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Samuelsson, J., Eiriksson, F. F., Åsberg, D., Thorsteinsdóttir, M. & Fornstedt, T. (2019). Determining gradient conditions for peptide purification in RPLC with machine-learning-based retention time predictions. Journal of Chromatography A, 1598, 92-100
Open this publication in new window or tab >>Determining gradient conditions for peptide purification in RPLC with machine-learning-based retention time predictions
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2019 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1598, p. 92-100Article in journal (Refereed) Published
Abstract [en]

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

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
In silico determination, Machine learning, Peptide, Preparative, Purification, Retention time prediction, Bioinformatics, Calibration, Errors, Forecasting, Learning systems, Pressure effects, Separation, Surface chemistry, In-silico, Linear solvent strengths, Numerical calculation, Preparative separation, Purification method, Support vector regression (SVR), Peptides
National Category
Materials Engineering Bioinformatics and Systems Biology Analytical Chemistry
Identifiers
urn:nbn:se:kau:diva-72137 (URN)10.1016/j.chroma.2019.03.043 (DOI)2-s2.0-85063889315 (Scopus ID)
Available from: 2019-05-28 Created: 2019-05-28 Last updated: 2019-07-04Bibliographically approved
Enmark, M., Rova, M., Samuelsson, J., Örnskov, E., Schweikart, F. & Fornstedt, T. (2019). Investigation of factors influencing the separation of diastereomers of phosphorothioated oligonucleotides. Analytical and Bioanalytical Chemistry, 411(15), 3383-3394
Open this publication in new window or tab >>Investigation of factors influencing the separation of diastereomers of phosphorothioated oligonucleotides
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2019 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 411, no 15, p. 3383-3394Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Springer, 2019
Keywords
IP-RPLC, Ion-pair, Oligonucleotide, Antisense, Phosphorothioate, Diastereomerr
National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-72453 (URN)10.1007/s00216-019-01813-2 (DOI)000469757300016 ()
Available from: 2019-06-12 Created: 2019-06-12 Last updated: 2019-07-01
Liangsupree, T., Multia, E., Metso, J., Jauhiainen, M., Forssén, P., Fornstedt, T., . . . Riekkola, M.-L. (2019). Rapid affinity chromatographic isolation method for LDL in human plasma by immobilized chondroitin-6-sulfate and anti-apoB-100 antibody monolithic disks in tandem. Scientific Reports, 9, Article ID 11235.
Open this publication in new window or tab >>Rapid affinity chromatographic isolation method for LDL in human plasma by immobilized chondroitin-6-sulfate and anti-apoB-100 antibody monolithic disks in tandem
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 11235Article in journal (Refereed) Published
Abstract [en]

Low-density lipoprotein (LDL) is considered the major risk factor for the development of atherosclerotic cardiovascular diseases (ASCVDs). A novel and rapid method for the isolation of LDL from human plasma was developed utilising affinity chromatography with monolithic stationary supports. The isolation method consisted of two polymeric monolithic disk columns, one immobilized with chondroitin-6-sulfate (C6S) and the other with apolipoprotein B-100 monoclonal antibody (anti-apoB-100 mAb). The first disk with C6S was targeted to remove chylomicrons, very-low-density lipoprotein (VLDL) particles, and their remnants including intermediate-density lipoprotein (IDL) particles, thus allowing the remaining major lipoprotein species, i.e. LDL, lipoprotein(a) (Lp(a)), and high-density lipoprotein (HDL) to flow to the anti-apoB-100 disk. The second disk captured LDL particles via the anti-apoB-100 mAb attached on the disk surface in a highly specific manner, permitting the selective LDL isolation. The success of LDL isolation was confirmed by different techniques including quartz crystal microbalance. In addition, the method developed gave comparable results with ultracentrifugation, conventionally used as a standard method. The reliable results achieved together with a short isolation time (less than 30 min) suggest the method to be suitable for clinically relevant LDL functional assays.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
Keywords
IMMUNOAFFINITY CHROMATOGRAPHY; FAST SEPARATION; LIPOPROTEINS; INSIGHTS; COLUMNS
National Category
Engineering and Technology
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-74335 (URN)10.1038/s41598-019-47750-z (DOI)000478575000022 ()31375727 (PubMedID)
Available from: 2019-08-15 Created: 2019-08-15 Last updated: 2019-08-16Bibliographically approved
Vera, C. M., Samuelsson, J., Fornstedt, T., Dennis, G. R. & Shalliker, R. A. (2019). Visualisation of axial temperature gradients and heat transfer process of different solvent compositions in ultra high performance liquid chromatography using thermography. Microchemical journal (Print), 145, 927-935
Open this publication in new window or tab >>Visualisation of axial temperature gradients and heat transfer process of different solvent compositions in ultra high performance liquid chromatography using thermography
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2019 (English)In: Microchemical journal (Print), ISSN 0026-265X, E-ISSN 1095-9149, Vol. 145, p. 927-935Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier, 2019
National Category
Physical Chemistry
Research subject
Physics
Identifiers
urn:nbn:se:kau:diva-71232 (URN)10.1016/j.microc.2018.11.037 (DOI)000457513000118 ()
Available from: 2019-02-21 Created: 2019-02-21 Last updated: 2019-03-06Bibliographically approved
Lin, G., Zhang, Y., Cheng, X., Gulliksson, M., Forssén, P. & Fornstedt, T. (2018). A regularizing Kohn-Vogelius formulation for the model-free adsorption isotherm estimation problem in chromatography. Applicable Analysis, 97(1), 13-40
Open this publication in new window or tab >>A regularizing Kohn-Vogelius formulation for the model-free adsorption isotherm estimation problem in chromatography
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2018 (English)In: Applicable Analysis, ISSN 0003-6811, E-ISSN 1563-504X, Vol. 97, no 1, p. 13-40Article in journal (Refereed) Published
Abstract [en]

