Comparison of chromatographic ion-exchange resins. III. Strong cation-exchange resins

A comparative study was performed on strong cation-exchangers to investigate the pH dependence, efficiency, binding strength, particle size distribution, static and dynamic capacity, and SEM pictures of chromatographic resins. The resins tested included: SP Sepharose XL, Poros 50 HS, Toyopearl SP 550c, SP Sepharose BB, Source 30S, TSKGel SP-5PW-HR20, and Toyopearl SP 650c. Testing was performed with four different proteins: anti-FVII Mab (IgG), aprotinin, lysozyme, and myoglobin. Dependence of pH on retention was generally very low for proteins with high pI. An unexpected binding at pH 7.5 of anti-FVII Mab with pI < 7.5 was observed on several resins. Efficiency results show the expected trend of higher dependence of the plate height with increasing flow rate of soft resins compared to resins for medium and high-pressure operation. Determination of particle size distribution by two independent methods, Coulter counting and SEM, was in very good agreement. The mono-dispersed nature of Source 30S was confirmed. Binding to cation-exchange resins as a function of ionic strength varies depending on the specific protein. Generally, binding and elution at high salt concentration may be performed with Toyopearl SP 550c and Poros 50 HS, while binding and elution at low salt concentration may be performed with Toyopearl SP 650c. A very high binding capacity was obtained with SP Sepharose XL. Comparison of static capacity and dynamic capacity at 10% break-through shows in general approximately 50-80% utilisation of the total available capacity during chromatographic operation. A general good agreement was obtained between this study and data obtained by others. The results of this study may be used for selection of resins for testing in process development. The validity of experiments and results with model proteins were tested using human insulin precursor in pure state and in real feed-stock on Toyopearl SP 550c, SP Sepharose BB, and Toyopearl SP 650c. Results showed good agreement with experiments with model proteins.     

Clean-up method for determination of established and emerging brominated flame retardants in dust

A clean-up method was developed to enable the determination of tri-decabrominated diphenyl ethers, isomer-specific hexabromocyclododecanes (HBCDs), 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE), decabromodiphenyl ethane (DBDPE), (2-ethylhexyl)tetrabromobenzoate (TBB), and bis(2-ethylhexyl)tetrabromophthalate (TBPH) in the same dust sample extract using reasonable amounts of solvents and without dividing the sample. After extraction, the sample was separated on a silica column into three fractions that were subsequently cleaned up individually. The polybrominated diphenyl ethers (PBDEs) and DBDPE were eluted in Fraction I, TBB, TBPH, and BTBPE in Fraction II, and HBCDs in Fraction III. Fractions I and II were analyzed using gas chromatography/mass spectrometry and Fraction III using liquid chromatography/mass spectrometry. The method gave good recoveries (60-120%), precise results using (13)?C-labelled internal standards and was accurate when comparing results to certified values (PBDEs in NIST SRM 2585). The method was applied to dust samples from the Stockholm (Sweden) area. All the emerging brominated flame retardants (BFRs) studied, except BTBPE, were present in all the samples in quantifiable concentrations, often higher than the PBDEs. BTBPE was quantified in only one sample. It is evident that emerging BFRs are present in Swedish homes, and these compounds should be included in the BFR analyses of indoor environments.     

The complete 51V MAS NMR spectrum of surface vanadia nanoparticles on anatase (TiO2): vanadia surface structure of a DeNOx catalyst

The first observations of the complete manifold of spinning sidebands (ssbs) including both the central and satellite transitions in (51)V MAS NMR spectra of surface vanadia nanoparticles on titania in DeNO(x) catalysts are presented. (51)V quadrupole coupling and chemical shift anisotropy parameters for the dominating vanadia structure are determined from (51)V MAS NMR spectra recorded at 9.4 and 14.1 T. Based on correlations previously established between (51)V NMR parameters and crystal structure data for inorganic vanadates, the NMR data are consistent with vanadium in a distorted octahedral oxygen coordination environment for the so-called strongly bonded vanadia species on the surface. The investigation includes two vanadia-titania model catalysts and six industrial-type DeNO(x) catalysts.     

Analysis of low molecular mass organic acids in natural waters by ion exclusion chromatography tandem mass spectrometry

A sensitive and selective method for the analysis of aliphatic low molecular mass organic acids (LMMOAs) in natural waters is presented. The method is based on separation with ion exclusion chromatography and detection with electrospray ionization tandem mass spectrometry (LC-MS/MS). The extra selectivity gained by applying MS/MS allows for a minimum of sample preparation and the use of a sub-optimal mobile phase regarding chromatographic resolution. Instead the mobile phase, comprising aqueous formic acid with methanol as organic modifier, was mainly optimized for maximum sensitivity and long term MS stability. Detection limits for malonic, fumaric, maleic, succinic, citraconic, glutaric, malic, alpha-ketoglutaric, tartaric, shikimic, trans-aconitic, cis-aconitic, isocitric and citric acid were in the range 1-50 nM, while the detection limits for pyruvic, oxalic and lactic acid were around 250 nM for an injection volume of 100 microL. Due to their metal-chelating properties, these LMMOAs are all considered to affect the bioavailability of metals and to be involved in soil forming processes. It is thus of interest to be able to monitor their presence in natural waters, and the method developed within this work was successfully applied for the analysis of LMMOAs in soil solution and stream water samples.     

