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• Explain the separation mechanism of ultrasound-assisted chromatography (UAC). • Comparatively analyzed ultrasound effects on C18 PEEK and SS columns. • Distinguished the differences between acoustic and temperature effects in UAC. • Ultrasound can be utilized as an external factor in chromatography. Based on the previous ultrasound-assisted chromatography (UAC) studies on plastic and stainless steel (SS) columns, this study explores the UAC mechanism by comparatively analyzing ultrasound effects on plastic and SS columns with C18 stationary phase when separating a mixture of polycyclic aromatic hydrocarbons (PAHs) under various ultrasound intensities. The results showed a substantial difference in H values between the PEEK and SS columns under the influence of ultrasound agitation. Specifically, for the pyrene peak, as the ultrasonic intensity increased from 0% to 100% of 900 W, the H values of the SS column slightly rose from 8.82 μm to 9.86 μm. Conversely, the corresponding values for the PEEK column exhibited a significant 12-fold increase from 11.5 μm to 134 μm. The findings demonstrated poor penetration of ultrasound energy through the SS column, and the temperature rise of the medium induced by the ultrasound was the primary contributing factor to PAH separation. However, ultrasound easily penetrated through the plastic column, resulting in acoustic cavitation within the C18 polyether ether ketone (PEEK) column. Cavitation induced heat generation and contributed to a decrease in retention time and the magnitude of peak broadening or distortion, depending on the specific ultrasonic energy. Based on the estimated change in inlet temperature of the PEEK column due to an acoustic effect, the comparison with temperature effects under non-sonic conditions consistently demonstrated a stronger acoustic effect in reducing the retention time, by 2–9%, depending on specific peaks and pairs. We revisited the previously described separation mechanism of ultrasound-assisted ion chromatography and conjoined with our findings to infer and establish a thorough explanation for the previously unexplained separation mechanism of chiral separation and size exclusion chromatography by UAC using SS columns. [ABSTRACT FROM AUTHOR]

• Capillary-channeled polymer fibers provide high throughput as 2D in 2DLC. • Fiber columns in 2D eliminates the need for UPLC hardware in the second dimension. • SEC x RP with fiber columns provides for potential implementation for ESI sampling. • PET and PP fibers exhibit different levels of selectivity, under RP conditions. • RP x RP with fiber columns provides orthogonality in chemical information. Polymeric materials are readily available, durable materials that have piqued the interest of many diverse fields, ranging from biomedical engineering to construction. The physiochemical properties of a polymer dictate the behavior and function, where large polydispersity among polymer properties can lead to problems; however, current polymer analysis methods often only report results for one particular property. Two-dimensional liquid chromatography (2DLC) applications have become increasingly popular due to the ability to implement two chromatographic modalities in one platform, meaning the ability to simultaneously address multiple physiochemical aspects of a polymer sample, such as functional group content and molar mass. The work presented employs size exclusion chromatography (SEC) and reversed-phase (RP) chromatography, through two coupling strategies: SEC x RP and RP x RP separations of the water-soluble polymers poly(methacrylic acid) (PMA) and polystyrene sulfonic acid (PSSA). Capillary-channeled polymer (C-CP) fiber (polyester and polypropylene) stationary phases were used for the RP separations. Particularly attractive is the fact that they are easily implemented as the second dimension in 2DLC workflows due to their low backpressure (<1000 psi at ∼70 mm sec−1) and fast separation times. In-line multi-angle light scattering (MALS) was also implemented for molecular weight determinations of the polymer samples, with the molecular weight of PMA ranging from 5 × 104 to 2 × 105 g mol−1, while PSSA ranges from 105 to 108 g mol−1. While the orthogonal pairing of SEC x RP addresses polymer sizing and chemistry, this approach is limited by long separation times (80 min), the need for high solute concentrations (PMA = 1.79 mg mL−1 and PSSA = 0.175 mg mL−1 to yield comparable absorbance responses) due to on-column dilution and subsequently limited resolution in the RP separation space. With RP x RP couplings, separation times were significantly reduced (40 min), with lower sample concentrations (0.595 mg mL−1 of PMA and 0.05 mg mL−1 of PSSA) required. The combined RP strategy provided better overall distinction in the chemical distribution of the polymers, yielding 7 distict species versus 3 for the SEC x RP coupling. [ABSTRACT FROM AUTHOR]

