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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.

In supercritical fluid chromatography (SFC), the retention of a solute depends on the temperature, density, pressure, and cosolvent fraction. Here, we investigate how the adsorption of the cosolvent MeOH changes with pressure and temperature and how this affects the retention of several solutes. The lower the pressure, the stronger the MeOH adsorption to the stationary phase; in addition, at low pressure, perturbing the pressure results in significant changes in the amounts of MeOH adsorbed to the stationary phase. The robustness of the solute retention was lowest when operating the systems at low pressures, high temperatures, and low cosolvent fractions in the eluent. Here, we found a clear relationship between the sensitivity of MeOH adsorption to the stationary phase and the robustness of the separation system. Finally, we show that going from classical SFC to ultrahigh-performance SFC (UHPSFC), that is, separations conducted with much smaller packing diameters, results in retention factors that are more sensitive to fluctuations in the flow rate than with traditional SFC. The calculated density profiles indicate only a slight density drop over the traditional SFC column (3%, visualized as lighter -> darker blue in the TOC), whereas the drop for the UHPSFC one was considerably larger (20%, visualized as dark red -> light green in the TOC). The corresponding temperature drops were calculated to be 0.8 and 6.5 degrees C for the SFC and UHPSFC systems, respectively. These increased density and temperature drops are the underlying reasons for the decreased robustness of UHPSFC., Artikeln ingick som manuskript i Glennes doktorsavhandling (2019), då med titeln: Impact of Methanol Adsorption on Robustness in Supercritical Fluid Chromatography

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

Thermal imaging was used to visualise axial temperature gradients in chromatography columns running solvents of water, methanol and acetonitrile at various compositions. The non-monotonic relationship between solvent composition and viscosity enabled the testing of solvent conditions with equivalent viscosities, but with different percentages of water. It was observed that at equivalent power the increase in water composition leads to an increase in the magnitude of the axial temperature gradients (Delta T-A). Observing the change in temperature at defined points (Delta T-p) with increasing power indicates that the relationship between Delta T-p and power is not linear but deviates at higher power. The degree of this deviation depends on what point along the column axis is observed as well what solvent composition is used. Being able to monitor the entire length of the column using thermography also allowed for the observation of different rates of heat transfer through to the bulky stainless steel end fittings from solvent to solvent. Revealing that water is able to transfer heat to the stainless steel surface in such a way that the temperature profile of transition point between columns to end fitting is more gradual compared to when methanol or acetonitrile is used. This evidence plus video data collected indicates that at constant flow rate an equilibrium between heat generated and heat dissipated through the stainless steel is formed, which differs from solvent to solvent. Moreover any disruption to this equilibrium, i.e. a change in flow rate will momentarily change the shape of the temperature profile.

A strategy for determining a suitable solvent gradient in silico in preparative peptide separations is presented. The strategy utilizes a machine-learning–based method, called ELUDE, for peptide retention time predictions based on the amino acid sequences of the peptides. A suitable gradient is calculated according to linear solvent strength theory by predicting the retention times of the peptides being purified at three different gradient slopes. The advantage of this strategy is that fewer experiments are needed to develop a purification method, making it useful for labs conducting many separations but with limited resources for method development. The preparative separation of met-enkephalin and leu-enkephalin was used as model solutes on two stationary phases: XBridge C18 and CSH C18. The ELUDE algorithm contains a support vector regression and is pre-trained, meaning that only 10–50 peptides are needed to calibrate a model for a certain stationary phase and gradient. The calibration is done once and the model can then be used for new peptides similar in size to those in the calibration set. We found that the accuracy of the retention time predictions is good enough to usefully estimate a suitable gradient and that it was possible to compare the selectivity on different stationary phases in silico. The absolute relative errors in retention time for the predicted gradients were 4.2% and 3.7% for met-enkephalin and leu-enkephalin, respectively, on the XBridge C18 column and 2.0% and 2.8% on the CSH C18 column. The predicted retention times were also used as initial values for adsorption isotherm parameter determination, facilitating the numerical calculation of overloaded elution profiles. Changing the trifluoroacetic acid (TFA) concentration from 0.05% to 0.15% in the eluent did not seriously affect the error in the retention time predictions for the XBridge C18 column, an increase of 1.0 min (in retention factor, 1.3). For the CSH C18 column the error was, on av

A protocol was developed for the visualisation of axial temperature gradients on a Kinetex column (1.3 μm C18 100 Å 50 × 2.1 mm) operated at near maximum pressure of the system (Pmax) using an infrared camera. Real time viscous frictional heating effects across the entire column length was observed, and showed that with increasing flow rate there was an increase in the maximum temperature of the column, and the difference between the inlet and outlet temperatures. Temperature profile data over the entire length of the column revealed the dynamics of heat exchange processes along different parts of the column, and raises the question on potential heating effects on eluents. The axial temperature gradients of eluents such as pure methanol, isopropyl alcohol and acetonitrile near Pmax were compared; finding that acetonitrile which had the highest flow velocity at Pmax gave the highest overall temperature increase for these eluents.

