Photografting of polymer monoliths by a crosslinking monomer.

The surface of fifteen polymethacrylate monolithic stationary phases has been modified by a post-polymerization UV-initiated grafting reaction with bifunctional poly(ethylene glycol)dimethacrylate monomers. An effect of crosslinking monomer length, its concentration in the modification mixture, and a time of the modification reaction have been selected to control the extent of modification by a design of experiments protocol. Hydrodynamic and kinetic properties of prepared columns were characterized by capillary liquid chromatography. Regression analysis of determined data revealed that there is only a minor effect of modification reaction on column permeability, as it is rather controlled by the composition of the polymerization mixture used to prepare generic monolith. On the other hand, the utilization of shorter crosslinking monomer increased the formation of small pores and minimized mass transfer resistance effect. Both column efficiency and mass transfer resistance also improved when a lower concentration of crosslinking monomer in the modification mixture was used. Photografting modification decreased a negative effect of mass transfer resistance related to a crosslink density gradient and allowed fast isocratic separations of dopamine metabolism-related compounds. Developed preparation protocol might be further utilized in the preparation of monolithic stationary phases in microfluidic devices.

Dual-retention mechanism of dopamine-related compounds on monolithic stationary phase with zwitterion functionality.

Seven retention models have been selected to describe a dual-retention behavior of ten dopamine-related compounds on polymer-based monolithic stationary phase with zwitterion sulfobetaine functionality. Regression quality, as well as a statistical significance of individual regression parameters, have been evaluated. Better regression performance showed two four-parameter models when compared to three-parameter models. On the other hand, limited number of experimental points disqualified statistical robustness of four-parameter models. Among three-parameter models, retention description introduced by Horváth and Liang provided comparable quality of regression at significantly improved robustness. Multivariate analysis of the best three-parameter models provided the description of physicochemical properties of dopamine precursors and metabolites. Principal component analysis and logistic regression allowed structural characterization of dopamine-related compounds based solely on regression parameters extracted from an isocratic elution data. Both polarity and type of functional groups has been correctly assigned for 3-methoxytyramine that has not been part of an evaluation study. Among applied dual-retention models, Horváth´s model, initially developed to describe a retention of ionic compounds on nonpolar stationary phases, provided robust regression of experimental data and allowed an extraction of structural characteristics of dopamine-related compounds.

Multichannel separation device with parallel electrochemical detection.

A device with four parallel channels was designed and manufactured by 3D printing in titanium. A simple experimental setup allowed splitting of the mobile phase in four parallel streams, such that a single sample could be analysed four times simultaneously. The four capillary channels were filled with a monolithic stationary phase, prepared using a zwitterionic functional monomer in combination with various dimethacrylate cross-linkers. The resulting stationary phases were applicable in both reversed-phase and hydrophilic-interaction retention mechanisms. The mobile-phase composition was optimized by means of a window diagram so as to obtain the highest possible resolution of dopamine precursors and metabolites on all columns. Miniaturized electrochemical detectors with carbon fibres as working electrodes and silver micro-wires as reference electrodes were integrated in the device at the end of each column. Experimental separations were successfully compared with those predicted by a three-parameter retention model. Finally, dopamine was determined in human urine to further confirm applicability of the developed device.

Controlling selectivity of polymer-based monolithic stationary phases.

In this work, we aimed to prepare a monolithic capillary column that allowed an isocratic separation of ten dopamine precursors and metabolites in a single run. Segments of five zwitterion sulfobetaine polymer monoliths have been modified by zwitterion phoshorylcholine by using an ultraviolet-initiated two-step photografting. Columns with 0, 33, 50, 66, and 100% of modified length were prepared. Effect of length of the modified segment and mobile phase composition has been tested. All columns provided dual-retention mechanism with reversed-phase retention in highly aqueous mobile phase and hydrophilic interaction mechanism in highly organic mobile phase. The retention mechanism was controlled by the composition of the mobile phase and has been described by a three-parameter model. We have used regression parameters to characterize the retention of analyzed compounds and to study individual pathways of dopamine metabolism. Comprehensive optimization of mobile phase composition allowed to find an optimal composition of the mobile phase and stationary phase surface chemistry arrangement to achieve desired separation. Optimized columns provided an isocratic separation of all tested compounds in less than nine min.

Monolithic capillary column with an integrated electrochemical detector.

The carbon fiber and silver microwire were used as working and pseudoreference electrode, respectively, and inserted into the ending of capillary to prepare monolithic capillary column with an integrated electrochemical detector. Prepared capillary devices offered stable and robust results with relative standard deviations of retention, resolution, and detection signal lower than 1.5, 5.5, and 5.0%, respectively. To further increase sensitivity of developed electrochemical microdetector, multiple pulse amperometry detection mode has been used. Optimized integrated device provided reliable chromatographic separation of mixture of neurotransmitters with calibration curve for dopamine linear from 0.5 to 20.0mgL-1 and an instrumental limit of detection as low as 24pg of injected dopamine. Finally, developed capillary column was applied to successful determination of dopamine in a human urine. By using both calibration curve and standard addition method, the dopamine level was determined to be 0.74±0.03mgL-1 and 0.71±0.02mgL-1, respectively. Triplicates of dopamine analysis provided relative standard deviations lower than 2.7% for intraday analyses, while interday relative standard deviations were lower than 3.6% for five consecutive days.

Retention of small molecules on polymethacrylate monolithic capillary columns.

In this paper, the concentration of N-isopropylacrylamide in the polymerization mixture has been varied to prepare several polymethacrylate monolithic capillary columns. Polymer monoliths combining N-isopropylacrylamide with zwitterion monomer, as well as various dimethacrylate crosslinking monomers have been prepared and characterized. Uracil, thiourea, phenol, toluene, ethylbenzene, propylbenzene, and butylbenzene have been used to characterize retention of prepared capillary columns in the mobile phases with 40-95% of acetonitrile and at working temperatures ranging from 25 to 60°C. By an optimization of six-parameter polynomial models we have found that the retention of small molecules is affected mainly by the concentration of the acetonitrile in the mobile phase with very low contribution of working temperature and combined effect of acetonitrile concentration and temperature. Concentration of the mobile phase controlled also enthalpy of the retention. On the other hand, entropic contribution was almost insensitive to the change of the mobile phase composition, especially for mobile phases containing more than 60% of acetonitrile.

Development of an online solid-phase extraction with liquid chromatography method based on polymer monoliths for the determination of dopamine.

Porous polymer monoliths have been used to develop an online solid-phase extraction with liquid chromatography method for determination of dopamine in urine as well as for a continuous monitoring of dopamine in flowing system. A polymerization mixture containing 4-vinylphenylboronic acid monomer has been used to prepare a trapping column based on specific ring formation reaction with dopamine cis-diol functionality. Additionally, a monolithic stationary phase with zwitterion functionality has been used to prepare capillary column for the separation of dopamine. Experimental conditions including molarity, pH, and flow rate of the loading buffer together with a valve switching time have been optimized to provide the highest recovery for dopamine. Experimental setup has been used to determine dopamine in a urine. By using both calibration curve and standard addition method, the dopamine level was determined to be 1.19 and 1.28 mg/L, respectively. Further, we have used experimental design to optimize coupling of two extraction monolithic loops to separation capillary column with monolithic phase for a comprehensive monitoring of dopamine. After multivariate analysis, sample loading flow-rate and a flow-rate of flushing buffer were selected as the most significant variables. Optimized experimental setup was applied to continuously monitor dopamine degradation.