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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
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
Samuelsson, J., Leśko, M., Enmark, M., Högblom, J., Karlsson, A. & Kaczmarski, K. (2018). Optimizing Column Length and Particle Size in Preparative Batch Chromatography Using Enantiomeric Separations of Omeprazole and Etiracetam as Models: Feasibility of Taguchi Empirical Optimization. Chromatographia, 81(6), 851-860
Open this publication in new window or tab >>Optimizing Column Length and Particle Size in Preparative Batch Chromatography Using Enantiomeric Separations of Omeprazole and Etiracetam as Models: Feasibility of Taguchi Empirical Optimization
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2018 (English)In: Chromatographia, Vol. 81, no 6, p. 851-860Article in journal (Refereed) Published
Abstract [en]

The overreaching purpose of this study is to evaluate new approaches for determining the optimal operational and column conditions in chromatography laboratories, i.e., how best to select a packing material of proper particle size and how to determine the proper length of the column bed after selecting particle size. As model compounds, we chose two chiral drugs for preparative separation: omeprazole and etiracetam. In each case, two maximum allowed pressure drops were assumed: 80 and 200 bar. The processes were numerically optimized (mechanistic modeling) with a general rate model using a global optimization method. The numerical predictions were experimentally verified at both analytical and pilot scales. The lower allowed pressure drop represents the use of standard equipment, while the higher allowed drop represents more modern equipment. For both compounds, maximum productivity was achieved using short columns packed with small-particle size packing materials. Increasing the allowed backpressure in the separation leads to an increased productivity and reduced solvent consumption. As advanced numerical calculations might not be available in the laboratory, we also investigated a statistically based approach, i.e., the Taguchi method (empirical modeling), for finding the optimal decision variables and compared it with advanced mechanistic modeling. The Taguchi method predicted that shorter columns packed with smaller particles would be preferred over longer columns packed with larger particles. We conclude that the simpler optimization tool, i.e., the Taguchi method, can be used to obtain “good enough” preparative separations, though for accurate processes, optimization, and to determine optimal operational conditions, classical numerical optimization is still necessary

Place, publisher, year, edition, pages
Springer, 2018
Keywords
Preparative chromatography, Omeprazole, Etiracetam, Optimization of productivity, Taguchi optimization, Equilibrium–dispersive model
National Category
Biochemistry and Molecular Biology Analytical Chemistry
Identifiers
urn:nbn:se:kau:diva-67311 (URN)10.1007/s10337-018-3519-z (DOI)2-s2.0-85046036669 (Scopus ID)
Available from: 2018-05-11 Created: 2018-05-11 Last updated: 2019-06-12Bibliographically approved
Forss, E., Haupt, D., Stalberg, O., Enmark, M., Samuelsson, J. & Fornstedt, T. (2017). Chemometric evaluation of the combined effect of temperature, pressure, and co-solvent fractions on the chiral separation of basic pharmaceuticals using actual vs set operational conditions. Journal of Chromatography A, 1499, 165-173
Open this publication in new window or tab >>Chemometric evaluation of the combined effect of temperature, pressure, and co-solvent fractions on the chiral separation of basic pharmaceuticals using actual vs set operational conditions
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2017 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1499, p. 165-173Article in journal (Refereed) Published
Abstract [en]

The need to determine the actual operational conditions, instead of merely using the set operational conditions, was investigated for in packed supercritical fluid chromatography (SFC) by design of experiments (DoE) using a most important type of compounds, pharmaceutical basics, as models. The actual values of temperature, pressure, and methanol levels were recorded and calculated from external sensors, while the responses in the DoE were the retention factors and selectivity. A Kromasil CelluCoat columh was used as the stationary phase, carbon dioxide containing varying methanol contents as the mobile phase, and the six racemates of alprenolol, atenolol, metoprolol, propranolol, clenbuterol, and mianserin were selected as model solutes. For the retention modeling, the most important term was the methanol fraction followed by the temperature and pressure. Significant differences (p<0.05) between most of the coefficients in the retention models were observed when comparing models from set and actual conditions. The selectivity was much less affected by operational changes, and therefore was not severely affected by difference between set and actual conditions. The temperature differences were usually small, maximum +/- 1.4 degrees C, whereas the pressure differences were larger, typically approximately +10.5 bar. The set and actual fractions of methanol also differed, usually by +/- 0.4 percentage points. A cautious conclusion is that the primary reason for the discrepancy between the models is a mismatch between the set and actual methanol fractions. This mismatch is more serious in retention models at low methanol fractions. The study demonstrates that the actual conditions should almost always be preferred. (C) 2017 Elsevier B.V. All rights reserved.