When using biosensors, analyte biomolecules of several different concentrations are percolated over a chip with immobilized ligand molecules that form complexes with analytes. However, in many cases of biological interest, e.g., in antibody interactions, complex formation steady-state is not reached. The data measured are so-called sensorgram, one for each analyte concentration, with total complex concentration vs time. Here we present a new four-step strategy for more reliable processing of this complex kinetic binding data and compare it with the standard global fitting procedure. In our strategy, we first calculate a dissociation graph to reveal if there are any heterogeneous interactions. Thereafter, a new numerical algorithm, AIDA, is used to get the number of different complex formation reactions for each analyte concentration level. This information is then used to estimate the corresponding complex formation rate constants by fitting to the measured sensorgram one by one. Finally, all estimated rate constants are plotted and clustered, where each cluster represents a complex formation. Synthetic and experimental data obtained from three different QCM biosensor experimental systems having fast (close to steady-state), moderate, and slow kinetics (far from steady-state) were evaluated using the four-step strategy and standard global fitting. The new strategy allowed us to more reliably estimate the number of different complex formations, especially for cases of complex and slow dissociation kinetics. Moreover, the new strategy proved to be more robust as it enables one to handle system drift, i.e., data from biosensor chips that deteriorate over time.

Two complementary instrumental techniques were used, and the data generated was processed with advanced numerical tools to investigate the interactions between anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) and apoB-100 containing lipoproteins. Partial Filling Affinity Capillary Electrophoresis (PF-ACE) combined with Adsorption Energy Distribution (AED) calculations provided information on the heterogeneity of the interactions without any a priori model assumptions. The AED calculations evidenced a homogenous binding site distribution for the interactions. Quartz Crystal Microbalance (QCM) studies were used to evaluate thermodynamics and kinetics of the Low-Density Lipoprotein (LDL) and anti-apoB-100 Mab interactions. High affinity and selectivity were observed, and the emerging data sets were analysed with so called Interaction Maps. In thermodynamic studies, the interaction between LDL and anti-apoB-100 Mab was found to be predominantly enthalpy driven. Both techniques were also used to study antibody interactions with Intermediate-Density (IDL) and Very Low Density (VLDL) Lipoproteins. By screening affinity constants for IDL-VLDL sample in a single injection we were able to distinguish affinity constants for both subpopulations using the numerical Interaction Map tool.

In electrostatic repulsive interaction chromatography, using a charged surface hybrid sorbent carrying positive charges can improve the peak shape of peptides in reversed-phase liquid chromatography (RP-LC), especially in overloaded conditions, compared with standard C-18 sorbents. However, the positive surface charges can interact with anionic additives commonly used in peptide separations, e.g., trifluoroacetic acid (TFA), complicating adsorption isotherm estimation. We investigated how the competition for available adsorption sites between TFA and two peptides influenced the adsorption isotherm in gradient elution. A model accounting for the competition with TFA was compared with a model neglecting TFA adsorption. We found that the two models predicted elution profiles with the same accuracy. We also found that the adsorption isotherms were extremely similar in shape, leading to the conclusion that neglecting the competition with TFA is a valid approximation enabling faster and more robust adsorption isotherm estimation for the studied type of sorbent.

Preparative chromatography is the best generic method today for the purification of small drugs and valuable chemical components at <10-kg level. Recent progress in computer technology and the development of new nonchiral/chiral stationary phases, as well as numerous improvements in reliability and economic performance, have considerably increased the interest in modeling in academic and industrial communities. This chapter serves as an introduction to modeling of preparative liquid chromatography where the aim is to improve process purification of valuable chemical components, such as drugs and chiral components. We go through the most important column and adsorption models and methods for determination of the essential thermodynamic adsorption data for both column characterization and process improvement. We also cover important operational modes, such as separation in gradient mode and cases where additives are present, and operational conditions sometimes neglected in the modeling process, such as the impact of injection profiles.

