The research in the Separation Science Group is driven by the overwhelming complexity experienced in especially natural but also in synthetic mixtures. Chromatography or electrophoresis is essential for qualitative and quantitative analysis of organic and inorganic molecules. After 100 year of development much open question remain and novel challenges continuously appear as users in all fields of chemistry and well beyond are confronted with new problems which can often only be addressed providing effective separation can be achieved. As progress is made new physical barriers are encountered which need be overcome. Research projects in the separation science group can roughly be grouped in two research types:
The development of novel column chemistry, sample preparation or system technology to overcome contemporary limitations of chromatography, electrophoresis or organic mass spectrometry. This typically requires synthetic chemistry of organic, inorganic or polymeric nature.
High- end implementation of state-of-the-art instrumentation to address problems in quantitative or qualitative analysis of organic molecules in complex mixtures in a targeted or untargeted way.
The current research projects are demonstrated below.
Temperature-responsive liquid chromatography (TRLC) uses smart polymers that react to a raise in temperature with an increase in retention. This effect can be exploited under purely water-based conditions, forgoing the need for organic solvents. Many of the practical hurdles people face with 2D-LC are based on the transfer of organic solvents from one to the other dimension; these hurdles are thus eliminated, leading to robust methods and simpler method development.
This project assesses the possibilities offered by TRLC×RPLC in terms of sensitivity, peak capacities and quantitative potential, such that optimal TRLCxRPLC platforms can be developed. Below, TRLC×RPLC was applied for the qualitative analysis of 17 small molecule pharmaceuticals, allowing the detection of impurities at a 0.05% level compared to selected main compounds, mimicking drug formulations comprising two active ingredients.
Figure 1: (left) Schematic demonstration of comprehensive LC×LC using a temperature responsive stationary phase in the first dimension and RPLC in the second deimension, (right) and a practical TRLC×RPLC application, whereby 17 steroid compounds were analysed. Of those, two were selected as active pharmaceutical ingredients and 15 were brought to the detection level of 0.05% for impurities in pharmaceutical drug products (ICH guidelines). All impurities were successfully detected and separated. (Picture taken from: Wicht et al. (2020). Journal of Chromatography A, 1630, 461561. doi.org/10.1016/j.chroma.2020.461561
In silico predictions of unknowns
Micropollutants, such as pharmaceuticals, pesticides, industrial chemicals, steroid hormones, etc. are defined as anthropogenic chemicals and can be found in water. It is seen as a serious threat, not just to aquatic life but also to humans, which requires the availability of tools allowing structural elucidation and ideally, fast identification of unknowns.
Over the past decade, high performance liquid chromatography, coupled with high resolution mass spectrometry (HPLC-HRMS), has been widely used in the analysis of environmental unknown samples. However, MS prediction software cannot accurately predict the elemental and structural composition of larger molecules (>200 Da), which makes it harder to be certain of identifying the unknown(s). Using a Quantitative Structure-Retention Relationship (QSRR) method, we are building an alternative predictive model which will help us to identify unknown compounds in water by predicting their retention time (tR).
Applying Temperature Responsive Liquid Chromatography
Thermo-responsive polymer-based columns offer the possibility to work with temperature gradients instead of solvent gradient during HPLC analysis. Around the cloud point temperature (Tcp) such polymer undergoes a globule-to-coil transition in aqueous media affecting the retention as a function of temperature. The methodology allows performing Temperature Responsive Liquid Chromatography (TRLC), a reversed phase type of separation using only water as mobile phase.
A possible application for TRLC is enhanced analysis of pharmaceuticals and of the the related oxidized products formed after treatment (with Advanced Oxidation Processes, AOPs) in waste water streams. The approach is exploited in 1D-TRLC and 2D-TRLCxRPLC based strategies. The temperature gradient possibility also shows potential also with Refractive Index Detection (RID), paving the way for the implementation of gradients in LC-RID.
Figure 1. Synthesis of the stationary phases and immobilization to the supporting material. Chromatograms obtained above and below the Tcp of the TR polymer illustrating the differences in retention with temperature (data collected with RID).
Use of biopartitioning chromatography as a high throughput surrogate measure for brain penetration
The ability to permeate across the blood-brain barrier (BBB) is essential for drugs acting on the central nervous system (CNS). Biopartitioning chromatography can be used as an in vitro system to model the biopartitioning process of drugs when there are no active processes. The technique uses micellar mobile phases at physiological conditions and reversed phase columns to determine retention times (retention factors). The retention factor of a drug, which is influenced by the adsorption of surfactant monolayer to the stationary phase and by micelles present in the mobile phase, gives an indication of the drug penetration across a membrane.
The target of this project is to obtain an in vitro biopartitioning chromatography experiment that can correlate the retention factors with measured BBB penetration as good as possible.
Application of comprehensive two-dimensional HPLC in separation of copolymers
Knowledge of the exact composition and purity of advanced copolymers is of utmost importance given their influence on the final physical properties. The development of analytical techniques to examine these impurities is therefore crucial with respect of large industrial productions. A comprehensive two-dimensional HPLC separation technique with a slow size exclusion (SEC) separation in the first dimension and a fast reversed phase liquid chromatography (RPLC) separation in the second dimension was therefore developed and will be further elaborated to improve and optimise it's overall peak capacity. Separation methods for a range of different copolymer types will be developed as an asset for the polymer chemist.
New strategy for fast chiral screening by high-performance liquid chromatography coupled with multivariate curve resolution-alternating least squares
A strategy aimed at developing rapid chiral screening technology was proposed in this paper with compressed screening time by mixing samples and screening the mixture of racemates. The data matrix of the mixture obtained by diode array detector or mass spectrometry was deconvoluted into resolved chromatograms and spectra by the multivariate curve resolution - alternating least squares algorithm. The individual racemate was then identified by the resolved spectra and its enantioselectivity was evaluated by the resolved chromatograms. Two example experiments were carried out to verify the feasibility of the strategy. A mixture consisting 5 racemates was successfully screened on Chiralcel OD column in one fifth of the conventional analysis time. Another mixture made by 10 racemates gained nine tenth of the original screening time on three CSPs with an prediction accuracy above 90%.
Selectivity optimization with commercial coupled columns by gradient stationary phase optimized selectivity liquid chromatography
The gradient SOSLC protocol is successfully extended to coupled columns of 4 different stationary phases. Fast and full baseline separations of the mixture composed of 12 compounds of phenones, benzoic acids and benzoates are demonstrated within analysis time of 15 minutes under both isocratic and linear gradient conditions. Although the extra column void volume is increased by the use of tubings, the average relative deviation of prediction on selectivity factor results less than 2% and the maximum relative deviations are below 5%. These results indicate that the proposed gradient SOSLC protocol on coupled columns can also bring us robust and promising solution for separation of a given mixture.
The benefits of extending the gradient SOSLC to coupled column chromatography include wider commercial availability of stationary phases / columns, full use of the present columns in the lab, more selection of column coupler and flexible applications.
Evaluation and optimization of Capillary ElectroChromatography as a high-efficient separation technique.
Capillary Electrochromatography (CEC) is theoretically a highly efficient technique but is not yet implemented in the industry as a standard separation technique. This research is focused on the development of a neutral comparison tool for CEC capillaries in terms of speed and efficiency. The tool will be further used to address the problems in this electrodriven separation technique and to compare capillaries packed with the newest packing materials (sub-um, mixed mode, fused-core particles...) and new packing methods.
