Application of microstructured membranes for increasing retention, selectivity and resolution in asymmetrical flow field-flow fractionation.

In the present proof-of-concept study, we demonstrate that retention time, selectivity and resolution can be increased in asymmetrical flow field-flow fractionation (AF4) by introducing microstructured ultrafiltration membranes. Evenly spaced micron-sized grooves, that are placed perpendicular to the channel flow on the accumulation wall of a field-flow fractionation system, cause a decrease in the zone velocity which is stronger for larger solutes. This has been demonstrated in thermal field-flow fractionation, and we prove that this is also the case in AF4. We examine the hypothesis theoretically and experimentally, by both computational and physical experiments. By means of moment analysis, we derive theoretically a set of equations which, under certain conditions, describe the mass transport and relate retention time, selectivity and plate height to the dimensions of the grooves. Physical experiments are carried out using microstructured polyethersulfone membranes fabricated by hot embossing, and the experimental results are compared with computational fluid dynamics experiments.

Recovery, overloading, and protein interactions in asymmetrical flow field-flow fractionation.

In asymmetrical flow field-flow fractionation (AF4), similar to other separation techniques, mass recovery and overloading require special attention in order to obtain quantitative results. We conducted a systematic study with five globular proteins of different molecular weight (36.7-669 kDa) and isoelectric point (4.0-6.5), and ultrafiltration membranes that are commonly used in aqueous AF4, regenerated cellulose (RC) and polyethersulfone (PES). Phosphate-buffered saline (PBS) with ionic strength 0.15 M and pH 7.2 was used as the carrier liquid in this study. The actual molecular weight cutoff (MWCO) was found to be higher than the nominal value and varied between membranes of different chemistry but the same nominal MWCO. Adsorption on the membrane was found to be dependent on the membrane chemistry (RC had lower adsorption compared to PES), and independent of the protein standard for the examined proteins. On the other hand, the mass overloading effects (i.e., higher retention times, peak broadening, and fronting peaks) were significantly more pronounced for γ-globulin than for the other proteins. The overloading effects could be rationalized with the increase of the local viscosity close to the membrane, depending on the properties of the proteins, and we derived theoretical equations that related the dependency of the migration velocity on the protein concentration through this non-ideal viscosity effect.

Probing selectivity of mixed-mode reversed-phase/weak-anion-exchange liquid chromatography to advance method development.

The current study comprises a systematic investigation to assess retention properties and selectivity of a mixed-mode reversed-phase/weak-anion-exchange (RP/WAX) stationary phase to aid method development. Retention was investigated for different compound classes which vary in hydrophobicity, van der Waals surface area, and charge as function of organic content, pH, and ionic strength of the mobile phase. The linear-solvent-strength model was successfully applied for aromatic hydrocarbons to obtain retention-time predictions based on log P values and van der Waals surface area values. For phenols, predictions were based on log P values and data from a single scouting run performed in isocratic mode to estimate the S parameter; the deviations between experimental and predicted retention times were smaller than 6%. To describe the mixed-mode (RP/WAX) retention behavior of singly and doubly negatively-charged aromatic acids, a novel model combining the linear-solvent-strength and the empirical stoichiometric-displacement-net-charge models is proposed and validated. Using combinations of three scouting runs that are not linearly dependent, the maximum prediction error was 11% and changes in selectivity were correctly forecasted when altering the mobile-phase composition, i.e., either organic modifier content or ionic strength. When using nine scouting runs in combination with a least-squares regression approach to determine the model parameters, the maximum prediction error was 6%.

Aggregation behavior of fullerenes in aqueous solutions: a capillary electrophoresis and asymmetric flow field-flow fractionation study.

