characterization methods to SFC conditions using several model compounds with several different methods for adsorption isotherm determination traditionally applied with success in LC, and now modified for SFC. We have limited our investigation to methanol as modifier and used the operational conditions, temperature and backpressure most typically observed in industrial settings; in addition, we have used commercial standard SFC-equipment. The results clearly shows that adsorption isotherm determinations in SFC are considerably more complicated than in LC; we will go through the most important pitfalls and give guidelines for more rigorous determinations of adsorption data in SFC. This is an industrial – academic cooperation in the Fundamental Separation Science Group www.separationscience.se
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| 29. |
- Enmark, Martin, 1984-, et al.
(författare)
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Analytical and preparative separation of phosphorothioated oligonucleotides : Columns and ion-pair reagents
- 2020
-
Ingår i: Analytical and Bioanalytical Chemistry. - : Springer. - 1618-2642 .- 1618-2650. ; 412:2, s. 299-309
-
Tidskriftsartikel (refereegranskat)abstract
- 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 520mers 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.
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| 30. |
- Enmark, Martin, 1984-, et al.
(författare)
-
Building machine-learning-based models for retention time and resolution predictions in ion pair chromatography of oligonucleotides
- 2022
-
Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1671
-
Tidskriftsartikel (refereegranskat)abstract
- 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 0 0 0 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.
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| 31. |
- Enmark, Martin, et al.
(författare)
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Characterization of the Adsorption of Racemic Methyl Mandelate on Tris-(3,5-dimethylphenyl)carbamoyl Cellulose Chiral Stationary Phase
- 2011
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Konferensbidrag (refereegranskat)abstract
- The adsorption equilibria of racemic methyl mandelate on a tris-(3,5-dimethylphenyl)carbamoyl cellulose chiral stationary phase (CSP) was investigated. This separation has previously been performed and analyzed on a methylcellulose tribenzoate coated CSP. In that case, both enantiomers could be described with a bi-Langmuir adsorption isotherm, convex upwards (type I). In our case, two fundamentally different properties were observed. First, the elution order was reversed. Furthermore, only the less retained enantiomer shows type I adsorption behavior, while the adsorption isotherm of the more retained compound contained an inflection point at low concentration. To analyze these differences, adsorption isotherms were determined and further analyzed using Scatchard plots and adsorption energy distribution calculations. The less retained enantiomer was best described by Tóth adsorption isotherm while the second enantiomer was best described with a bi-Moreau adsorption isotherm. The Moreau model is an extension of the Langmuir model including non-ideal adsorbate-adsorbate interactions; here the unusual model provided an explanation to the non-ideal adsorption of the more retained enantiomer. Furthermore, the possibility of using the Moreau model as a local model for adsorption in AED calculations was evaluated by synthetically generated raw adsorption slope data. It was demonstrated that the AED accurately could predict the number of adsorption sites for the generated data. The adsorption behavior of both enantiomers was also studied at several different temperatures and it was found to be exothermic; in addition, the non-idealadsorbate-adsorbate interaction strength decreases with increasing temperature. Stochastic analysis of the adsorption process could identify a single kinetic site for each enantiomer. The average amount of adsorption/desorption events increases and the sojourn time decreases with increasing temperature.
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| 32. |
- Enmark, Martin, et al.