Plant polysaccharides have numerous medicinal functions. Due to the differences in their origins, regions of production, and cultivation conditions, the quality and the functions of polysaccharides can vary significantly. They are macromolecules with large molecular weight (MW) and complex structure, and pose great challenge for the analytical technology used. Taking Dendrobium officinale (DO) from various origins and locations as model samples. In this investigation, mechanochemical extraction method was used to successfully extract polysaccharides from DO using water as solvent, the process is simple, fast (40 s) and with high yield. The MWs of the intact saccharides from calibration curve and light scattering measurement were determined and compared after separation with size exclusion chromatography (SEC). The large polysaccharide was acid hydrolyzed to oligosaccharides and the products were efficiently separated and identified using liquid chromatography coupled to a high resolution tandem mass spectrometry (LC–MS 2 ). Obvious differences were observed among LC–MS 2 chromatograms of digested products, and the chemical structures for the products were proposed based on accurate mass values. More importantly, isomeric digested carbohydrate compounds were explored and characterized. All the chromatographic and mass spectrometric results in this study provided a multi-dimensional characterization, fingerprint analysis, and molecular structure level assessment of plant polysaccharides. [ABSTRACT FROM AUTHOR]

• Simulated moving bed chromatography can be performed at a microliter scale. • Custom-made 3D printed valve reduces overall dead volume to 358 µL. • Pressure-driven closed-loop flow control is a suitable flow source. • Desalting level of up to 99% for continuous protein desalting • Feed flow rate as low as 15 µL/min. In the 1960s, chromatography processes were revolutionized by the invention of simulated moving bed chromatography. This method not only enhances the separation performance and resin utilization in comparison to batch-chromatography, it has also a much lower buffer consumption. While simulated moving bed chromatography nowadays is applied for a wide range of industrial applications, it was never transferred to the micro-scale (in regards to column and system volume). In our opinion a micro simulated moving bed chromatography system (µSMB) would be a useful tool for many applications, ranging from early process development and long term studies to downstream processing of speciality products. We implemented such a µSMB with a 3D printed central rotary valve and a microfluidic flow controller as flow source. We tested the system with a four zone open loop setup for the separation of bovine serum albumin and ammonium sulfate with size exclusion chromatography. We used four process points and could achieve desalting levels of BSA ranging from 94% to 99%, with yields ranging form 65% to 88%. Thus, we were able to achieve comparable results to common lab scale processes. With a total dead volume of 358 µL, including all sensors, connections and the valve, this is, to the best of our knowledge, the smallest SMB system that was ever built and we were able to perform experiments with feed flow rates reaching as low as 15 µL/min. [ABSTRACT FROM AUTHOR]

A comparison is made using size-exclusion chromatography (SEC) of synthetic polymers between fully porous particles (FPPs) and superficially porous particles (SPPs) with similar particle diameters, pore sizes and equal flow rates. Polystyrene molecular weight standards with a mobile phase of tetrahydrofuran are utilized for all measurements conducted with standard HPLC equipment. Although it is traditionally thought that larger pore volume is thermodynamically advantageous in SEC for better separations, SPPs have kinetic advantages and these will be shown to compensate for the loss in pore volume compared to FPPs. The comparison metrics include the elution range (smaller with SPPs), the plate count (larger for SPPs), the rate production of theoretical plates (larger for SPPs) and the specific resolution (larger with FPPs). Advantages to using SPPs for SEC are discussed such that similar separations can be conducted faster using SPPs. SEC using SPPs offers similar peak capacities to that using FPPs but with faster operation. This also suggests that SEC conducted in the second dimension of a two-dimensional liquid chromatograph may benefit with reduced run time and with equivalently reduced peak width making SPPs advantageous for sampling the first dimension by the second dimension separator. Additional advantages are discussed for biomolecules along with a discussion of optimization criteria for size-based separations. [ABSTRACT FROM AUTHOR]