In this work, the electrophoretic behavior of hydrophobic fullerenes [buckminsterfullerene (C60), C70, and N-methyl-fulleropyrrolidine (C60-pyrr)] and water-soluble fullerenes [fullerol (C60(OH)24); polyhydroxy small gap fullerene, hydrated (C120(OH)30); C60 pyrrolidine tris acid (C60-pyrr tris acid); and (1,2-methanofullerene C60)-61-carboxylic acid (C60CHCOOH)] in micellar electrokinetic capillary chromatography (MECC) was evaluated. The aggregation behavior of the water-soluble compounds in MECC at different buffer and sodium dodecyl sulfate (SDS) concentrations and pH values of the background electrolyte (BGE) was studied by monitoring the changes observed in the electrophoretic pattern of the peaks. Broad and distorted peaks that can be attributed to fullerene aggregation were obtained in MECC which became narrower and more symmetric by working at low buffer and SDS concentrations (below the critical micelle concentration, capillary zone electrophoresis (CZE) conditions). For the characterization of the suspected aggregates formed (size and shape), asymmetrical flow field-flow fractionation (AF4) and transmission electron microscopy (TEM) were used. The results showed that the increase in the buffer concentration promoted the aggregation of the particles, while the presence of SDS micelles revealed multiple peaks corresponding to particles of different aggregation degrees. Furthermore, MECC has been applied for the first time for the analysis of C60 in two different cosmetic products (i.e., anti-aging serum and facial mask).

Characterization of aggregates of surface modified fullerenes by asymmetrical flow field-flow fractionation with multi-angle light scattering detection.

Fullerenes are carbon nanoparticles with widespread biomedical, commercial and industrial applications. Attributes such as their tendency to aggregate and aggregate size and shape impact their ability to be transported into and through the environment and living tissues. Knowledge of these properties is therefore valuable for their human and environmental risk assessment as well as to control their synthesis and manufacture. In this work, asymmetrical flow-field flow fractionation (AF4) coupled to multi-angle light scattering (MALS) was used for the first time to study the size distribution of surface modified fullerenes with both polyhydroxyl and carboxyl functional groups in aqueous solutions having different pH (6.5-11) and ionic strength values (0-200mM) of environmental relevance. Fractionation key parameters such as flow rates, flow programming, and membrane material were optimized for the selected fullerenes. The aggregation of the compounds studied appeared to be indifferent to changes in solution pH, but was affected by changes in the ionic strength. Polyhydroxy-fullerenes were found to be present mostly as 4nm aggregates in water without added salt, but showed more aggregation at high ionic strength, with an up to 10-fold increase in their mean hydrodynamic radii (200mM), due to a decrease in the electrostatic repulsion between the nanoparticles. Carboxy-fullerenes showed a much stronger aggregation degree in water (50-100nm). Their average size and recoveries decreased with the increase in the salt concentration. This behavior can be due to enhanced adsorption of the large particles to the membrane at high ionic strength, because of their higher hydrophobicity and much larger particle sizes compared to polyhydroxy-fullerenes. The method performance was evaluated by calculating the run-to-run precision of the retention time (hydrodynamic radii), and the obtained RSD values were lower than 1%. MALS measurements showed aggregate sizes that were in good agreement with the AF4 data. A comparison of the scattering radii from the MALS with the hydrodynamic radii obtained from the retention times in AF4 indicated that the aggregate shapes are far from spherical. TEM images of the fullerenes in the dry state also showed branched and irregular clusters.

Visualization procedures for proteins and peptides on flat-bed monoliths and their effects on matrix-assisted laser-desorption/ionization time-of-flight mass spectrometric detection.

The present study concerns the application of visualization methods, i.e. coomassie-brilliant-blue-R staining (CBB-R), silver-nitrate staining, and fluorescamine labeling, and subsequent MALDI-MS analysis of intact proteins and peptides on the surface of flat-bed monoliths, intended for spatial two-dimensional chromatographic separations. The use of 100-µm thick macroporous poly(butyl methacrylate-co-ethylene dimethacrylate) flat-bed monoliths renders a fixation step obsolete, so that CBB-R and silver-nitrate staining and destaining could be achieved in 10-15 min as opposed to up to 24h, as is typical on 2D-PAGE gels. The detection limits remained comparable. The compatibility of the monolithic layer with subsequent MALDI-MS analysis of individual proteins and peptide spots was investigated with regards to mass accuracy, mass precision, resolution, and signal intensity. When comparing results from MALDI-MS analysis of proteins and peptides on a flat-bed monolith to results obtained directly on stainless-steel target plates, significant losses in mass precision, signal intensity, and an increased variation in resolution were observed. In addition, a loss in signal intensity up to two orders of magnitude was observed when using monolithic layers. After CCB-R and silver-nitrate staining and destaining to disrupt the protein-dye complexes no MALDI spectra with significant S/N ratios could be achieved. After fluorescamine labeling heterogeneous signals were observed, which resulted from a distribution in the number of fluorescence-labeled lysine groups and from the presence of labeled derivatives that had undergone condensation reactions.