(författare)
-
Computer-assisted optimization in preparative SFC, Challenges, Pitfalls and Solutions
- 2010
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Konferensbidrag (refereegranskat)abstract
- Preparative packed column SFC (pSFC) is an environmentally favorable alternative to normal phase HPLC. We are participating in a larger research project where the overreaching goal is to make an impact in the recovery of high-value compounds from agricultural byproducts and waste materials using environmentally sustainable techniques. Our specific task is to isolate large amounts of valuable compounds from discarded biomaterials using pSFC. It is still not possible to accurately model, simulate and optimize a preparative SFC proess, although SFC has been in use for almost fifty years. This is mainly because of the compressibility of the mobile phase and how this influences the physical parameters of the system. More particular, if pSFC is operated under combinations of back pressures and flow rates that give negligible pressure drop, it has been demonstrated and implied, that the tools developed and used within HPLC, can be transferred and used successfully [1, 2]. However, when the pressure drop becomes noticeable, the induced density gradient significantly increases the complexity. An earlier study has clearly described the effect of this pressure gradient on the retention factor, and also the column efficiency [3]. However, it remains to be investigated how the entire adsorption isotherm depends on the density.In this study we will investigate the use of the inverse and the perturbation peak methods, originally developed within LC, to determine the nonlinear adsorption isotherm for i) negligible, and ii) non-negligible pressure drop conditions respectively. To do this, we will use a simple racemic substance and a cellulose based CSP with a column equipped with pressure sensors at the inlet and outlet. Using obtained parameters we will validate the methods by simulating the separation using a variable coefficient version of the Equilibrium-Dispersive model for cases i and ii. [1] S. Ottiger, J. Kluge, A. Rajendran , M. Mazzotti, J. Chromatogr., A 1162 (2007) 74-82[2] C. Wenda, A. Rajendran, J. Chromatogr., A 1216 (2009) 8750-8758[3]A. Rajendran, O. Kräuchi, M. Mazzotti, M. Morbidelli, J Chromatogr., A, 1092 (2005) 149-160[4] G. Guiochon, A. Felinger, D. G. Shirazi, A. M. Katti, Fundamentals of Preparative and Nonlinear Chromatography 2nd Ed, Elsevier Academic Press (2006).
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| 33. |
- Enmark, Martin, et al.
(författare)
-
Computer Assisted Optimization of Pharmaceutical Purification - The Impact of Algorithms and Experimental Approach
- 2010
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Konferensbidrag (refereegranskat)abstract
- Computer assisted process optimization of chromatographic separations requires the selection of computer algorithms and measurement of relevant parameters. In this study, we will investigate how the choice of algorithms and the number of performed initial experiments affect the estimated optimal separations conditions. We will focus on conditions typically encountered for slurry packing coated 20 µm CSP in large-scale columns used for pharmaceutical intermediate purification.Probably the most essential for parameters in this context are the adsorption isotherm parameters for the components. The rapid "inverse method" is commonly used and this method requires that one numerically solve the mass balance equations describing the chromatographic process. Here we thoroughly investigate how different algorithms that solve the Equilibrium-Dispersive (ED) mass balance equations will affect the estimated adsorption isotherm parameters.Furthermore, we will investigate and compare how different strategies affect the prediction of the optimal separation conditions. First, we will use a more rapid approach that requires a minimum of experiments and uses standard algorithms to estimate optimal conditions. Secondly, we will use a more exact approach that requires more experiments and uses more advanced simulation- and optimization algorithms
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| 34. |
- Enmark, Martin, 1984-, et al.
(författare)
-
Determination of adsorption isotherms in supercritical fluid chromatography
- 2013
-
Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1312, s. 124-133
-
Tidskriftsartikel (refereegranskat)abstract
- Abstract In this study we will demonstrate the potential of modern integrated commercial analytical SFC-systems for rapid and reliable acquisition of thermodynamic data. This will be done by transferring the following adsorption isotherm determination methods from liquid chromatography (LC) to supercritical fluid chromatography (SFC): Elution by Characteristic Points (ECP), the Retention Time Method (RTM), the Inverse Method (IM) and the Perturbation Peak (PP) method. In order to transfer these methods to SFC in a reliable, reproducible way we will demonstrate that careful system verification using external sensors of mass flow, temperature and pressure are needed first. The adsorption isotherm data generated by the different methods were analyzed and compared and the adsorption isotherms ability to predict new experimental elution profiles was verified by comparing experiments with simulations. It was found that adsorption isotherm data determined based on elution profiles, i.e., ECP, IM and RTM, were able to accurately predict overloaded experimental elution profiles while the more tedious and time-consuming PP method, based on small injections on concentration plateaus, failed in doing so.
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| 35. |
- Enmark, Martin, 1984-, et al.
(författare)
-
Development of a unified gradient theory for ion-pair chromatography using oligonucleotide separations as a model case
- 2023
-
Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1691
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 36. |
- Enmark, Martin, et al.
