Asymmetrical flow field-flow fractionation to probe the dynamic association equilibria of ß-D-galactosidase.

Protein dynamics play a significant role in many aspects of enzyme activity. Monitoring of structural changes and aggregation of biotechnological enzymes under native conditions is important to safeguard their properties and function. In this work, the potential of asymmetrical flow field-flow fractionation (AF4) to study the dynamic association equilibria of the enzyme ß-D-galactosidase (ß-D-Gal) was evaluated. Three commercial products of ß-D-Gal were investigated using carrier liquids containing sodium chloride or ammonium acetate, and the effect of adding magnesium (II) chloride to the carrier liquid was assessed. Preservation of protein structural integrity during AF4 analysis was essential and the influence of several parameters, such as the focusing step (including use of frit-inlet), cross flow, and injected amount, was studied. Size-exclusion chromatography (SEC) and dynamic light scattering (DLS) were used to corroborate the in-solution enzyme oligomerization observed with AF4. In contrast to SEC, AF4 provided sufficiently mild separation conditions to monitor protein conformations without disturbing the dynamic association equilibria. AF4 analysis showed that ammonium acetate concentrations above 40 mM led to further association of the dimers ("tetramerization") of ß-D-Gal. Magnesium ions, which are needed to activate ß-D-Gal, appeared to induce dimer association, raising justifiable questions about the role of divalent metal ions in protein oligomerization and on whether tetramers or dimers are the most active form of ß-D-Gal.

Evaluation of particle and bed integrity of aqueous size-exclusion columns packed with sub-2 µm particles operated at high pressure.

The performance of columns packed with 1.7 µm particles for aqueous size-exclusion chromatography was assessed at high-pressure conditions and linked to particle- and column-bed integrity. Decreasing the particle size from 3.5 µm to 1.7 µm increases the resolution due to the improved mass-transfer characteristics, allowing to significantly speed-up analysis without compromising the selectivity. A sub-minute separation of intact proteins was realized on a 4.6 mm i.d × 75 mm long column packed with 1.7 µm SEC particles applying a flow rate of 1.8 mL/min, corresponding to a column pressure of 530 bar. Ultra-high pressure operation (exceeding manufacturer's recommendations) resulted in peak deformation, a shift towards earlier retention times, and an alteration in selectivity. To gain insights in the mechanisms of column deterioration, short 30 mm long columns were operated at UHPLC conditions, maximizing the pressure drop over individual particles. This resulted in the presence of fractured particles situated at the column outlet, as verified by scanning electron micrographs. Mercury-intrusion porosimetry and argon-adsorption measurements did not reveal significant differences in intraparticle volume between particle batches sampled before and after pressure stress testing. As particles at the column outlet fracture (but not collapse) at high pressure operation, a void was formed at the column inlet. The degradation of the separation performance appeared to be the result of a decrease in interparticle pore volume.

Generic approach to the method development of intact protein separations using hydrophobic interaction chromatography.

We describe a liquid chromatography method development approach for the separation of intact proteins using hydrophobic interaction chromatography. First, protein retention was determined as function of the salt concentration by isocratic measurements and modeled using linear regression. The error between measured and predicted retention factors was studied while varying gradient time (between 15 and 120 min) and gradient starting conditions, and ranged between 2 and 15%. To reduce the time needed to develop optimized gradient methods for hydrophobic interaction chromatography separations, retention-time estimations were also assessed based on two gradient scouting runs, resulting in significantly improved retention-time predictions (average error < 2.5%) when varying gradient time. When starting the scouting gradient at lower salt concentrations (stronger eluent), retention time prediction became inaccurate in contrast to predictions based on isocratic runs. Application of three scouting runs and a nonlinear model, incorporating the effects of gradient duration and mobile-phase composition at the start of the gradient, provides accurate results (improved fitting compared to the linear solvent-strength model) with an average error of 1.0% and maximum deviation of -8.3%. Finally, gradient scouting runs and retention-time modeling have been applied for the optimization of a critical-pair protein isoform separation encountered in a biotechnological sample.

Aqueous size-exclusion chromatographic separations of intact proteins under native conditions: Effect of pressure on selectivity and efficiency.

The selectivity and separation efficiency of aqueous size-exclusion chromatographic separations of intact proteins were assessed for different flow rates, using columns packed with 3 and 5 µm silica particles containing 150 and 290 Å stagnant pores. A mixture of intact proteins with molecular weights ranging between 17 000 and 670 000 Da was used to construct the calibration curves. Both the model fit and the predictive properties, using a leave-one-out strategy, of different polynomial models (up to fifth order) were evaluated for different flow rates. The best compromise between model fit and predictive properties was obtained using a third-order polynomial model. The accuracy of the predictive properties decreased with 10% with an eightfold increase in the flow rate. No changes in retention factors (hence selectivity) were observed in the flow-rate range applied. A strong correlation between molecular weight and plate height was observed. Exclusion of large-molecular-weight proteins led to a significant reduction in the stationary-phase mass-transfer contribution to the total plate-height value, and this effect was also independent of the flow rate applied. The kinetic-performance limits, in terms of plate number and time, and optimal column-length particle-size combinations were determined at the maximum recommended operating pressure of the size-exclusion chromatography columns (20 MPa). Finally, the possibilities of method speed-up using ultra-high-pressure size-exclusion chromatography in combination with columns packed with sub-2 µm particles are discussed.