Determination of ribavirin in human serum using liquid chromatography tandem mass spectrometry.

A method has been developed for the determination of ribavirin in human serum for therapeutic drug monitoring purposes, using liquid chromatography electrospray ionization mass spectrometry. Separation was obtained with a mobile phase gradient starting and ending in 100% aqueous conditions using a Waters Atlantis® T3 column (100×2mm, 3µm). The entire sample preparation consisted of dilution, followed by ultrafiltration. From the clear ultrafiltrate 5µL was injected on the LC-MS/MS system. The calibration curves were linear in the range of 0.2-10mg/L with within-run and between-run precisions (CVs) in the range of 0-10%. The method was validated with respect to specificity, selectivity, linearity, accuracy, precision, recovery and stability and meets the requirements of the FDA. The method was extensively tested for matrix effects by determining the variation of the slopes of calibration curves in different sources of serum and plasma. This method is suitable for the determination of ribavirin in human serum for therapeutic drug monitoring.

Advancing liquid/liquid extraction through a novel microfluidic device: theory, instrumentation and applications in gas chromatography.

A new chip-based liquid-liquid extraction technique for sample preparation of aqueous samples for GC was developed. Extraction is performed in a segmented flow system with additional mixing provided by an etched channel structure. The dimensions of the device are optimized to allow benefiting of the advantages of chip technology without suffering from the limitations of over-miniaturization. Phase separation is performed with a novel phase separator developed in house. The instrumental set-up is simple. The results obtained for selected test analytes show that the extraction is quantitative (recoveries = 92-110%, RSD < 6%) for a wide range of hydrophobicities (LogK(o/w) = 0.86-4.79). The performance at different flow rates (0.5-6.0 mL/min) and flow ratios (ß = 1-10) was evaluated, confirming the flexibility and the possibility to perform enrichment. The results obtained for a few selected applications demonstrate the suitability of the method to perform quick, simple and reliable sample preparation for analytes of interest in real samples.

Hydrodynamic chromatography of macromolecules using polymer monolithic columns.

The selectivity window of size-based separations of macromolecules was tailored by tuning the macropore size of polymer monolithic columns. Monolithic materials with pore sizes ranging between 75 nm and 1.2 µm were prepared in situ in large I.D. columns. The dominant separation mechanism was hydrodynamic chromatography in the flow-through pores. The calibration curves for synthetic polymers matched with the elution behavior by HDC separations in packed columns with 'analyte-to-pore' aspect ratios (λ) up to 0.2. For large-macropore monoliths, a deviation in retention behavior was observed for small polystyrene polymers (M(r)<20 kDa), which may be explained by a combined HDC-SEC mechanism for λ<0.02. The availability of monoliths with very narrow pore sizes allowed investigation of separations at high λ values. For high-molecular weight polymers (M(r)>300,000 Da) confined in narrow channels, the separation strongly depended on flow rate. Flow-rate dependent elution behavior was evaluated by calculation of Deborah numbers and confirmed to be outside the scope of classic shear deformation or slalom chromatography. Shear-induced forces acting on the periphery of coiled polymers in solution may be responsible for flow-rate dependent elution.

Determination of cholesterol and triglycerides in serum lipoproteins using flow field-flow fractionation coupled to gas chromatography-mass spectrometry.