(författare)
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Enantioseparation of omeprazole effect of different packing particle size on productivity
- 2012
-
Ingår i: Journal of Chromatography A. - Amsterdam : Elsevier. - 0021-9673. ; 1240:1, s. 123-131
-
Tidskriftsartikel (refereegranskat)abstract
- Enantiomeric separation of omeprazole has been extensively studied regarding both product analysis and preparation using several different chiral stationary phases. In this study, the preparative chiral separation of omeprazole is optimized for productivity using three different columns packed with amylose tris (3,5-dimethyl phenyl carbamate) coated macroporous silica (5, 10 and 25 Όm) with a maximum allowed pressure drop ranging from 50 to 400 bar. This pressure range both covers low pressure process systems (50–100 bar) and investigates the potential for allowing higher pressure limits in preparative applications in a future. The process optimization clearly show that the larger 25 Όm packing material show higher productivity at low pressure drops whereas with increasing pressure drops the smaller packing materials have substantially higher productivity. Interestingly, at all pressure drops, the smaller packing material result in lower solvent consumption (L solvent/kg product); the higher the accepted pressure drop, the larger the gain in reduced solvent consumption. The experimental adsorption isotherms were not identical for the different packing material sizes; therefore all calculations were recalculated and reevaluated assuming identical adsorption isotherms (with the 10 Όm isotherm as reference) which confirmed the trends regarding productivity and solvent consumption.
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| 37. |
- Enmark, Martin, et al.
(författare)
-
Evaluation of scale-up from analytical to preparative supercritical fluid chromatography
- 2015
-
Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1425, s. 280-286
-
Tidskriftsartikel (refereegranskat)abstract
- An approach for reliable transfer from analytical to preparative scale supercritical fluid chromatography was evaluated. Here, we accounted for the conditions inside the columns as well as to the fact that most analytical instruments are volume-controlled while most preparative scale units are mass-controlled. The latter is a particular problem when performing pilot scale experiments and optimizations prior to scaling up to production scale. This was solved by measuring the mass flow, the pressure and the temperature on the analytical unit using external sensors. Thereafter, it was revealed with a design of experiments approach that the methanol fraction and the pressure are the two most important parameters to control for preserved retention throughout the scale-up; for preserved selectivity the temperature was most important in this particular system. Using this approach, the resulting chromatograms from the preparative unit agreed well with those from the analytical unit while keeping the same column length and particles size. A brief investigation on how the solute elution volume varies with the volumetric flow rate revealed a complex dependency on pressure, density and apparent methanol content. Since the methanol content is a parameter of great importance to control during the scale up, we must be careful when changing operational and column design conditions which generates deviations in pressure, density and methanol content between different columns. (C) 2015 Elsevier B.V. All rights reserved.
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| 38. |
- Enmark, Martin, 1984-
(författare)
-
Fundamental Investigations of Supercritical Fluid Chromatography
- 2015
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis aims at a deeper understanding of Supercritical FluidChromatography (SFC). Although preparative SFC has started to replace Liquid Chromatography (LC) in the pharmaceutical industry - because of its advantages in speed and its less environmental impact - fundamental understanding is still lacking. Therefore there is no rigid framework to characterize adsorption or to understand the impact of changes in operational conditions. In Paper I we demonstrated, after careful system verification, that most methods applied to determine adsorption isotherms in LC could not be applied directly in SFC. This was mainly due to operational differences and to the fact that the fluid is compressible which means that everything considered constant in LC varies in SFC. In Paper II we showed that the most accurate methods for adsorption isotherm determination in LC, the so called plateau methods, do not work properly for SFC. Instead, methods based on overloaded profiles should be preferred. In Paper III a Design of Experiments approach was successfully used to quantitatively describe the retention behavior of several solutes and the productivity of a two component separation system. This approach can be used to optimize SFC separations or to provide information about the separation system. In Paper IV severe peak distortion effects, suspected to arise from injection solvent and mobile phase fluid mismatches, were carefully investigated using experiments and simulations. By this approach it was possible to examine the underlying reasons for the distortions, which is vital for method development. Finally, in Paper V, the acquired knowledge from Paper I-IV was used to perform reliable scale-up in an industrial setting for the first time. This was done by carefully matching the conditions inside the analytical and preparative column with each other. The results could therefore provide the industry with key knowledge for further implementation of SFC.
