Online hyphenation of size-exclusion chromatography and gas-phase electrophoresis facilitates the characterization of protein aggregates.

Gas-phase electrophoresis yields size distributions of polydisperse, aerosolized analytes based on electrophoretic principles. Nanometer-sized, surface-dry, single-charged particles are separated in a high laminar sheath flow of particle-free air and an orthogonal tunable electric field. Additionally, nano Electrospray Gas-Phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA) data are particle-number based. Therefore, small particles can be detected next to larger ones without a bias, for example, native proteins next to their aggregates. Analyte transition from the liquid to the gas phase is a method inherent prerequisite. In this context, nonvolatile sample buffers influence results. In the worst case, the (bio-)nanoparticle signal is lost due to an increased baseline and unspecific clustering of nonvolatile components. We present a novel online hyphenation of liquid chromatography and gas-phase electrophoresis, coupling a size-exclusion chromatography (SEC) column to an advanced nES GEMMA. Via this novel approach, it is possible to (i) separate analyte multimers already present in liquid phase from aggregates formed during the nES process, (ii) differentiate liquid phase and spray-induced multimers, and (iii) to remove nonvolatile buffer components online before SEC-nES GEMMA analysis. Due to these findings, SEC-nES GEMMA has the high potential to help to understand aggregation processes in biological buffers adding the benefit of actual size determination for noncovalent assemblies formed in solution. As detection and characterization of protein aggregation in large-scale pharmaceutical production or sizing of noncovalently bound proteins are findings directly related to technologically and biologically relevant situations, we proposed the presented method to be a valuable addition to LC-MS approaches.

Analysis of exhaled breath from smokers, passive smokers and non-smokers by solid-phase microextraction gas chromatography/mass spectrometry.

In this study, 38 samples of expired air were collected and analyzed from 20 non-smoking volunteers, four passive smokers and 14 smokers (21 women and 17 men). Measurements were carried out using solid-phase microextraction (SPME) as an isolation and preconcentration technique. The determination and identification were accomplished by gas chromatography coupled with mass spectrometry (GC/MS). Our data showed that ca 32% of all identified compounds in the breath of healthy non-smokers were saturated hydrocarbons. In the breath of smoking and passive smoking volunteers hydrocarbons were predominant, but also present were more exogenous analytes such as furan, acetonitrile and benzene than in the breath of non-smokers. Acetonitrile, furan, 3-methylfuran, 2,5-dimethylfuran, 2-butanone, octane and decane were identified in breath of smoking and passive smoking persons.

Using of molecularly imprinted polymers for determination of gallic and protocatechuic acids in red wines by high performance liquid chromatography.

The sorption capacities of gallic- and protocatechuic acid-molecularly imprinted polymers (GA-MIP and PCA-MIP, respectively) and non-imprinted polymer (NIP) have been determined on the piston columns by the frontal analyses (FAs). Mobile phases consisted of MeOH, MeOH/H2O (1:1), 12.5% EtOH or ACN. Solutes concentrations used in FAs were 1µg/mL and 50µg/mL. All sorption capacities were depended on analyte and solvent used. Results obtained from the FAs have shown that both imprinted polymers almost always were preferentially recognized PCA molecule. Only in MeOH, the GA-MIP had ability to recognize its template molecule positively. Surprisingly, in some cases, also the NIP exhibited higher sorption capacities than the MIPs for their templates, e.g. in ACN for GA or in MeOH for PCA. This behaviour indicates that in some solvents, the low affinity sites of the blank polymer can act as strong interacting sites. In the next, prepared MIPs were successfully used as the SPE-sorbents for the extraction and purification of chosen phenolic acids from red wine samples. The recoveries both of MIPs were the highest for PCA, what was in agreement with the experiments carried out in 12.5% EtOH during FAs. Prepared MIP-beads allowed the purification of chosen red wine samples with satisfactory selectivities and high recoveries. The linearity of the method was in the range from 10µg/mL to 70µg/mL and 0.1µg/mL to 4.5µg/mL for GA and PCA, respectively, with the determination coefficients ranging from 0.996-0.999. The LODs (S/N=3) ranged from 0.1µg/mL to 0.4µg/mL. The RSDs for the recoveries varied from 4.0% to 8.1%. The PAs-MIPs and corresponding NIP were also characterized by attenuated total reflectance analysis Fourier transform infrared spectroscopy (ATR-FTIR) and scanning electron analysis (SEM).