Asymmetric flow field flow fractionation (AsFlFFF) was combined with pyrolysis-gas chromatography mass spectrometry for a sized based fractionation and a detailed compositional study of the triglycerides and cholesterol associated with the various lipoprotein subclasses present in human serum. Serum samples were injected in the AsFlFFF instrument and fractionated with a time-delayed exponential decay cross flow program. The fractions collected after AsFlFFF elution were injected into a programmable temperature vaporizer (PTV) GC-injector, containing a fritted liner. A temperature and split-flow program for the PTV injector was optimized for the thermally assisted hydrolysis and methylation of the compounds of interest. The resulting fatty acid and cholesterol methyl esters were separated by GC and characteristic fragment ions were detected by MS. The system was optimized and calibrated with triglyceride and cholesterol standards for quantitative analysis. The possible interference by phospholipids with the quantitative results was investigated and found to be of minor importance. The concentrations and lipoprotein profiles of triglycerides and cholesterol were determined in a pooled serum sample of healthy volunteers and a serum sample of a sepsis patient. The results obtained with the GC-MS approach were compared with those of a previously developed method based on AsFlFFF with a dual enzymatic reaction detection system. A good agreement of the profiles was found, for cholesterol as well as for the triglycerides, even when the GC-MS method quantifies the fatty acids while with the enzymatic reaction method the glycerol concentrations are determined. Total cholesterol and triglyceride concentration values for the serum samples showed good agreement with the results of the standard enzymatic method as used in practice in the university hospital.

Methacrylate monolithic stationary phases for gradient elution separations in microfluidic devices.

Methacrylate monolithic stationary phases were produced in fused-silica chips by UV initiation. Poly(butyl methacrylate-co-ethylene dimethacrylate) (BMA) and poly(lauryl methacrylate-co-ethylene dimethacrylate) (LMA) monoliths containing 30, 35 and 40% monomers were evaluated for the separation of peptides under gradient conditions. The peak capacity was used as an objective tool for the evaluation of the separation performance. LMA monoliths of the highest density gave the highest peak capacities (≈40) in gradients of 15 min and all LMA monoliths gave higher peak capacities than the BMA monoliths with the same percentage of monomers. Increasing the gradient duration to 30 min did not increase the peak capacity significantly. However, running fast (5 min) gradients provides moderate peak capacities (≈20) in a short time. Due to the system dead volume of 1 µL and the low bed volume of the chip, early eluting peptides migrated over a significant part of the column during the dwell time under isocratic conditions. It was shown that this could explain an increased band broadening on the monolithic stationary phase materials used. The effect is stronger with BMA monoliths, which partly explains the inferior performance of this material with respect to peak capacity. The configuration of the connections on the chip appeared to be critical when fast analyses were performed at pressures above 20 bar.

Construction and initial evaluation of an apparatus for spatial comprehensive two-dimensional liquid-phase separations.

Spatial comprehensive two-dimensional chromatography is discussed as a potentially alternative to the conventional column-based approach. In "spatial" separations each analyte ends up in a specific location, rather than being eluted at a specific time. Ultimately, higher peak-capacity-production rates (peak capacity per unit time) may be attained by spatial two- and three-dimensional separations. While low-pressure planar chromatography is well developed, the high-pressure equivalent is still in its infancy. We discuss the requirements for a device for high-pressure spatial two-dimensional chromatography and we describe a possible design. A prototype instrument has been constructed in-house. The preparation of a polymer monolithic separation body and a valve configuration that allows manual sample injection are described. Initial tests of this study included the investigation of the homogeneity of the monolith and the flow profile through the separation body. Furthermore, in order to evaluate the current chromatographic performance of the device, a mixture of dyes was separated in one dimension within 30 s.

Use of kinetic plots for the optimization of the separation time in ultra-high-pressure LC.

The kinetic-plot approach, in which experimental t(0) and N-values are extrapolated to the performance at maximum system pressure by increasing the column length, was validated by coupling 250×3 mm columns packed with 3 µm particles. The extra-column volume introduced by coupling columns could be neglected with respect to the peak volumes. Plate numbers of up to 132,000 were experimentally achieved by coupling four columns. The maximum deviation between the experimental and predicted plate numbers was 7% for two coupled columns, and decreasing to 0.1% for four coupled columns. Kinetic plots were used to find the conditions to separate a critical pair, with a preset value for the effective plate number, in the shortest possible time. For high-efficiency separations yielding 100,000 effective plates, the optimum critical-pair retention factor was around 4.5. Kinetic plots are presented to find the optimal column length to obtain the fastest possible 100,000 effective-plate separation, taking into account the effect of mobile-phase viscosity on column pressure, and consequently the optimum column length.