Place, publisher, year, edition, pages
Elsevier, 2017
Keywords
Supercritical fluid chromatography, Basic pharmaceuticals, Chiral separations, Design of experiments, Actual conditions, Set conditions
National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-71672 (URN)10.1016/j.chroma.2017.03.077 (DOI)000401394200017 ()28389095 (PubMedID)
Note

Dan Haupt och Olle Stålberg har affiliering KAU på artikeln men hittar dem inte via personalkatalogen./BHJ

Available from: 2019-03-29 Created: 2019-03-29 Last updated: 2019-04-04Bibliographically approved
Vera, C. M., Shock, D., Dennis, G. R., Samuelsson, J., Enmark, M., Fornstedt, T. & Shalliker, R. A. (2015). A preliminary study on the selectivity of linear polynuclear aromatic hydrocarbons in SFC using phenyl-type stationary phases. Microchemical journal (Print), 121, 136-140
Open this publication in new window or tab >>A preliminary study on the selectivity of linear polynuclear aromatic hydrocarbons in SFC using phenyl-type stationary phases
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2015 (English)In: Microchemical journal (Print), ISSN 0026-265X, E-ISSN 1095-9149, Vol. 121, p. 136-140Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier, 2015
Keywords
SFC; HPLC; Polynuclear aromatic hydrocarbons; Selectivity; Organic modifier
National Category
Polymer Technologies Other Chemical Engineering Organic Chemistry Analytical Chemistry
Identifiers
urn:nbn:se:kau:diva-37302 (URN)10.1016/j.microc.2015.02.011 (DOI)000354589700016 ()
Funder
Swedish Research Council
Available from: 2015-08-06 Created: 2015-08-06 Last updated: 2017-12-06Bibliographically approved
Lesko, M., Åsberg, D., Enmark, M., Samuelsson, J., Fornstedt, T. & Kaczmarski, K. (2015). Choice of Model for Estimation of Adsorption Isotherm Parameters in Gradient Elution Preparative Liquid Chromatography. Chromatographia, 78(19-20), 1293-1297
Open this publication in new window or tab >>Choice of Model for Estimation of Adsorption Isotherm Parameters in Gradient Elution Preparative Liquid Chromatography
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2015 (English)In: Chromatographia, ISSN 0009-5893, E-ISSN 1612-1112, Vol. 78, no 19-20, p. 1293-1297Article in journal (Refereed) Published
Abstract [en]

The inverse method is a numerical method for fast estimation of adsorption isotherm parameters directly from a few overloaded elution profiles and it was recently extended to adsorption isotherm acquisition in gradient elution conditions. However, the inverse method in gradient elution is cumbersome due to the complex adsorption isotherm models found in gradient elution. In this case, physicochemically correct adsorption models have very long calculation times. The aim of this study is to investigate the possibility of using a less complex adsorption isotherm model, with fewer adjustable parameters, but with preserved/acceptable predictive abilities. We found that equal or better agreement between experimental and predicted elution profiles could be achieved with less complex models. By being able to select a model with fewer adjustable parameters, the calculation times can be reduced by at least a factor of 10. 