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| 39. |
- Enmark, Martin, 1984-, et al.
(författare)
-
Investigation of factors influencing the separation of diastereomers of phosphorothioated oligonucleotides
- 2019
-
Ingår i: Analytical and Bioanalytical Chemistry. - : Springer. - 1618-2642 .- 1618-2650. ; 411:15, s. 3383-3394
-
Tidskriftsartikel (refereegranskat)abstract
- This study presents a systematic investigation of factors influencing the chromatographic separation of diastereomers of phosphorothioated pentameric oligonucleotides as model solutes. Separation was carried out under ion-pairing conditions using an XBridge C18 column. For oligonucleotides with a single sulfur substitution, the diastereomer selectivity was found to increase with decreasing carbon chain length of the tertiary alkylamine used as an ion-pair reagent. Using an ion-pair reagent with high selectivity for diastereomers, triethylammonium, it was found the selectivity increased with decreased ion-pair concentration and shallower gradient slope. Selectivity was also demonstrated to be dependent on the position of the modified linkage. Substitutions at the center of the pentamer resulted in higher diastereomer selectivity compared to substitutions at either end. For mono-substituted oligonucleotides, the retention order and stereo configuration were consistently found to be correlated, with Rp followed by Sp, regardless of which linkage was modified. The type of nucleobase greatly affects the observed selectivity. A pentamer of cytosine has about twice the diastereomer selectivity of that of thymine. When investigating the retention of various oligonucleotides eluted using tributylammonium as the ion-pairing reagent, no diastereomer selectivity could be observed. However, retention was found to be dependent on both the degree and position of sulfur substitution as well as on the nucleobase. When analyzing fractions collected in the front and tail of overloaded injections, a significant difference was found in the ratio between Rp and Sp diastereomers, indicating that the peak broadening observed when using tributylammonium could be explained by partial diastereomer separation.
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| 40. |
- Enmark, Martin, 1984-, et al.
(författare)
-
Investigation of plateau methods for adsorption isotherm determination in supercritical fluid chromatography
- 2014
-
Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1354, s. 129-138
-
Tidskriftsartikel (refereegranskat)abstract
- The Perturbation Peak (PP) method and Frontal analysis (FA) are considered as the most accurate methods for adsorption isotherms determination in liquid chromatography. In this study we investigate and explain why this is not the case in Supercritical Fluid Chromatography (SFC), where the PP method does not work at all, using a modern analytical system. The main reason was found to be that the solute to be studied must be dissolved in the MeOH reservoir before it is mixed with CO2. Since the solute occupies a certain partial volume in the reservoir, the larger the solute content the larger this fractional volume will be, and the final MeOH fraction in the mobile phase will then be smaller compared to the bulk mobile phase without solute in the modifier. If the retention of small injections on the concentration plateaus, i.e., “analytical-size” perturbation peaks, is sensitive to small variations of MeOH in the eluent, this will seriously decrease the accuracy of the PP method. This effect was verified and compensated for and we also demonstrated that the same problem will occur in frontal analysis, another concentration plateau method.
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| 41. |
- Enmark, Martin, 1984-, et al.
(författare)
-
Investigation of robustness for supercritical fluid chromatography separation of peptides : Isocratic vs gradient mode
- 2018
-
Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1568, s. 177-187
-
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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| 42. |
- Enmark, Martin, et al.