Comprehensive 2-D chromatography of random and block methacrylate copolymers.

A comprehensive 2-D separation method was developed for the characterization of methacrylate copolymers. In both dimensions conditions were employed that give a critical separation for the homopolymer of one of the monomers in the copolymer, and exclusion behaviour for the other. The 2-D separation was realized by using a normal-phase column in one dimension and a reversed phase column in the other, and by precisely tuning the compositions of the two mobile phases employed. In the normal-phase dimension mixtures of THF and n-hexane or n-heptane were used as mobile phase, and in the reversed-phase dimension mixtures of ACN and THF. Moreover, stationary phase particles had to be selected for both columns that gave an exclusion window appropriate for the molecular size of the sample polymers to be characterized. The 2-D critical chromatography principle was tested with a polystyrene (PS)-polymethylmethacrylate (PMMA) block copolymer and with block and random polybutylmethacrylate (PBMA)-PMMA copolymers. Ideally, the retention time for a copolymer in both dimensions of this system would depend on the size of only one of the blocks, or on the contribution of only one of the monomers to the size of a random copolymer. However, it was found that the elution of the PS-PMMA block copolymer depended on the size of both blocks, even when the corresponding homopolymer of one of the monomers showed critical elution behaviour. Therefore, the method could not be calibrated for block sizes by using homopolymer standards alone. Still, it was shown that the method can be used to determine differences between samples (PS-PMMA and PBMA-PMMA) with respect to total molecular size or block sizes separately, or to average size and chemical composition for random copolymers. Block and random PBMA-PMMA copolymers showed a distinctly different pattern in the 2-D plots obtained with 2-D critical chromatography. This difference was shown to be related to the different procedures followed in the polymerization process, and the different molecular distributions resulting from these.

Optimal gradient operation in comprehensive liquid chromatography x liquid chromatography systems with limited orthogonality.

A novel strategy is described for designing optimal second dimension (2D) gradient conditions for a comprehensive two-dimensional liquid chromatography system where the two dimensions are not fully orthogonal. Using the approach developed here, the initial and final organic modifier content values resulting in the highest coverage of separation space can be derived for each 2D gradient run. Theory indicates that these values can be determined by adapting 2D gradient operation to the degree of orthogonality. The new method is tested on a comprehensive two-dimensional liquid chromatography system that uses reversed phase (RP) columns showing different selectivities in the two dimensions. A comparison between analyses carried out using normal and optimized 2D gradients showed that the latter allow a more efficient use of analysis time. This can result either in an improved peak capacity or in decreasing total analysis time, depending on the final goal of the experiment. In the latter scenario, the number of separated peaks is comparable to that obtained using gradients spanning a wide range of organic modifier but, now, in half the time. As test samples complex mixtures of peptides were analyzed.

Fractionation of human serum lipoproteins and simultaneous enzymatic determination of cholesterol and triglycerides.

A method based on Asymmetric Flow Field-Flow Fractionation (AF4) was developed to separate different types of lipoproteins from human serum. The emphasis in the method optimization was on the possibilities to characterize the largest lipoprotein fractions (LDL and VLDL), which is usually not possible with the size-exclusion chromatography methods applied in routine analysis. Different channel geometries and flow programs were tested and compared. The use of a short fractionation channel was shown to give less sample dilution at the same fractionation power compared to a conventional, long channel. Different size selectivities were obtained with an exponential decay and a linear cross flow program. The ratio of the UV absorption signal to the light scattering signal was used to validate the relation between retention time and size of the fractionated particles. An experimental setup was developed for the simultaneous determination of the cholesterol and triglycerides distribution over the lipoprotein fractions, based on enzymatic reactions followed by UV detection at 500 nm. Coiled and knitted PTFE tubing reactors were compared. An improved peak sharpness and sensitivity were observed with the knitted tubing reactor. After optimization of the experimental conditions a satisfactory linearity and precision (2-3% rsd for cholesterol and 5-6% rsd for triglycerides) were obtained. Finally, serum samples, a pooled sample from healthy volunteers and samples of sepsis patients, were analyzed with the method developed. Lipoprotein fractionation and cholesterol and triglyceride distributions could be correlated with the clinical background of the samples.