Two graft copolymer samples of identical average composition were synthesized by grafting living polystyrene anions onto a broadly distributed PMMA backbone. Size exclusion chromatography (SEC) with only RI-detection, SEC with viscometry and light scattering detection, SEC with UV and RI dual detection, gradient chromatography and 2-dimensional chromatography were applied to compare the information that can be obtained by the different techniques. While only limited information was retrieved by conventional SEC or SEC with molar mass sensitive detection, SEC with UV and RI revealed different chemical heterogeneity of the samples. Using gradient chromatography and 2-dimensional chromatography it was possible to identify non-grafted side chains and unreacted parent PMMA besides the actual graft copolymer molecules. While in one sample a heavily grafted product was formed besides non-grafted PMMA, the second sample did not contain ungrafted PMMA but a graft product of lower grafting density. The different product distributions were explained by the different synthetic procedures. [ABSTRACT FROM AUTHOR]

• SEC packing materials with three different bondings were prepared. • The three materials were characterized and compared. • Non-specific interactions were systematically studied. • The new HO-PEO phase was clearly the most favourable one. • A fast platform method (3 min analysis time) has been developed for protein SEC. Size exclusion chromatography has become an essential tool for the protein therapeutics industry. Conceptually, it is a simple form of chromatography that is driven by entropy and sieving effects. An ideal size exclusion column would exhibit no adsorptive interactions between its internal surfaces and the solutes being analysed, but that is not easily achieved. To this end, we have studied the utility of three unique packing materials in pursuit of additional column chemistries that might be less prone to interacting with proteins. These packing materials were each prepared from bridged ethylene hybrid organic/inorganic particles but uniquely derivatized into either hydroxy terminated PEO bonded, methoxy terminated PEO bonded, or diol bonded packing materials. All three materials were packed into column hardware modified with hydrophilic hybrid surface technology (h-HST) so that packing material effects could be more clearly observed without any influence from the secondary interactions that can originate from metal hardware. Non-specific interactions were compared for various challenging protein samples in the presence of ammonium acetate (volatile) and phosphate buffered saline (non-volatile) buffers. It was reconfirmed that the h-HST column hardware mitigates a majority of non-desired secondary interactions. However, during studies on hydrophobic interactions, the new hydroxy terminated PEO packing material showed clear benefit to obtaining higher apparent recoveries to better ensure accurate aggregate quantitation. Further experiments were explored to show that a hydroxy terminated PEO column could be effectively paired with a mobile phase comprised of standard strength phosphate buffered saline to make a fast platform method capable of baseline resolving monoclonal antibody monomer and aggregate peaks within a 3 min analysis time. [ABSTRACT FROM AUTHOR]

Surface plasmon resonance (SPR) is an optical technique that measures biomolecular interactions. Stand-alone SPR cannot distinguish different binding components present in one sample. Moreover, sample matrix components may show non-specific binding to the sensor surface, leading to detection interferences. This study describes the development of coupled size-exclusion chromatography (SEC) SPR sensing for the separation of sample components prior to their on-line bio-interaction analysis. A heterogeneous polyclonal human serum albumin antibody (anti-HSA) sample, which was characterized by proteomics analysis, was used as test sample. The proposed SEC-SPR coupling was optimized by studying system parameters, such as injection volume, flow rate and sample concentration, using immobilized HSA on the sensor chip. Automated switch valves were used for on-line regeneration of the SPR sensor chip in between injections and for potential chromatographic heart cutting experiments, allowing SPR detection of individual components. The performance of the SEC-SPR system was evaluated by the analysis of papain-digested anti-HSA sampled at different incubation time points. The new on-line SEC-SPR methodology allows specific label-free analysis of real-time interactions of eluting antibody sample constituents towards their antigenic target. [ABSTRACT FROM AUTHOR]