(författare)
-
On the Reproducibility between Different Modern Supercritcal Fluid Chromatographic Systems
- 2013
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Three different, commercially available, Supercritical Fluid Chromatography (SFC) systems were investigated: Thar Super Pure Discovery Series SFC, Waters UPC2 and Agilent 1260 Infinity SFC. The reason for choosing the two analytical systems from Agilent and Waters is that they represent two of the latest commercial systems available while the semi-preparative instrument was added to widen the study to include instruments also used for preparative purposes. With identical operational conditions set these systems were used to acquire analytical retention data and adsorption isotherms from overloaded injections. The investigation was limited to the use of methanol as modifier and operational conditions, temperature and back pressure most typically observed when utilizing SFC to separate polar compounds. The results clearly show that both analytical retention times and elution profiles are system dependent. Since the overloaded elution profiles are system dependent the adsorption isotherm will also be different. However, this do not mean that the adsorption is different, instead this it is due to the fact that identical instrumental settings, especially pressure and modifier composition settings, does not necessarily mean that the conditions inside the column are identical. This means that it is not possible to transfer an established separation method from one system to another, even if one is using the same column and identical instrument settings. Understanding of SFC-systems will be of fundamental importance for successful transfer of methods between systems, reliable adsorption isotherm determination, and analytical quality work and scaling up from analytical to preparative mode. These issues can probably be solved by measuring mass flow, pressure and temperature along the column, together with a sound understanding of the density variations of the mobile phase. However, the work of finding acceptable applications or guidelines to remove the tedious need for these measurements is currently investigated in our lab. This is a contribution from the Fundamental Separation Science Group www.FSSG.se
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| 43. |
- Enmark, Martin, et al.
(författare)
-
Preparative Separation of Chiral Pharmaceutical Compounds - The Effects of Packing Particle Size, Pressure Limit and Column Geometry on Productivity and Solvent Consumption
- 2011
-
Konferensbidrag (refereegranskat)abstract
- In this study, omeprazole was used as a model compound. Omeprazole and other related sulfoxidebenzimidazolesare used against gastric ulcersand have been extensively studied regarding chromatographic resolution techniques using several different chiral stationary phases.First, AstraZeneca launched Losec, a racemic mixture of RS-omeprazole. Facing loss of patent, the more potent S-enantiomer was developed and marketed as Nexium. Now the patent of Nexium is close expiration why methods for isolation of the pure S-enantiomer will be of importance for the generic pharmaceutical companies.The experimental model separation system represents a system with good selectivity and high solubility of the solute in the eluent. In this investigation the productivity optima for three different particle sizes (5, 10 and 25 µm) at maximum system pressure ranging from 50 to 400 bars are studied. Two different optimizations cases were studied in depth. First,a process optimization with fixed column geometry is studied. The results clearly show that larger packing materials have higher productivity at low pressure drops on the analytical size column.With increasing allowed pressure drops, over 200 bar, the smaller packing materials have substantially higher productivity. The results also show that smaller packing material will always have much lower solvent consumption compared to larger particles.The second process optimization was performed with a fixed column volume, but the column geometry was variable. The results shows that the productivity obtained for the smaller packing particles materials was higher compared to the large for all allowed pressure drops. The productivity obtained for the small particle compared to the large increased by 25-300 % while maintaining 50-300 % less solvent consumption for the purification of the first enantiomer.The addition of TEA seems to be unfavorable for all tested conditions.In conclusion, the optimization of the enantioseparation of omeprazole has been shown to be dependent on column packing particle size as well as column geometry. It has been demonstrated that all parameters need to be simultaneously optimized to reach a global productivity optima.
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| 44. |
- Enmark, Martin, et al.