An automated method for the analysis of phenolic acids in plasma based on ion-pairing micro-extraction coupled on-line to gas chromatography/mass spectrometry with in-liner derivatisation.

A new method is presented for the analysis of phenolic acids in plasma based on ion-pairing 'Micro-extraction in packed sorbent' (MEPS) coupled on-line to in-liner derivatisation-gas chromatography-mass spectrometry (GC-MS). The ion-pairing reagent served a dual purpose. It was used both to improve extraction yields of the more polar analytes and as the methyl donor in the automated in-liner derivatisation method. In this way, a fully automated procedure for the extraction, derivatisation and injection of a wide range of phenolic acids in plasma samples has been obtained. An extensive optimisation of the extraction and derivatisation procedure has been performed. The entire method showed excellent repeatabilities of under 10% and linearities of 0.99 or better for all phenolic acids. The limits of detection of the optimised method for the majority of phenolic acids were 10ng/mL or lower with three phenolic acids having less-favourable detection limits of around 100 ng/mL. Finally, the newly developed method has been applied in a human intervention trial in which the bioavailability of polyphenols from wine and tea was studied. Forty plasma samples could be analysed within 24h in a fully automated method including sample extraction, derivatisation and gas chromatographic analysis.

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.

Evaluation of comprehensive on-line liquid chromatography thermally assisted hydrolysis and methylation-gas chromatography-mass spectrometry for characterization of sulfonated lignins.

Sulfonated lignins, used as dispersants in agrochemical formulations, have been characterized by hyphenation of ion-pair RPLC and thermally assisted hydrolysis and methylation-GC-MC (THM-GC-MS). The chemical structure of a series of selected lignins was evaluated, both in terms of overall composition and in terms of composition as a function of molecular size. Some sulfonated lignins give rise to unstable formulations. In the compositional analysis, these samples were found to yield additional fragments. A newly developed comprehensive method made it possible to monitor the occurrence of these fragments as a function of molecular size. In addition to differences in the molecular size between "good" and "bad" batches, clear differences in chemical composition were established.

A fast method for the identification of Mycobacterium tuberculosis in sputum and cultures based on thermally assisted hydrolysis and methylation followed by gas chromatography-mass spectrometry.

A fast gas chromatography-mass spectrometry (GC-MS) method with minimum sample preparation is described for early diagnosis of tuberculosis (TB). The automated procedure is based on the injection of sputum samples which are then methylated inside the GC injector using thermally assisted hydrolysis and methylation (THM). The THM-GC-MS procedure was optimized for the injection of sputum samples. For the identification of Mycobacterium tuberculosis the known marker tuberculostearic acid (TBSA) and other potential markers were evaluated. Hexacosanoic acid in combination with TBSA was found to be specific for the presence of M. tuberculosis. For validation of the method several sputum samples with different viscosities spiked with bacterial cultures were analyzed. Finally, 18 stored sputum samples collected in Vietnam from patients suspected to suffer from TB were re-analyzed in Amsterdam by microscopy after decontamination/concentration and using the new THM-GC-MS method. No false positives were found by THM-GC-MS and all patients who were diagnosed with TB were also found positive using our newly developed THM-GC-MS method. These results show that the new fast and sensitive THM-GC-MS method holds great potential for the diagnosis of TB.

Fully automated system for the gas chromatographic characterization of polar biopolymers based on thermally assisted hydrolysis and methylation.

Pyrolysis-gas chromatography (Py-GC) is a powerful tool for the detailed compositional analysis of polymers. A major problem of Py-GC is that polar (bio)polymers yield polar pyrolyzates which are not easily accessible to further GC characterization. In the present work, a newly developed fully automated procedure for thermally assisted hydrolysis and methylation (THM) of biopolymers is described. Drying of the sample, addition of the reagent, incubation and pyrolysis are performed inside the liner of a programmable temperature vaporizer injector. The new system not only allows efficient analysis of large series of samples, but also allows automated optimization of the experimental parameters based on an experimental design approach. The performance of the automated THM-procedure was evaluated by performing THM-GC of a poly(acrylic acid)-poly(maleic anhydride) copolymer (PAA/PMAH) and several polysaccharides. The optimized THM-procedure was applied for the structural characterization and differentiation of several lignins and hydroxypropylmethyl-celluloses. It was also applied to proteins. Here myoglobin and cytochrome c were used as the model compounds. Both conventional GC-mass spectrometry (MS) and comprehensive two-dimensional gas chromatography (GCxGC)-time-of-flight (TOF) MS were used for separation and identification of the species formed. The information obtained can aid in structure elucidation of polar biopolymers as well as in providing detailed compositional information which can be used to differentiate structurally similar biopolymers.