Methacrylate monolithic capillary columns for gradient peptide separations.

For the separation of peptides with gradient-elution liquid chromatography a poly(butyl methacrylate-co-ethylene dimethacrylate) (BMA) monolithic capillary column was prepared and tested. The conditional peak capacity was used as a metric for the performance of this column, which was compared with a capillary column packed with C18-modified silica particles. The retention of the peptides was found to be smaller on the BMA column than on the particulate C18 column. To obtain the same retention in isocratic elution an approximately 15% (v/v) lower acetonitrile concentration had to be used in the mobile phase. The retention window in gradient elution was correspondingly smaller with the BMA column. The relation between peak width and retention under gradient conditions was studied in detail. It was found that in shallow gradients, with gradient times of 30min and more, the peak widths of the least retained compounds are strongly increased with the BMA column. This was attributed to the fact that these compounds migrate and elute with an unfavorable high retention factor. More retained compounds are eluted later in the gradient, but with a lower effective retention factor. With shallow gradients the peak capacity of the BMA column ( approximately 90) was clearly lower than that of a conventional packed column ( approximately 150). On the other hand, with steep gradients, when components elute with a low effective retention factor, the performance of the BMA column is relatively good. With a gradient time of 15min similar peak widths and thus similar peak capacities ( approximately 75) were found for the packed and the monolithic column. Two strategies were investigated to obtain higher peak capacities with methacrylate monolithic columns. The use of lauryl methacrylate (LMA) instead of butyl methacrylate (BMA) gave an increase in retention and narrower peaks for early eluting peptides. The peak capacity of the LMA column was approximately 125 in a 60min gradient. Another approach was to use a longer BMA column which resulted in a peak capacity of approximately 135 could be obtained in 60min.

Recent applications in CEC.

In this review, research papers on CEC are summarized that have been published between May 2005 and May 2007. Application-oriented research is discussed in which CEC is used in biochemical and pharmaceutical studies, in the analysis of food and natural products, and in industrial, environmental, and forensic analysis. Some trends and developments in separation science that may increase the applicability of the separation technique CEC are highlighted: 2-D separation systems and the application of nano- and microfluidic devices in separations.

Topographic structures and chromatographic supports in microfluidic separation devices.

A review is given of the literature on the design, development and use of micromachined devices for separations in the liquid phase. The emphasis is on those devices that offer more than just an empty channel for, e.g., electrophoretic separation. Topographic structures have been incorporated in the channels during their microfabrication, offering a variety of possibilities for the separation of (mainly) DNA molecules based on different principles. Supports for a stationary phase for chromatographic separations have been introduced in the channels in different ways: by packing of the channels with stationary phase particles, by polymerization of monolithic structures, or by lithographic machining of pillars in the channels. It is shown that the latter strategy gives the highest potential for increasing the separation power of the devices. Still, more conventional approaches are closer to a routine application.

Efficiency of methacrylate monolithic columns in reversed-phase liquid chromatographic separations.

Butyl-methacrylate-based porous monoliths were prepared inside fused-silica capillaries as reversed-phase separation media for liquid chromatography (LC) and capillary electrochromatography (CEC). During our previous research on methacrylate-based monoliths for reversed-phase separations, we noticed that a separation efficiency of up to 300,000 plates/m can easily be obtained in the CEC mode for unretained compounds. However, the efficiencies for retained compounds were much lower in reversed-phase systems, especially in pressure-driven LC. In this work methacrylate-based columns were prepared and characterized in terms of efficiency and retention in reversed-phase (pressure-driven) LC and in CEC. Much attention has been paid to the mass-transfer mechanism in the stationary phase. Factors that affect the plate heights for specific compounds have been investigated. A possible explanation for the relatively low separation efficiency of retained compounds and suggestions to improve molecular mass transfer are provided.