(författare)
-
Preparative Separation of Omeprazole: Predictions of the Optimal Experimental Conditions by Computer Simulations
- 2011
-
Konferensbidrag (refereegranskat)abstract
- In this study, omeprazole was used as a model compound. Omeprazole and other related sulfoxide benzimidazoles are used against gastric ulcers and have been extensively studied regarding chromatographic resolution techniques using several different chiral stationary phases.First, AstraZeneca launched Losec, a racemic mixture of RS-omeprazole. Facing loss of patent, the more potent S-enantiomer was developed and marketed as Nexium. Now the patent of Nexium is close expiration why methods for isolation of the pure S-enantiomer will be of importance for the generic pharmaceutical companies.The experimental model separation system represents a system with good selectivity and high solubility of the solute in the eluent. In this investigation the productivity optima for three different particle sizes (5, 10 and 25 µm) at maximum system pressure ranging from 50 to 400 bars are studied. Two different optimizations cases were studied in depth. First,a process optimization with fixed column geometry is studied. The results clearly show that larger packing materials have higher productivity at low pressure drops on the analytical size column.With increasing allowed pressure drops, over 200 bar, the smaller packing materials have substantially higher productivity.The results also show that smaller packing material will always have much lower solvent consumption compared to larger particles.The second process optimization was performed with a fixed column volume, but the column geometry was variable. The results shows that the productivity obtained for the smaller packing particles materials was higher compared to the large for all allowed pressure drops. The productivity obtained for the small particle compared to the large increased by 25-300 % while maintaining 50-300 % less solvent consumption for the purification of the first enantiomer.The addition of TEA seems to be unfavorable for all tested conditions.In conclusion, the optimization of the enantioseparation of omeprazole has been shown to be dependent on column packing particle size as well as column geometry. It has been demonstrated that all parameters need to be simultaneously optimized to reach a global productivity optima.
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| 45. |
- Enmark, Martin, 1984-, et al.
(författare)
-
Selectivity limits of and opportunities for ion pair chromatographic separation of oligonucleotides
- 2021
-
Ingår i: Journal of Chromatography A. - : Elsevier. - 0021-9673 .- 1873-3778. ; 1651
-
Tidskriftsartikel (refereegranskat)abstract
- A B S T R A C T 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 anti sense oligonucleotide separation problems, and it was shown that the ion-pair reagent gradient increases the selectivity of non-charge & ndash;based separation problems while maintaining charge-difference & ndash;based selectivity. (c) 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )
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| 46. |
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| 47. |
- Enmark, Martin, et al.
(författare)
-
Using Modern SFC Systems for Adsorption Characterization
- 2013
-
Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Recently the pharmaceutical industry has started to replace preparative HPLC with preparative SFC to lower the environmental impact and to increase performance. Reliable characterization of the adsorption processes in SFC is therefore of utmost importance. The key thermodynamic phase system information is obtained by rigorous determination of adsorption isotherm data over a broad concentration range. If properly processed, this data gives not only correct information about the degree of heterogeneity but also the energy of interactions and mono layer capacities of each individual type of adsorption site in the phase system. Ultimately, this can result in identification of the types of interactions, i.e., dipole-dipole, van der Waals interactions etc. In this study we will present transfer of selected adsorption characterization methods, traditionally applied with success in LC, to SFC. We have here transferred all available knowledge from LC – from model selection to validation. We will also, using recent findings, explain the effects of pressure and temperature variations as well as how to accurately measure the volumetric flow rate on a modern analytical SFC system. We will demonstrate how the latest SFC instruments, with some critical modifications; have the potential for rapid and reliable acquisition of thermodynamic data using the ECP method. Finally we will elaborate on how the adsorption depends on density, temperature and modifier content in the mobile phase. This is a contribution from the Fundamental Separation Science Group www.FSSG.se
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| 48. |
- Fornstedt, Torgny, et al.
(författare)
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A more reliable procedure for estimating interactions between drugs and biomolecules using biosensors : a comparison with chromatography
- 2012
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- This poster serves as background information to the corresponding lecture. Adsorption isotherms are essential in order to understand the interaction between small molecules such as pharmaceutical compounds and larger biomolecules. An adsorption isotherm describes the relationship of free substance in a solution with adsorbed substance to a surface, at a specific and constant temperature. Adsorption isotherms could be determined using several different method, all method have their pros and cons. In this study we are using two modern but principally different biosensors to determine interactions: quarts micro-balance (QCM) and Surface plasmon resonance (SPR) to determine interactions. For a long time adsorption isotherms has been determined solely by the chromatographic community. In this study we will present transformation of adsorption analysis tools from chromatography to biosensors, especially calculation of adsorption energy distribution prior adsorption model fit. We will also discuss how the experiments should be conducted. Guidelines will be given for the experimental setup and for when the chromatographic or a biosensor technique is to be preferred. This is a contribution from the Fundamental Separation Science Group in Karlstad www.separationscience.se
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