| 1. |
- Samuelsson, Jörgen, 1971-
(author)
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Development of Methods for Phase System Characterization in Liquid Chromatography
- 2008
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Doctoral thesis (other academic/artistic)abstract
- The aim of this thesis is first and foremost to improve the fundamental knowledge of nonlinear and preparative separation theory by focusing on some of the remaining “white spots” on the theoretical chromatographic map. Secondly, the acquired knowledge is used to develop, validate and execute new methods for phase characterization in liquid chromatography. The methodology used in this thesis is a combination of experiments, fundamental nonlinear theory and systematic computer simulations. A fundamental knowledge of the molecular interactions between the compounds to be separated and the separation media requires the determination of adsorption isotherms over a broad concentration range to give a complete picture of all interactions in the separation system - weak as well as strong. In addition, such adsorption data is essential for optimization in preparative chromatography. For the first time, it has been experimentally shown that the injected molecules are not present in the detected peak when a small excess of molecules are injected into a chromatographic system equilibrated with a constant stream of identical molecules. Several experimental procedures for this method were developed such as (i) the optimal injection strategy and (ii) different labeling methods for visualizing the injected molecules. Remarkable phenomena in the single-component case, such as invisible peak deformation and deformed (invisible) frontal chromatograms, are reported, investigated, and explained. This phenomenon has asides from its future practical implementation, also a large didactic value. The accuracy of the ECP method is experimentally improved, and used to characterize the separation of protolytic compounds at different pH on modern commercially available silica and hybrid silica column packing materials. That investigation enables us to answer why basic compounds give a much more compact preparative peak profile at pH 11 than they yields at lower pH.
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| 2. |
- Enmark, Martin, 1984-
(author)
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Fundamental Investigations of Supercritical Fluid Chromatography
- 2015
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Doctoral thesis (other academic/artistic)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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| 3. |
- Forssén, Patrik, 1966-
(author)
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Adsorption Isotherm Parameter Estimation in Nonlinear Liquid Chromatography
- 2005
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Doctoral thesis (other academic/artistic)abstract
- This thesis concerns the development and validation of methods for the industrially important area of adsorption isotherm parameter estimation in preparative, nonlinear high performance liquid chromatography (HPLC). Preparative chromatography is a powerful separation method to get pure compounds from more or less complex liquid mixtures, e.g., mixtures of mirror-image molecules. Computer simulations can be used to optimize preparative chromatography, but then competitive adsorption isotherm parameters are usually required. Here two methods to estimate adsorption isotherm parameters are treated: (i) the perturbation peak (PP) method and (ii) the inverse method (IM).A new theory for the PP method was derived and led to a new injection technique which was validated experimentally. This injection technique solved the severe problem with vanishing peaks and enabled us to use the PP method to estimate binary competitive adsorption isotherms valid over a broad concentration range. Also, the injection technique made it possible to estimate competitive adsorption isotherms for a quaternary mixture for the first time. Finally, an interesting perturbation peak phenomenon, known as the “Helfferich Paradox”, was experimentally verified for the first time.The IM is a relatively new method to determine adsorption isotherm parameters. It has the advantage of requiring very small samples, but also requires an advanced computer algorithm. An improved implementation of this computer algorithm was developed and tested experimentally. Also, a variant of the IM called “the inverse method on plateaus” was tested experimentally and the estimated adsorption isotherm parameters were shown to be valid over a broader concentration range than those estimated with the standard IM.
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| 4. |
- Glenne, Emelie
(author)
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Fundamental Investigations of Adsorption in SFC
- 2019
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Doctoral thesis (other academic/artistic)abstract
- In supercritical fluid chromatography (SFC), the eluent is composed by carbon dioxide, often with additional components, in a condition between gas and liquid. This thesis aims to reach a deeper understanding of SFC by revealing the function of the additional eluent components through systematic adsorption studies.In Paper I, investigation of surface excess adsorption isotherms of methanol revealed that a monolayer of methanol was formed. In Paper II, severe peak deformation effects due to this adsorption were shown. The findings in these papers revealed that a competitive additive model best predicts the solute retention at low methanol fractions whereas at higher fractions, methanol acts just as a modifier. In Paper III, the generality of the effects was proven by investigation of several co-solvent and stationary phase combinations. In Paper IV it was investigated how the robustness of SFC separations depend on the co-solvent adsorption, pressure, and temperature. In Paper V, the impact of the addition of amine additives was investigated. Two different mechanisms for solute peak deformations were observed.The knowledge achieved about SFC in this theses provides guidelines for development of more robust SFC methods where peak deformations/distortions can be avoided.