Application of the van't Hoff dependences in the characterization of molecularly imprinted polymers for some phenolic acids.

Thermodynamic analysis was used to quantify the contribution of entropic and enthalpic terms of the binding processes of selected phenolic acids (PAs), quercetin and diperodon on series of molecularly imprinted polymers (MIPs). All polymers were prepared using acrylamide as functional monomer and acetonitrile as a porogen. The following PAs were used as templates - gallic (GA), gentisic (GeA), syringic (SyrA), protocatechuic (PCA), 4-hydroxybenzoic (pHBA) and vanillic (VA). The assessment was based on quantification by HPLC measurement of the analytes tested at temperature range from 20°C to 60°C in two mobile phases - methanol and porogen. There were determined van't Hoff curves - dependences between logarithms of the retention factors (lnk) and the inverse value of the temperature (1/T). All plots fall along straight lines, what suggests that there were no changes in the sorption mechanisms over the studied temperature range. Determined thermodynamic characteristics helped to specify the nature of molecular recognition on the PAs-MIPs. We found that preferred eluent for analytes sorption on the PAs-MIPs and the NIP was porogen. When methanol as the mobile phase was used there was not documented sorption of the investigated compounds on the NIP. Calculated imprinting factors (IFs) in porogen were highest in the dominant advantage of template molecules used, what confirmed a good molecular imprinting effect. The IF values for PAs studied were as follows: GA=21.98±2.62, PCA=6.07±0.13, pHBA=3.58±0.25, SyrA=2.80±0.17, GeA=2.37±0.34 and VA=2.07±0.10. The results of thermodynamic studies demonstrated that enthalpic term was the dominating driving force for the predominant part of investigated analytes. The exceptions were: SyrA on the NIP and on the GA-MIP, diperodon on the PCA-MIP in acetonitrile and quercetin on the GA-MIP in methanol where a favourable driving force was to be found an entropic term. The PAs-MIPs and NIP were also characterized by attenuated total reflectance analysis Fourier transform infrared spectroscopy (ATR-FTIR) and scanning electron microscopy (SEM).

Thermodynamic study of molecularly imprinted polymer used as the stationary phase in high performance liquid chromatography.

Molecularly imprinted polymer (MIP) and non-imprinted polymer (NIP) on the base of methacrylic acid prepared by a bulk polymerization were used as stationary phases for the HPLC analysis. The thermodynamic processes were carried out to investigate the temperature effects during sorption processes of potential local anaesthetics - morpholinoethyl esters of alkoxy-substituted phenylcarbamic acid (MEsP), local anaesthetic - diperodon, flavonoid - quercetin in methanol, acetonitrile and toluene (porogen) as mobile phases. Mobile phases and corresponding solvents were selected according to the solubility of each analyte. The template was chosen from the set of homologous of MEsP - 2-(morpholin-4-yl)ethyl (2-methoxyphenyl)carbamate. Values of retention factors were measured over the temperature range of 20-60°C. There were determined van't Hoff curves - dependences between logarithms of the retention factors (lnk) and the inverse value of the temperature (1/T). Observed graphs were linear directly indicating that there were no changes of interaction mechanisms in the studied range of temperature. Selectivities (evaluated by the separation factors, α) and sorption selectivities (evaluated by the imprinting factors, IFs) of the MIP and the NIP toward template, related and not-related structures with the template were evaluated chromatographically. The highest separation factors and the imprinting factors (IF=4.73 ± 0.35 for the template) were observed in methanol, not in porogen. Only in the case of quercetin the highest IF was observed in ACN (1.88 ± 0.13). Contrary to expectations, the driving force for the affinity of the target molecules for both of polymers was enthalpic term (with an average of 54%, 82% and 84% contribution of enthalpic term for MeOH, ACN and toluene, respectively on the MIP and 53%, 57% and 65% for MeOH, ACN and toluene, respectively on the NIP). The MIP and NIP were also characterized by attenuated total reflectance analysis Fourier transform infrared spectroscopy (ATR-FTIR) and thermogravimetric analysis (TGA).