Band broadening in size-exclusion chromatography (SEC) is always present to some extent. Broadening effects on averages such as the weight- and number average molecular weights ( M W ¯ and M n ¯ respectively) are minimal with modern SEC systems. However, broadening distorts the shape of the true molecular weight distribution (MWD), which causes problems if one wants to compare the detailed form of the MWD to a model. An addition to current methods for overcoming this problem is presented. One starts with a sufficiently wide range of samples whose exact values of M n ¯ and M W ¯ have been measured by non-SEC methods (e.g. by fluorimetry and light scattering, respectively, of the sample without size separation). A true (unbroadened) molecular weight distribution for a sample can be obtained by deconvolution (here using a maximum-entropy algorithm) by fitting SEC data for these samples to these exact M n ¯ and M W ¯ values to find the values of the parameters in a sufficiently flexible assumed broadening function. This was modelled using simulated band broadening and subsequent deconvolution, with the broadening parameters least-squares fitted to the “exact” sets of values of M n ¯ and M W ¯ . The results show that if these M n ¯ and M W ¯ values are for a series of broad (not narrow) standards covering a sufficient range of molecular weight, then after deconvolution, a good representation of the original molecular weight distribution used in the simulation is obtained. The method should prove useful for water-soluble polymers, for which it is often difficult to obtain narrow standards of a wide range of molecular weight, as required in a number of well-established methods for correcting for band broadening. [ABSTRACT FROM AUTHOR]

Average molecular weights and polydispersity indexes are some of the most important parameters considered in the polymer characterization. Usually, gel permeation chromatography (GPC) and multi angle light scattering (MALS) are used for this determination, but GPC values are overestimated due to the dispersion introduced by the column separation. Several procedures were proposed to correct such effect usually involving more complex calibration processes. In this work, a new method of calculation has been considered including diffusion effects. An equation for the concentration profile due to diffusion effects along the GPC column was considered to be a Fickian function and polystyrene narrow standards were used to determine effective diffusion coefficients. The molecular weight distribution function of mono and poly disperse polymers was interpreted as a sum of several Fickian functions representing a sample formed by only few kind of polymer chains with specific molecular weight and diffusion coefficient. Proposed model accurately fit the concentration profile along the whole elution time range as checked by the computed standard deviation. Molecular weights obtained by this new method are similar to those obtained by MALS or traditional GPC while polydispersity index values are intermediate between those obtained by the traditional GPC combined to Universal Calibration method and the MALS method. Values for Pearson and Lin coefficients shows improvement in the correlation of polydispersity index values determined by GPC and MALS methods when diffusion coefficients and new methods are used. [ABSTRACT FROM AUTHOR]

• 2D SEC-CEX-MS allows multiplex analysis of mAb aggregates and charge variants in a single method. • 2D SEC-CEX-MS streamlines product development in industry. • 2D SEC-CEX-MS is applied to understand mAb forced degradation pathways. Monoclonal antibodies (mAbs) are extremely complex due to the presence of structural modifications resulting from enzymatic and chemical reactions such as glycosylation, glycation, deamidation, isomerisation, oxidation, aggregation and fragmentation. Size and charge variants analysis are carried out from the early stages of drug development throughout product lifetime to investigate product degradation pathways and optimise process conditions. However, conventional analytical workstreams for size and charge variant characterization are both time and sample demanding, requiring the application of multiple analytical methods. This study presents the development of a novel 2D-LC/MS approach combining both aggregate and charge variant profiling of a mAb candidate in a single method. Aggregate quantification was performed in the first dimension (1D) by size exclusion chromatography SEC, followed by online fraction transfer of the monomer peak to the second dimension (2D) by a heart-cutting for charge variant analysis by cation exchange chromatography (CEX). Aiming to maximise the information obtained from minimal sample and time required for analysis, a salt-based separation with UV detection was developed for supporting the processing of a large number of samples to facilitate high-throughput process development (HTPD). In addition, a mass spectrometry (MS) compatible SEC-CEX separation was developed enabling online charge variant peak identification. This study presented the ability to multiplex mAb size and charge variants analysis by coupling SEC with CEX in a 2D-LC set-up. To date, this is the first 2D SEC-CEX-UV(-MS) application for intact mAb analysis. [ABSTRACT FROM AUTHOR]

Highlights: [•] Application of the capability indices to the SEC/LS/RI method using BAS as model. [•] The X- and moving range chart confirmed that the method is in-control and stable. [•] The method meets the capability requirement and run under conditions preset by the analyst. [•] Method was used in stability study of bevacizumab pharmaceutical preparations. [•] Method does not comply with the specification limits and a precision control is required. [ABSTRACT FROM AUTHOR]