Extending the molecular application range of gas chromatography.

Gas chromatography is an important analytical technique for qualitative and quantitative analysis in a wide range of application areas. It is fast, provides a high peak capacity, is sensitive and allows combination with a wide range of selective detection methods including mass spectrometry. However, the application area of GC is limited because the molecules to be analysed have to be thermally stable and sufficiently volatile. Numerous molecules do not meet these requirements and hence are not amenable to direct GC analysis. Recent research has resulted in better chromatographic columns and methods for sample preparation that enable a significant expansion of the molecular application range of GC. The strategies exploited include conversion of (macro)molecules into smaller species and approaches to reduce the polarity of molecules. In this review we identify four generic routes for extending the applicability of GC. These include high-temperature GC, derivatisation, pyrolysis and thermochemolysis. The principles, recent developments and future perspectives of these routes are discussed and examples of applications using the different options will be shown. Life sciences, metabonomics and profiling strategies for sample characterization are identified as important future drivers for the continued development of GC.

Characterization of olive oil volatiles by multi-step direct thermal desorption-comprehensive gas chromatography-time-of-flight mass spectrometry using a programmed temperature vaporizing injector.

The feasibility of a versatile system for multi-step direct thermal desorption (DTD) coupled to comprehensive gas chromatography (GC x GC) with time-of-flight mass spectrometric (TOF-MS) detection is studied. As an application the system is used for the characterization of fresh versus aged olive oil after treatment at 70, 175, 250 and 600 degrees C.

Hyphenation of aqueous liquid chromatography to pyrolysis-gas chromatography and mass spectrometry for the comprehensive characterization of water-soluble polymers.

A recently developed hyphenated system for "organic" size-exclusion chromatography-pyrolysis-gas chromatography-mass spectrometry (SEC-Py-GC-MS) is adapted to allow the use of aqueous LC eluents as applied in the characterization of water-soluble polymers. The system uses syringe-based transfer of multiple LC-fractions to the GC instrument with solvent elimination and subsequent pyrolysis in a programmed temperature vaporization injector. The problems of the large-volume injections of aqueous, salt containing eluents into the Py-GC-MS are solved by using a 'sintered-bed liner' for elimination of the water at a high temperature, a volatile salt and the installation of a back-flush option. After optimization, the system was applied for the determination of the combined molecular weight-chemical composition of a polyethylene glycol-polypropylene glycol block copolymer. This analysis was done with the system in the aqueous SEC-Py-GC-MS mode. Also demonstrated is the automated at-line characterization of a random polystyrene-polymethylmethacrylate copolymer, now with the system in the gradient reversed-phase LC-Py-GC-MS mode. The methods proposed in the present work are very useful for the detailed characterization of water-soluble copolymers.

On-line size exclusion chromatography-pyrolysis-gas chromatography-mass spectrometry for copolymer characterization and additive analysis.

On-line coupled size exclusion chromatography-pyrolysis gas chromatography mass spectrometry (SEC-Py-GC-MS) is studied as a novel tool for the characterization of complex polymer samples. An automated system for on-line SEC-Py-GC-MS allowing transfer of multiple fractions was developed based on stop-flow operation of the SEC dimension, syringe-based transfer of the SEC fraction to the GC instrument and solvent elimination with subsequent pyrolysis in a programmed temperature vaporization (PTV) injector. After optimization the system was applied to the characterization of a complex terpolymer composed of very similar monomers. The use of the system for combined pyrolysis and additive analyses in polycarbonate was also demonstrated. Results obtained with the new method indicate the interesting potentials of the method for detailed characterization of polymeric materials.

Hyphenation of infrared spectroscopy to liquid chromatography for qualitative and quantitative polymer analysis: degradation of poly(bisphenol A)carbonate.

Hyphenation of infrared spectroscopy (IR) to liquid chromatography (LC) has been applied to study chemical changes in poly(bisphenol A)carbonate (PC) as a result of degradation. Especially coupling of LC to FTIR through solvent elimination is a sensitive approach to identify changes in functionality observed in the LC chromatograms as has been demonstrated by coupling of liquid chromatography under critical conditions (LCCC) to IR. Furthermore, an example is shown in which two-dimensional liquid chromatography, i.e. LCCC x SEC, was coupled to IR by means of a flow cell. This resulted in data sets containing most probably valuable data, but extracting relevant information from these large data sets is not straightforward at all. Therefore, multivariate data analysis (MVDA) of SEC-FTIR data was used to extract relevant data from large data sets. This approach revealed chemical differences due to degradation that could not be detected by other means. Spectral features could be identified that allowed to quantitatively predict the degradation of poly(bisphenol A)carbonate as a function of degradation conditions.