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| 5. |
- Olsson, Petter, 1981-
(author)
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Essentially Lipids : Analytical methods for the characterization of lipid materials
- 2015
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Doctoral thesis (other academic/artistic)abstract
- This thesis describes analytical methods for chromatographic characterizations of lipids in biological and technical systems.Lipids are a group of compounds with a central role in all known forms of life. In addition to the biological roles, lipids are also components in many products of our daily usage. The application areas include food, pharma, cosmetics, biofuels, etc.Analytical characterization of lipids is challenging since a typical lipid extract often contains several hundred unique compounds which also could vary significantly in chemical properties. Separation is in many cases crucial for identification and quantification of individual lipids and here high performance liquid chromatography (HPLC) is regarded as the technique of first choice today.In the present work, methodologies for both normal phase (NP) and reversed phase (RP) HPLC are presented. The focus has been to develop methods that are fast, have low analytical complexity and are compatible with several detection systems. Cyanopropyl coated silica was evaluated as stationary phase for NP-HPLC with the aim to separate all the lipid classes in common lipid extracts. The analytes were eluted with a binary mobile phase system based on hexane, toluene and methanol which yielded in a total runtime of 30 minutes. In a subsequent study, additional stationary phases for NP-HPLC were evaluated for online sample cleanup of polycyclic aromatic hydrocarbons (PAHs) in lipid matrixes. In the demonstrated methodology for RP-HPLC, a binary system consisting of methanol/water was utilized with phenylpropyl coated silica to elute all tested lipid classes in 30 minutes.The methodologies were compatible with various detection techniques including evaporative light scattering detection (ELSD), charged aerosol detection (CAD) as well as electrospray mass spectrometry (ESI-MS) and were applied to characterize lipid materials of both biological and technical nature.
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| 6. |
- Shamshir, Adel, 1986-
(author)
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Study of retention mechanisms in Hydrophilic Interaction Chromatography by Nuclear Magnetic Resonance Spectroscopy
- 2020
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Doctoral thesis (other academic/artistic)abstract
- This thesis deals with investigations of the retention mechanisms in hydrophilic interaction liquid chromatography (HILIC) using nuclear magnetic resonance spectroscopy. The aims are to understand how the different types of stationary phases can influence the retention of various solutes and the adsorption abilities of solvent on stationary phases.The thesis encompasses the development of a saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy method by which the mechanisms in HILIC were probed on hydrophilic and polar stationary phases with varying charge properties and water-retaining abilities under high-resolution magic angle spinning (HR-MAS) conditions. By applying the developed method, results show that toluene is indeed capable of traversing the water-enriched layers of all the three tested stationary phases. In addition, the STD-NMR method was applied to study interaction mechanisms using set hydrophilic compounds ranging from small organic acids, nucleobases, nucleosides, and other neutral molecules to further elucidate the differences in HILIC selectivity caused by a dipolar interaction, hydrogen bonding, and electrostatic interaction.Finally, the solvent adsorption on the stationary surface was studied by applying a nuclear magnetic relaxation dispersion (NMRD) technique in combination with a relaxation model in order to resolve the deuterium T1-NMRD profile of acetonitrile-d3 in aqueous solutions confined in the pores of modified HILIC silicas with nominal pore diameters ranging from of 6 to 10 nm. It was found that the acetonitrile-d3 had a strong field dependence at low magnetic fields, which was attributed to surface sites at which the molecules were trapped with residence times in the range of 0.1–3 µs.