Highlights: [•] Size exclusion separation of polysaccharide with reverse phase column. [•] Use the entire separation window before and after void volume to tackle complex separation problem. [•] Combined use of size and hydrophobicity based separation in RPLC. [ABSTRACT FROM AUTHOR]

Highlights: [•] A chromatographic refolding method which saves up to 99% refolding buffer. [•] The methods shifts refolding equilibrium. [•] The method can be connected to a purification step. [Copyright &y& Elsevier]

Highlights: [•] A method was optimized for extracting PAHs from silicone rubber passive samplers. [•] Pressurized liquid extraction (PLE) with gel permeation chromatography was used. [•] Selective PLE with Florisil removed silicone oligomers but not sufficiently. [•] Recoveries of spiked PAHs ranged from 81% to 102%, with a mean of 93%. [•] The method saves considerable solvent and time compared to conventional methods. [ABSTRACT FROM AUTHOR]

Highlights: [•] Light and X-ray scattering methods combined with SEC are of complementarily useful. [•] A shark cartilage proteoglycan was first characterized using these methods. [•] The molecular weight (distribution) was estimated by SEC-LALLS. [•] The chain conformation (persistence length) was estimated by SEC-SAXS. [Copyright &y& Elsevier]

This study investigated the experimental conditions needed to obtain molecular weight distribution (MWD) of ultrahigh MW poly(ethylene oxide) (PEO) using size exclusion chromatography (SEC) hyphenated with a multiangle light scattering photometer (MALS) and a differential refractive index detector (RI). Ultrahigh MW components yielded non-linear angular dependency in scattered light intensities. The first-order linear fitting using Zimm formalism resulted in significant differences in MW depending on if the signals from detector 4 to 16 were used in the fitting or only five low-angles from 4 to 8 were used. In contrast, no significant differences in MW were obtained for lower MW PEO samples (equal or less than 1000 KDa) between the two fitting approaches. It was thus proposed to use only the five low-angles to derive MW for a sample with broad polydispersity including both ultrahigh and low MW components. The SEC separation was done using one column designed for ultrahigh MW polymer separation connected with another mixed-bed column. The ultrahigh MW column allowed separation and characterization of polymeric components in the MW range between 10 and 50 million Dalton (MDa) and the size range between 300 and 600 nm in radius of gyration (R g). Online calibration curves were obtained from the linear fittings of MW as a function of elution volume. MW polydispersity was derived from the online calibration curve showing that the ultrahigh MW PEO had higher polydispersity than the lower MW samples. The double logarithmic plot of radius of gyration versus MW indicated that both ultrahigh MW and low MW PEO would adopt expanded coil conformations in the aqueous solution. [ABSTRACT FROM AUTHOR]

Abstract: The SEC separation of a randomly branched polymer, in particular local dispersity due to branching, are theoretically examined. A model of the SEC separation of randomly branched polymer with tetrafunctional branch points enabling the estimation of local dispersity was developed. Measurable quantities (branching indices) were compared with real data. Local dispersity due to branching is demonstrated to depend on elution volume and degree of branching and in the area of the beginning of the elution curve it can reach non-negligible values. [Copyright &y& Elsevier]

Abstract: Size exclusion chromatography (SEC) is often applied for characterization of protein–polyethylene glycol (PEG) conjugates regarding the number of attached PEG chains (PEGamers). SEC analysis is advantageous as it is precise, robust, and straightforward to establish. However, most SEC based assays have a maximal throughput of a few samples per hour. We present a strategy to increase analytical throughput based on combining a short column with a fast flow rate, and finally multivariate calibration in order to compensate for the resolution lost in the trade off for speed. Different multivariate approaches were compared and multilinear regression was shown to result in the most precise calibrations. Further, a dynamic calibration approach was developed in order to account for changes in column performance over time. In this way, it was possible to establish a highly precise assay for protein PEGamer quantification with a throughput of 30 samples per hour. [Copyright &y& Elsevier]