Place, publisher, year, edition, pages
Springer Berlin/Heidelberg, 2015
Keywords
cycloheptanone; cyclohexanone; ketone; unclassified drug, adsorption; adsorption isotherm; Article; calculation; controlled study; elution; isotherm; liquid chromatography; model; physical chemistry; predictive value; priority journal; simulation; theory
National Category
Analytical Chemistry
Research subject
Chemistry; Chemical Engineering
Identifiers
urn:nbn:se:kau:diva-42370 (URN)10.1007/s10337-015-2949-0 (DOI)000361798500007 ()2-s2.0-84942368375 (Scopus ID)
Funder
Swedish Research Council, 621-2012-3978
Available from: 2016-06-07 Created: 2016-05-23 Last updated: 2018-11-22Bibliographically approved
Vera, C. M., Shock, D., Dennis, G. R., Samuelsson, J., Enmark, M., Fornstedt, T. & Shalliker, R. A. (2015). Contrasting selectivity between HPLC and SFC using phenyl-type stationary phases: A study on linear polynuclear aromatic hydrocarbons. Microchemical journal (Print), 119, 40-43
Open this publication in new window or tab >>Contrasting selectivity between HPLC and SFC using phenyl-type stationary phases: A study on linear polynuclear aromatic hydrocarbons
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2015 (English)In: Microchemical journal (Print), ISSN 0026-265X, E-ISSN 1095-9149, Vol. 119, p. 40-43Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier, 2015
Keywords
Supercritical fluid chromatography, Polynuclear aromatic hydrocarbons, Phenyl stationary phases, Synergi polar, Cosmosil
National Category
Chemical Engineering
Research subject
Chemical Engineering
Identifiers
urn:nbn:se:kau:diva-41634 (URN)10.1016/j.microc.2014.10.008 (DOI)000348957800007 ()
Available from: 2016-04-11 Created: 2016-04-11 Last updated: 2017-10-31Bibliographically approved
Enmark, M., Asberg, D., Leek, H., Ohlen, K., Klarqvist, M., Samuelsson, J. & Fornstedt, T. (2015). Evaluation of scale-up from analytical to preparative supercritical fluid chromatography. Journal of Chromatography A, 1425, 280-286
Open this publication in new window or tab >>Evaluation of scale-up from analytical to preparative supercritical fluid chromatography
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2015 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1425, p. 280-286Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
SFC, Chiral separation, Transfer of method, Scale-up, Operational parameters, Design of experiments
National Category
Chemical Engineering
Research subject
Chemical Engineering
Identifiers
urn:nbn:se:kau:diva-40985 (URN)10.1016/j.chroma.2015.11.001 (DOI)000366770400032 ()
Available from: 2016-03-08 Created: 2016-03-08 Last updated: 2019-07-12Bibliographically approved
Enmark, M. (2015). Fundamental Investigations of Supercritical Fluid Chromatography. (Doctoral dissertation). Karlstad: Karlstads universitet
Open this publication in new window or tab >>Fundamental Investigations of Supercritical Fluid Chromatography
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis aims at a deeper understanding of Supercritical Fluid

Chromatography (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.

Abstract [en]

This thesis aims at a deeper understanding of Supercritical Fluid

Chromatography (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-II it was demonstrated why most methods applied to determine adsorption isotherms in LC could not be applied directly for SFC. Methods based on extracting data from overloaded profiles should be preferred.

 

In Paper III a Design of Experiments approach was successfully used to quantitatively describe the behavior of several solutes in a

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 often observed in SFC were carefully investigated and explained using experiments and simulations.

 

Finally, in Paper V, the prerequisites for performing reliable and predictable scale-up of SFC were investigated by small and large scale experiments.

Place, publisher, year, edition, pages
Karlstad: Karlstads universitet, 2015. p. 63
Series
Karlstad University Studies, ISSN 1403-8099 ; 2015:45
Keywords
Supercritical Fluid Chromatography, SFC, Modeling, Adsorption Isotherms, Scale-up, Peak Distortion, Injection, Design of Experiments, Perturbation Peak Method, Elution by Characteristic Points Method, Method transfer
National Category
Analytical Chemistry Chemical Engineering
Research subject
Chemistry
Identifiers
urn:nbn:se:kau:diva-37913 (URN)978-91-7063-663-9 (ISBN)
Public defence
2015-10-16, Nyquistsalen 9C 203, Karlstads universitet, Karlstad, 10:15 (English)
Opponent
Supervisors
Note

Paper 4 ("Evaluation of scale-up from analytical to preparative...") ingick som manuskript med samma titel i avhandlingen. Nu publicerad. 

Available from: 2015-09-25 Created: 2015-09-11 Last updated: 2019-07-12Bibliographically approved
Enmark, M., Samuelsson, J., Forss, E., Forssén, P. & Fornstedt, T. (2014). Investigation of plateau methods for adsorption isotherm determination in supercritical fluid chromatography. Journal of Chromatography A, 1354, 129-138
Open this publication in new window or tab >>Investigation of plateau methods for adsorption isotherm determination in supercritical fluid chromatography
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2014 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1354, p. 129-138Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
Adsorption isotherms, Antipyrine, Elution by Characteristic Points, Perturbation Peak method, Supercritical fluid chromatography
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kau:diva-34392 (URN)10.1016/j.chroma.2014.05.070 (DOI)000339144000015 ()24931444 (PubMedID)
Available from: 2014-10-15 Created: 2014-10-15 Last updated: 2019-07-12Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-8943-6286

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