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| 7. |
- Thewalim, Yasar, 1976-
(author)
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Retention time predictions in Gas Chromatography
- 2011
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Doctoral thesis (other academic/artistic)abstract
- In gas chromatography, analytes are separated by differences in their partition between a mobile phase and a stationary phase. Temperature-program, column dimensions, stationary and mobile phases, and flow rate are all parameters that can affect the quality of the separation in gas chromatography. To achieve a good separation (in a short amount of time) it is necessary to optimize these parameters. This can often be quite a tedious task.Using computer simulations, it is possible to both gain a better understanding of how the different parameters govern retention and separation of a given set of analytes, and to optimize the parameters within minutes. In the research presented here, this was achieved by taking a thermodynamic approach that used the two parameters ΔH (enthalpy change) and ΔS (entropy change) to predict retention times for gas chromatography. By determining these compound partition parameters, it was possible to predict retention times for analytes in temperature-programmed runs. This was achieved through the measurement of the retention times of n-alkanes, PAHs, alcohols, amines and compounds in the Grob calibration mixture in isothermal runs. The isothermally obtained partition coefficients, together with the column dimensions and specifications, were then used for computer simulation using in-house software.The two-parameter model was found to be both robust and precise and could be a useful tool for the prediction of retention times. It was shown that it is possible to calculate retention times with good precision and accuracy using this model. The relative differences between the predicted and experimental retention times for different compound groups were generally less than 1%.The scientific studies (Papers I-IV) are summarized and discussed in the main text of this thesis.
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| 8. |
- Undin, Torgny, 1975-
(author)
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Development and improvement of methods for characterization of HPLC stationary phases
- 2011
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Licentiate thesis (other academic/artistic)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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| 9. |
- Åsberg, Dennis, 1988-
(author)
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Fundamental and Regulatory Aspects of UHPLC in Pharmaceutical Analysis
- 2017
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Doctoral thesis (other academic/artistic)abstract
- Ultra-high performance liquid chromatography (UHPLC) provides a considerable increase in throughput compared to HPLC and a reduced solvent consumption. The implementation of UHPLC in pharmaceutical analysis, e.g. quality control, has accelerated in recent years and there is currently a mix of HPLC and UHPLC instrumentation within pharmaceutical companies. There are, however, technical and regulatory challenges converting a HPLC method to UHPLC making it difficult to take full advantage of UHPLC in regulatory-focused applications like quality control in pharmaceutical production.Using chromatographic modelling and fundamental theory, this thesis investigated method conversion between HPLC and UHPLC. It reports on the influence of temperature gradients due to viscous heating, pressure effects and stationary phase properties on the separation performance. It also presents a regulatory concept for less regulatory interaction for minor changes to approved methods to support efficient life cycle management.The higher pressure in UHPLC gave a retention increase of up to 40% as compared to conventional HPLC while viscous heating, instead, reduced retention and the net result was very solute dependent. Selectivity shifts were observed even between solutes with similar structure when switching between HPLC and UHPLC and an experimental method to predict such selectivity shifts was therefore developed. The peak shape was negatively affected by the increase in pressure for some solutes since secondary interactions between the solute and the stationary phase increased with pressure.With the upcoming ICH Q12 guideline, it will be possible for the industry to convert existing methods to UHPLC in a more flexible way using the deeper understanding and the regulatory concept presented here as a case example.
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| 10. |
- Lindholm, Johan, 1974-
(author)
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Development and Validation of HPLC Methods for Analytical and Preparative Purposes
- 2004
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Doctoral thesis (other academic/artistic)abstract
- This thesis concerns the development and validation of high performance liquid chromatography (HPLC) methods aimed for two industrially important areas: (i) analysis of biotechnological synthesis and (ii) determination of adsorption isotherm parameters. There is today a lack of detailed recommendations for analytical procedures in the field of biotechnological production of drugs. Therefore, guidelines were given for analytical development and validation in this field; the production of 9α-hydroxyprogesterone was used as model. In addition, a rapid method using HPLC coupled with diode-array-detection (DAD) and mass spectrometry (MS), was developed for the preliminary identification and quantification of the product. In addition, requirements and recommendations were developed for the selection of the internal standard and for its inclusion in the process liquid. By using this approach the precision and accuracy of the quantitative method were considerably improved. Preparative chromatography is a powerful separation method for the purification of pure compounds from more or less complex sample mixtures. One such mixture can be the process liquid from a fermentation, another example can be a racemic mixture of compounds whose enantiomeric constituents must be isolated. Computer-assisted modeling can be used to optimize preparative chromatography. However, competitive adsorption isotherm parameters are required as input data for the computer simulations. In this thesis, a new injection technique, based on a firm theoretical basis, was developed for the peak perturbation (PP) method allowing the determination of binary competitive adsorption isotherm parameters from a broad concentration range. With the new method the determination of adsorption isotherm parameters from a quaternary mixture could be done for the first time. The profiles simulated with these parameters showed excellent agreement with the corresponding experimental profiles, validating the accuracy of the adsorption isotherm parameters derived by the new method.
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