Abstract: Polymers, especially copolymers, are highly complex samples and, therefore, require various setups for their thorough characterization. In this work, one- and two-dimensional chromatographic approaches were applied to characterize two homopolymers and two dissimilar copolymers prepared by rhodium-mediated carbene polymerization using ethyl and benzyl diazoacetate as the monomers: poly-(EA-ran-BnA) and poly((EA)b-(BnA-ran-EA)b). Different strategies of synthesis suggested that poly-(EA-ran-BnA) was an approximately 1:1 random copolymer and that poly((EA)b-(BnA-ran-EA)b) was a block-type copolymer of which the (BnA-ran-EA)b block is dominated by BnA. The hydrodynamic volume of the investigated polymers turned out to be comparable, i.e. size exclusion chromatography (SEC) did not separate them. Temperature-gradient interaction chromatography was not feasible and one-dimensional (solvent-)gradient-elution liquid chromatography (GELC) suffered from coelution problems. Pyrolysis-gas-chromatography (Py-GC–MS) experiments allowed determining the monomer ratio. SEC with UV and refractive-index detection indicated the presence of molecular-weight-dependent chemical heterogeneity for the poly((EA)b-(BnA-ran-EA)b) copolymer. This finding was confirmed with off-line SEC//Py-GC–MS. Only a comprehensive two-dimensional GELC×SEC setup enabled the separation of high-molecular-weight material of the less-retained homopolymer from low-molecular-weight copolymeric material. In this way it was possible to detect some high-molecular-weight homopolymeric material in one of the copolymers. [Copyright &y& Elsevier]

Abstract: “Obstruction factor” is a generic rubric under which are usually gathered the interparticle, intraparticle, stationary phase, and total obstruction factors, γ e , γ p , γ s , and γ t , respectively. These, in turn, affect longitudinal diffusion and stationary, mobile phase, and stagnant mobile phase mass transfer. We conclude here our investigation into the various obstruction factors operative in size-exclusion chromatography (SEC). Stop-flow experiments were employed to determine either the interparticle (for analytes with K SEC =0) or the total (for analytes with K SEC >0) obstruction factor, and these results were combined with those from variable-flow-rate experiments which provided the intraparticle obstruction factor. Because of minimal enthalpic interactions between the analytes and stationary phase, in SEC γ s ≈0, which allows for isolation of the other obstruction factors. A relationship between γ t , γ e , and γ p was proposed for SEC, based on previous independent work and dependent upon the various column porosities. This relationship was extended to hydrodynamic chromatography, a technique in which, ideally, both γ s and γ p are equal to zero. [Copyright &y& Elsevier]

Abstract: We report the first of a series of studies on the obstruction factor γ in size-exclusion chromatography (SEC). Here, using narrow dispersity polymer standards we examine how the intraparticle obstruction factor γ p depends individually on a number of analyte properties, column characteristics, and user-defined parameters. Far from being constant, γ p is seen to vary with analyte molar mass and solvent, as well as with the pore size and particle size of the column packing material, sometimes in seemingly counterintuitive manner. Over the limited temperature range accessible to our equipment, however, no statistically significant change in γ p with temperature was discovered. The results presented should be applicable to forms of packed column chromatography other than SEC. The latter technique, however, presents a convenient test bed for quantitative determination of the obstruction factor, due to minimized sorptive mass transfer and longitudinal diffusion contributions to band broadening in most forms of SEC. [ABSTRACT FROM AUTHOR]

Abstract: Gradient-elution liquid chromatography (GELC) is a powerful tool for the characterization of synthetic polymers. However, gradient-elution chromatograms often suffer from breakthrough phenomena. Breakthrough can be averted by using a sample solvent as weak as the mobile phase. However, this approach is only applicable to polymers for which a sufficiently strong solvent exists which is at the same time a weak eluent. Finding such a solvent for a given polymer can be laborious or may even be impossible. Besides, when working with comprehensive two-dimensional LC the effluent of the first dimension is the injection solvent of the second dimension. In this case, it is not possible to avoid breakthrough by adjusting the eluent strength of the second-dimension injection solvent. Therefore, another strategy to avert breakthrough has to be implemented. In this work, we successfully avoided breakthrough in GELC by mixing the mobile phase not before, but after the autosampler. This was demonstrated measuring a blend of poly(methyl methacrylate) standards with different molecular-weights as model mixture with comprehensive two-dimensional GELC×size-exclusion chromatography. The strategy is thought to be applicable to all substances with a sufficiently strong dependence of retention on mobile-phase composition. This typically applies to large molecules (synthetic and natural polymers) and allows efficient refocusing. Unretained and barely retained substances are not refocused and therefore suffer in the proposed setup from peak broadening. [ABSTRACT FROM AUTHOR]