Competitive adsorption isotherms must be estimated in order to simulate and optimize modern continuous modes of chromatography in situations where experimental trial-and-error approaches are too complex and expensive. The inverse method is a numeric approach for the fast estimation of adsorption isotherms directly from overloaded elution profiles. However, this identification process is usually ill-posed. Moreover, traditional model-based inverse methods are restricted by the need to choose an appropriate adsorption isotherm model prior to estimate, which might be very hard for complicated adsorption behavior. In this study, we develop a Kohn-Vogelius formulation for the model-free adsorption isotherm estimation problem. The solvability and convergence for the proposed inverse method are studied. In particular, using a problem-adapted adjoint, we obtain a convergence rate under substantially weaker and more realistic conditions than are required by the general theory. Based on the adjoint technique, a numerical algorithm for solving the proposed optimization problem is developed. Numerical tests for both synthetic and real-world problems are given to show the efficiency of the proposed regularization method.

National Category
Chemical Sciences Geophysics Analytical Chemistry
Research subject
Chemistry; Physics
Identifiers
urn:nbn:se:kau:diva-65938 (URN)10.1080/00036811.2017.1284311 (DOI)000417831700003 ()
Available from: 2018-01-25 Created: 2018-01-25 Last updated: 2019-03-28Bibliographically approved
Zhang, Y., Forssén, P., Fornstedt, T., Gulliksson, M. & Dai, X. (2018). An adaptive regularization algorithm for recovering the rate constant distribution from biosensor data. Inverse Problems in Science and Engineering, 26(10), 1464-1489
Open this publication in new window or tab >>An adaptive regularization algorithm for recovering the rate constant distribution from biosensor data
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2018 (English)In: Inverse Problems in Science and Engineering, ISSN 1741-5977, E-ISSN 1741-5985, Vol. 26, no 10, p. 1464-1489Article in journal (Refereed) Published
Abstract [en]

We present here the theoretical results and numerical analysis of a regularization method for the inverse problem of determining the rate constant distribution from biosensor data. The rate constant distribution method is a modern technique to study binding equilibrium and kinetics for chemical reactions. Finding a rate constant distribution from biosensor data can be described as a multidimensional Fredholm integral equation of the first kind, which is a typical ill-posed problem in the sense of J. Hadamard. By combining regularization theory and the goal-oriented adaptive discretization technique, we develop an Adaptive Interaction Distribution Algorithm (AIDA) for the reconstruction of rate constant distributions. The mesh refinement criteria are proposed based on the a posteriori error estimation of the finite element approximation. The stability of the obtained approximate solution with respect to data noise is proven. Finally, numerical tests for both synthetic and real data are given to show the robustness of the AIDA.

Place, publisher, year, edition, pages
Oxon, UK: Taylor & Francis, 2018
Keywords
Rate constant distribution, inverse problem, regularization, adaptive finite element, a posteriori error estimation
National Category
Computational Mathematics Geophysics Chemical Engineering
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-68754 (URN)10.1080/17415977.2017.1411912 (DOI)000438638300005 ()
Note

Zhang, Yue saknar cas! 20181018

Available from: 2018-08-16 Created: 2018-08-16 Last updated: 2019-03-28Bibliographically approved
Forssén, P. & Fornstedt, T. (2018). Impact of column and stationary phase properties on the productivity in chiral preparative LC. Journal of Separation Science, 41(6), 1346-1354
Open this publication in new window or tab >>Impact of column and stationary phase properties on the productivity in chiral preparative LC
2018 (English)In: Journal of Separation Science, ISSN 1615-9306, E-ISSN 1615-9314, Vol. 41, no 6, p. 1346-1354Article in journal (Refereed) Published
Abstract [en]