Mobility of Thermomyces lanuginosus lipase on a trimyristin substrate surface

We have studied the mobility of active and inactive Thermomyces lanuginosus lipase (TLL) on a spin-coated trimyristin substrate surface using fluorescence recovery after photobleaching (FRAP) in a confocal microscopy setup. By photobleaching a circular spot of fluorescently labeled TLL adsorbed on a smooth trimyristin surface, both the diffusion coefficient D and the mobile fraction f could be quantified. FRAP was performed on surfaces with different surface density of lipase and as a function of time after adsorption. The data showed that the mobility of TLL was significantly higher on the trimyristin substrate surfaces compared to our previous studies on hydrophobic model surfaces. For both lipase variants, the diffusion decreased to similar rates at high relative surface density of lipase, suggesting that crowding effects are dominant with higher adsorbed amount of lipase. However, the diffusion coefficient at extrapolated infinite surface dilution, D0, was higher for the active TLL compared to the inactive (D0 = 17.9 x 10(-11) cm2/s vs D0 = 4.1 x 10(-11) cm2/s, data for the first time interval after adsorption). Moreover, the diffusion decreased with time after adsorption, most evident for the active TLL. We explain the results by product inhibition, i.e., that the accumulation of negatively charged fatty acid products decreased the diffusion rate of active lipases with time. This was supported by sequential adsorption experiments, where the adsorbed amount under flow conditions was studied as a function of time after adsorption. A second injection of lipase led to a significantly lower increase in adsorbed amount when the trimyristin surface was pretreated with active TLL compared to pretreatment of inactive TLL.     

Analysis of pesticide residues in fruit and vegetables with ethyl acetate extraction using gas and liquid chromatography with tandem mass spectrometric detection

A multiresidue method based on extraction with ethyl acetate has been used at the Swedish National Food Administration since 1989 to monitor pesticide residues in fruit and vegetables. The method has been continuously adjusted, resulting in simple and quick analyses of pesticide residues. To recover basic pesticides, the addition of an alkali is necessary. The addition of sodium hydrogen carbonate has been shown to recover all pesticides effectively without any degradation. The liquid chromatography (LC) with tandem mass spectrometry (MS/MS) technique has made it possible to analyse more polar pesticides and to replace many single methods. The latest development in the multiresidue method, comprising the use of gas chromatography (GC) with MS/MS, has further improved the analysis by replacing the conventional GC detectors. The need for cleanup has been reduced or eliminated entirely. Consequently, the method has been simplified in a way that makes it possible to recover all included analytes in many different matrices in one single extraction and to detect them either with GC-MS/MS or with LC-MS/MS.     

A comparison between dual polarization interferometry (DPI) and surface plasmon resonance (SPR) for protein adsorption studies

This work was performed with the aim of comparing protein adsorption results obtained from the recently developed dual polarization interferometry (DPI) with the well-established surface plasmon resonance (SPR) technique. Both techniques use an evanescent field as the sensing element but completely different methods to calculate the adsorbed mass. As a test system we used adsorption of the lipase from Thermomyces lanuginosus (TLL) on C18 surfaces. The adsorbed amount calculated with both techniques is in good agreement, with both adsorption isotherms saturating at 1.30–1.35 mg/m² at TLL concentrations of 1000 nM and above. Therefore, this supports the use of both SPR and DPI as tools for studying protein adsorption, which is very important when comparing adsorption data obtained from the use different techniques. Due to the spot sensing in SPR, this technique is recommended for initial kinetic studies, whereas DPI is more accurate when the refractive index and thickness of the adsorbed layer is of more interest.     

Lipase surface diffusion studied by fluorescence recovery after photobleaching

We have analyzed surface diffusion properties of a variant of Thermomyces lanuginosa lipase (TLL) on hydrophilic silica and silica methylated with dichlorodimethylsilane (DDS) or octadecyltrichlorosilane (OTS). For this study a novel method for analysis of diffusion on solid surfaces was developed. The method is based on fluorescence recovery after photobleaching using confocal microscopy. When a rectangular area of the sample was photobleached, fluorescence recovery could be analyzed as one-dimensional diffusion, resulting in simplified mathematical expressions for fitting the data. The method was initially tested by measuring bovine serum albumin diffusion on glass, which led to a diffusion coefficient in good correspondence to earlier reports. For the analysis of TLL diffusion, ellipsometry data of TLL adsorption were used to calibrate fluorescence intensity to surface density of lipase, enabling measurements of the diffusion coefficient at different surface densities. The average diffusion coefficient was calculated in two time intervals after adsorption. Mobile fraction and diffusion coefficient were lowest on the OTS surface, when extrapolated to infinite surface dilution. Moreover, the diffusion rate decreased with time on the hydrophobic surfaces. Our observations can be explained by the surface dependence on the distribution of orientations and conformations of adsorbed TLL, where the transition from the closed to the catalytically active open and more hydrophobic structure is important.