Abstract: The determination of target analytes such as nonyl- and octylphenols and 17β-estradiol in fish homogenate require of solid–liquid extraction step. In this work microwave-assisted extraction (MAE) and focused-ultrasound liquid extraction (FUSLE) were studied as two different alternatives for extraction of the target compounds in zebrafish (Danio rerio) homogenate. In this work solid phase extraction (SPE) using 5-g and 10-g Florisil cartridges and gel permeation chromatography (GPC) were studied for the clean-up of the MAE and FUSLE extracts due to the non-selective extraction step. Although good recoveries were obtained both for SPE (106% and 126% range) and GPC (79% and 100% range) clean-up procedures, cleaner chromatograms were obtained after SPE and finally 5-g Florisil cartridges were tested since no improvement was observed when 10-g Florisil cartridges were used. Under optimized clean-up conditions, MAE and FUSLE provided comparable results for 4nOP and NP, while more accurate results were obtained for 4tOP and E2 after FUSLE. Finally, the method was applied to the determination of alkylphenols and 17β-estradiol in zebrafish homogenate that had been exposed to known concentrations of the target analytes. In the case of alkylphenols two different isomers of nonyl- and octylphenol (4-(3′,6′-dimethyl-3′-hepthyl)phenol, 363-NP, and 4-(3′-methyl-3′-hepthyl)phenol, 33-OP) were studied. [ABSTRACT FROM AUTHOR]

Abstract: Interparticle void volumes and porosities of packed capillaries have been determined using intraparticle Donnan exclusion of a small, unretained, co-ionic tracer (nitrate ions). The operational domain of this approach has been characterized for bare silica, reversed-phase, and strong cation-exchange materials (with different particle sizes and intraparticle pore sizes) in dependence of the mobile phase ionic strength. Interparticle porosities agree well with those analyzed by inverse size-exclusion chromatography (ISEC). Limitations to the use of Donnan exclusion (electrostatic exclusion) and ISEC (mechanical exclusion) arise as either type of exclusion becomes noticeable also in the cusp regions between the particles, or as the intraparticle pores are so large that complete electrostatic and size-exclusion are difficult to realize. Our data confirm that intraparticle Donnan exclusion presents a most simple, fast, and reliable approach for the analysis of packing densities. [Copyright &y& Elsevier]

Abstract: Columns switch recycling size exclusion chromatography (csrSEC) was proposed to achieve high resolution protein separation with good biocompatibility. Proteins were firstly separated by two serially coupled SEC columns, and fractions were in sequence switched back to the first column by two-position valves for further separation in terms of close-loop recycling until satisfactory resolution was achieved. Compared to SEC, the separation window was broadened by increasing column length via cycling without further increase on back pressure. Compared to recycling SEC (rSEC), the overtaking of later eluted components by early eluted ones after several cycles could be avoided for complex sample analysis, by parking fractions in the second SEC column before transferred in turn back to the first one for cycling ordinally. In our experiments, the baseline separation of five proteins with molecular weight ranging from 10kDa to 80kDa was achieved by csrSEC. Furthermore, a multidimensional csrSEC–RPLC platform was constructed, and peak capacity up to 3600 was obtained for protein separation. All these results demonstrated that csrSEC is a promising protein separation mode with good biocompatibility, broadened separation window and improved resolution. [Copyright &y& Elsevier]