Betulin from birch bark was extracted using two principally different extraction methodologies - classical Reflux Boiling (RB) and Pressurized Liquid Extraction (PLE). The extraction methods were analyzed based on both recovery and purity as well as for RB industrial feasibility. The purity and recovery for the different extraction methods were analyzed using High Performance Liquid Chromatography (HPLC) coupled with three different detection principles: Diode Array Detection (DAD), Mass Spectrometry (MS) and Charged Aerosol Detection (CAD). The chromatographic purity was determined by all detections whereas the DAD was used also for complementary gravimetric calculations of the purity of the extracts. The MS detection (in MS and MS/MS modes) was mainly used to characterize the impurities. Two steps to increase the purity of RB extracts were evaluated - pre-boiling the bark in water and precipitation by adding water to the extract. Finally, the methods were compared in terms of amounts of betulin produced and solvent consumed. The RB method including a precipitation step produced the highest purity of betulin. However, results indicate that PLE using three cycles with the precipitation step gives similar purities as for RB. The PLE method produced up to 1.6 times higher amount of extract compared to the RB method. However, the solvent consumption (liter solvent per gram product) for PLE was around 4.5 times higher as compared to the classical RB. PLE performed with only one extraction cycle gave results more similar to RB with 1.2 times higher yield and 1.4 times higher solvent consumption. The RB process was investigated on an industrial scale using a model approach and several important key-factors could be identified. The most energy demanding step was the recycling of extraction solvent which motivates that solvent consumption should be kept low and calculations show a great putative energy reduction by decreasing the ethanol concentration used in the RB process

A fundamental investigation of the pressure effect on individual adsorption sites was undertaken based on adsorption energy distribution and adsorption isotherm measurements. For this purpose, we measured adsorption equilibrium data at pressures ranging from 100 to 1000 bar at constant flow and over a wide concentration range for three low-molecular-weight solutes, antipyrine, sodium 2-naphthalenesulfonate, and benzyltriethylammonium chloride, on an Eternity C18 stationary phase. The adsorption energy distribution was bimodal for all solutes, remaining clearly so at all pressures. The bi-Langmuir model best described the adsorption in these systems and two types of adsorption sites were identified, one with a low and another with a high energy of interaction. Evidence exists that the low-energy interactions occur at the interface between the mobile and stationary phases and that the high-energy interactions occur nearer the silica surface, deeper in the C18 layer. The contribution of each type of adsorption site to the retention factor was calculated and the change in solute molar volume from the mobile to stationary phase during the adsorption process was estimated for each type of site. The change in solute molar volume was 2-4 times larger at the high-energy site, likely because of the greater loss of solute solvation layer when penetrating deeper into the C18 layer. The association equilibrium constant increased with increasing pressure while the saturation capacity of the low-energy site remained almost unchanged. The observed increase in saturation capacity for the high-energy site did not affect the column loading capacity, which was almost identical at 50- and 950-bar pressure drops over the column. (C) 2016 Elsevier B.V. All rights reserved.

Background: The caudal zona incerta (cZi) and posterior subthalamic area (PSA) are an emerging deep brain stimulation (DBS) target for essential tremor (ET). Objectives: To evaluate the efficacy of tremor control in relation to the anatomical locations of stimulation fields in 50 patients with ET and DBS of the cZi. Methods: A total of 240 contacts were evaluated separately with monopolar stimulation, and amplitudes were optimized for improvement of tremor and hand function. Stimulation fields, i.e., volumes of neural activation, were simulated for each optimized setting and assembled into probabilistic stimulation maps (PSMs). Results: There were differences in the anatomical distribution of PSMs associated with good versus poor tremor control. The location of PSMs which achieved good and excellent tremor control corresponded well with the PSM for the clinically used settings, and they were located within the superior part of the PSA. Conclusions: PSMs may serve as a useful tool for defining the most efficacious anatomical location of stimulation. The best tremor control in this series of cZi DBS was achieved with stimulation of the superior part of the PSA, which corresponds to the final part of the cerebellothalamic projections before they reach the ventral lateral thalamus., Funding agencies: Umea University; Foundation for Clinical Neuroscience at the University Hospital of Umea; Foundation for Clinical Research of the Stockholm County Council; European Unions Seventh Framework Program IMPACT [305814]

Two complementary instrumental techniques were used, and the data generated was processed with advanced numerical tools to investigate the interactions between anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) and apoB-100 containing lipoproteins. Partial Filling Affinity Capillary Electrophoresis (PF-ACE) combined with Adsorption Energy Distribution (AED) calculations provided information on the heterogeneity of the interactions without any a priori model assumptions. The AED calculations evidenced a homogenous binding site distribution for the interactions. Quartz Crystal Microbalance (QCM) studies were used to evaluate thermodynamics and kinetics of the Low-Density Lipoprotein (LDL) and anti-apoB-100 Mab interactions. High affinity and selectivity were observed, and the emerging data sets were analysed with so called Interaction Maps. In thermodynamic studies, the interaction between LDL and anti-apoB-100 Mab was found to be predominantly enthalpy driven. Both techniques were also used to study antibody interactions with Intermediate-Density (IDL) and Very Low-Density (VLDL) Lipoproteins. By screening affinity constants for IDL-VLDL sample in a single injection we were able to distinguish affinity constants for both subpopulations using the numerical Interaction Map tool.