By generating 1500 random chiral separation systems, assuming two-site Langmuir interactions, we investigated numerically how the maximal productivity (P-R,P-max) was affected by changes in stationary phase adsorption properties. The relative change in P-R,P-max, when one adsorption property changed 10%, was determined for each system and for each studied parameter the corresponding productivity change distribution of the systems was analyzed. We could conclude that there is no reason to have columns with more than 500 theoretical plates and larger selectivity than 3. More specifically, we found that changes in selectivity have a major impact on P-R,P-max if it is below similar to 2 and, interestingly, increasing selectivity when it is above similar to 3 decreases P-R,P-max. Increase in relative saturation capacity will have a major impact on P-R,P-max if it is below similar to 40%, but only modest above this percent. Increasing total monolayer saturation capacity, or decreasing the first eluting enantiomer's retention factor, will have a modest effect on P-R,P-max and increased efficiency will have almost no effect at all on P-R,P-max unless it is below similar to 500 theoretical plates. Finally, we showed that chiral columns with superior analytic performance might have inferior preparative performance, or vice versa. It is, therefore, not possible to assess columns based on their analytical performance alone.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2018
Keywords
enantiomers, preparative chromatography, process optimization, productivity
National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-67070 (URN)10.1002/jssc.201701435 (DOI)000428797000019 ()29359510 (PubMedID)
Available from: 2018-04-19 Created: 2018-04-19 Last updated: 2018-04-26Bibliographically approved
Enmark, M., Glenne, E., Leśko, M., Langborg Weinmann, A., Leek, T., Kaczmarski, K., . . . Fornstedt, T. (2018). Investigation of robustness for supercritical fluid chromatography separation of peptides: Isocratic vs gradient mode. Journal of Chromatography A, 1568, 177-187
Open this publication in new window or tab >>Investigation of robustness for supercritical fluid chromatography separation of peptides: Isocratic vs gradient mode
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2018 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1568, p. 177-187Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
Gramicidin, Method transfer, Peptide, Robustness, SFC, Water, Design of experiments, Effluent treatment, Gas chromatography, Oceanography, Peptides, Robustness (control systems), Supercritical fluids, Coriolis mass flow meter, Experimental system, Gradient separations, Method transfers, Operational conditions, Super critical fluid chromatography, Volumetric flow rate, Solvents
National Category
Chemical Engineering
Research subject
Chemical Engineering
Identifiers
urn:nbn:se:kau:diva-69063 (URN)10.1016/j.chroma.2018.07.029 (DOI)000443669600019 ()
Available from: 2018-09-06 Created: 2018-09-06 Last updated: 2018-11-22Bibliographically approved
Vera, C. M., Samuelsson, J., Fornstedt, T., Dennis, G. R. & Shalliker, R. A. (2018). Protocol for the visualisation of axial temperature gradients in ultra high performance liquid chromatography using infrared cameras. Microchemical journal (Print), 141, 141-147
Open this publication in new window or tab >>Protocol for the visualisation of axial temperature gradients in ultra high performance liquid chromatography using infrared cameras
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2018 (English)In: Microchemical journal (Print), ISSN 0026-265X, E-ISSN 1095-9149, Vol. 141, p. 141-147Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
Frictional heating, High pressure, Thermal effects, Thermal imagining, UHPLC
National Category
Biochemistry and Molecular Biology Chemical Sciences
Identifiers
urn:nbn:se:kau:diva-67492 (URN)10.1016/j.microc.2018.05.004 (DOI)2-s2.0-85047094080 (Scopus ID)
Available from: 2018-06-07 Created: 2018-06-07 Last updated: 2018-07-11Bibliographically approved
Forssén, P., Multia, E., Samuelsson, J., Andersson, M., Aastrup, T., Altun, S., . . . Fornstedt, T. (2018). Reliable Strategy for Analysis of Complex Biosensor Data. Analytical Chemistry, 90(8), 5366-5374
Open this publication in new window or tab >>Reliable Strategy for Analysis of Complex Biosensor Data
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2018 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 90, no 8, p. 5366-5374Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2018
Keywords
Biosensors, Dissociation, Kinetics, Molecular biology, Analyte concentration, Complex concentration, Complex formation reactions, Dissociation kinetics, Experimental system, Global fitting procedures, Heterogeneous interactions, Numerical algorithms, Rate constants
National Category
Chemical Sciences Organic Chemistry Biochemistry and Molecular Biology
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-67280 (URN)10.1021/acs.analchem.8b00504 (DOI)000430512200059 ()2-s2.0-85045627010 (Scopus ID)
Available from: 2018-05-04 Created: 2018-05-04 Last updated: 2018-11-22Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0002-7123-2066

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