In this review we discuss how viscosity contrasts between the injection plug and the mobile phase may lead to loss in separation performance, especially in UHPLC columns or SFC environments. Firstly, the wall effect is discussed, and how it can amplify viscosity contrast effects. We then illustrate how viscosity contrasts lead to the phenomenon known as viscous fingering, and we detail the destructive effects of this phenomenon. We expand on the viscous fingering component, however, demonstrating that viscosity contrast effects begin to deteriorate performance long before the conditions are such that viscous fingering occurs. Subtle changes in band-shape are apparent even with very low viscosity contrasts. Lastly we illustrate how viscosity contrast effects lead to severe peak distortions in SFC. Analysts who seek high efficiency separations must make every effort to eliminate, or at least minimise the viscosity contrast between the injection plug and the mobile phase.

A selectivity study was undertaken using two types of phenyl-type (Synergi polar RP and Cosmosil 5PBB) stationary phases in supercritical fluid chromatography. The mobile phases used in the SFC environment employed CO2 with methanol as the modifier. These columns were tested using linear polynuclear aromatic hydrocarbons as the test solutes. The retention behaviour of these PAHs in SFC was compared to their behaviour in HPLC and there were very distinct differences. On the Synergi-polar stationary phase the PAH selectivity was highly dependent on the methanol modifier concentration, but in comparison on the Cosmosil column, the selectivity between the members of this PAH test group showed almost no change as a function of the methanol concentration, even though the retentivity on the Cosmosil stationary phase was far greater than the Synergi stationary. In fact, the mobile phase played a very passive role in the separations observed on the Cosmosil stationary phase. (C) 2014 Elsevier B.V. All rights reserved.

In this work, a method to study and analyze the interaction data in free solution by exploiting partial filling affinity capillary electrophoresis (PF-ACE) followed by adsorption energy distribution calculations (AED) prior model fit to adsorption isotherms will be demonstrated. PF-ACE-AED approach allowed the possibility to distinguish weak and strong interactions of the binding processes between the most common apolipoprotein E protein isoforms (apoE2, apoE3, apoE4) of high density lipoprotein (HDL) and apoE-containing HDL2 with major glycosaminoglycan (GAG) chain of proteoglycans (PGs), chondroitin-6-sulfate (C6S). The AED analysis clearly revealed the heterogeneity of the binding processes. The major difference was that they were heterogeneous with two different adsorption sites for apoE2 and apoE4 isoforms, whereas interestingly for apoE3 and apoE-containing HDL2, the binding was homogeneous (one site) adsorption process. Moreover, our results allowed the evaluation of differences in the binding process strengths giving the following order with C6S: apoE-containing HDL2 > apoE2 > apoE4 > apoE3. In addition, the affinity constant values determined could be compared with those obtained in our previous studies for the interactions between apoE isoforms and another important GAG chain of PGs - dermatan sulfate (DS). The success of the combination of AED calculations prior to non-linear adsorption isotherm model fit with PF-ACE when the concentration range was extended, confirmed the power of the system in the clarification of the heterogeneity of biological processes studied.

The retention behaviour of a homologous series of polyaromatic hydrocarbons was evaluated on two phenyl-type stationary phases in reversed phase supercritical fluid chromatography (SFC). These phases were the Synergi polar-RP phase and the Cosmosil 5PBB phase, both of which are polar end-capped and incorporate an ether in a propyl chain that tethers the phenyl ring to the silica surface. The Cosmosil 5PBB phase also has five bromine atoms on the phenyl ring. The retention capacity of the Cosmosil column was substantially greater than the Synergi column. However, selectivity on the Cosmosil column was effectively independent of the acetonitrile modifier composition in the CO2 mobile phase, whereas, selectivity on the Synergi column was greatly affected by the acetonitrile modifier in the CO2 mobile phase. The results from this study showed that selectivity and retention studies in HPLC cannot be used to predict selectivity and retention behaviour in SFC. (C) 2015 Elsevier B.V. All rights reserved.