Abstract: Although the medicinal plant and food Nigella glandulifera Freyn has been researched for decades, isobenzofuranones have never been isolated before. Two isobenzofuranone derivatives and two saponins were successfully separated and purified from seeds of N. glandulifera Freyn by high-speed counter-current chromatography (HSCCC) with the optimized two-phase solvent system, n-hexane-ethyl acetate–methanol–water (7:3:5:5, v/v). Salfredin B11 (22.1mg, HPLC purity 95.3%), 5, 7-dihydroxy-6-(3-methybut-2-enyl) isobenzofuran-1(3H)-one (18.9mg, HPLC purity 97.3%) and crude sample 2 (555mg) were separated from 600mg of ethyl acetate extract of N. glandulifera Freyn. Following a cleaning-up step by chromatography on Sephadex LH-20, hederagenin (12mg) and 3-O-[β-d-xylopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl]-hederagenin (45mg) were separated from sample 2. All of the fractions before peak II were collected and subjected to a Sephadex LH-20 column and eluted by methanol, two of triterpene saponins (12mg of hederagenin and 45mg of 3-O-[β-d-xylopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl]-hederagenin) were isolated. The structures of peak fractions were identified by IR, electron ionization MS, 1H NMR and 13C NMR. 5, 7-Dihydroxy-6-(3-methybut-2-enyl) isobenzofuran-1(3H)-one was isolated for the first time from higher plant and salfredin B11 was isolated for the first time in this plant. [Copyright &y& Elsevier]

Abstract: A method combining gel filtration chromatography (GFC), protease digestion, and ion pair chromatography with inductively coupled plasma mass spectrometry detection was developed for the determination of arsenic species bound to proteins. The method was first established by examining the interactions of two model proteins, metallothionein (MT) and hemoglobin, with three reactive trivalent arsenic species. It was then successfully applied to the speciation of arsenic in red blood cells of rats. Inorganic arsenite (iAsIII), monomethylarsonous acid (MMAIII), and dimethylarsinous acid (DMAIII) were efficiently released from the proteins by protease digestion at pH 8.0, with the recovery ranging from 93% to 106%. There was no oxidation of iAsIII or MMAIII during the protease digestion process. Up to 61% DMAIII (the least stable arsenic species) was unchanged, and the rest was oxidized to the pentavalent dimethylarsinic acid (DMAV). The arsenic species in the red blood cells of control rats was present as DMAIII complex with hemoglobin. The method enabling the determination of the specific arsenic species that bind to cellular proteins is potentially useful for studying arsenic distribution, metabolism, and toxicity. [Copyright &y& Elsevier]

Abstract: We have evaluated a process incorporating aqueous two-phase extraction, hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for the purification of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cell supernatant. These unit operations were chosen not only for allowing the removal of target impurities but also for facilitating the integration of different process units without the need for any conditioning step. Extraction in aqueous two-phase systems (ATPSs), composed of polyethylene glycol (PEG) and sodium citrate, allowed the concentration of the antibodies in the citrate-rich phase and the removal of the most hydrophobic compounds in the PEG-rich phase. An ATPS composed of 10% (w/w) PEG 3350 and 12% (w/w) citrate, at pH 6, allowed the recovery of IgG with a 97% yield, 41% HPLC purity and 72% protein purity. This bottom phase was then directly loaded on a phenyl-Sepharose HIC column. This intermediate purification step allowed the capture of the antibodies using a citrate mobile phase with 99% of the antibody recovered in the elution fractions, with 86% HPLC purity and 91% protein purity. Finally, SEC allowed the final polishing by removing IgG aggregates. HIC-eluted fractions were directly injected in a Superose 6 size-exclusion column affording a 100% pure IgG solution with 90% yield. [Copyright &y& Elsevier]

Abstract: A method based on ultra-performance liquid chromatography–electrospray ionisation-tandem mass spectrometry (UPLC–MS/MS) for the determination of six thyreostatic drugs in thyroid tissue has been optimised and validated in accordance with Decision 2002/657/EC. Sample extraction was evaluated in methanol and in ethyl acetate, the latter which gave better results. Two clean-up strategies were compared: one based on silica cartridges (SPE), and the other, on gel permeation chromatography (GPC). Recoveries ranged from 40% to 79% for the SPE approach and from 80% to 109% for GPC. Quantification was performed with blank tissue samples spiked with the analytes in the range 50–500μgkg−1. 5,6-Dimethyl-2-thiouracil and 2-mercaptobenzimidazole-d 4 were used as internal standards. Decision limit (CCα) and detection capability (CCβ) ranged from 1 to 15μgkg−1 and from 6 to 25μgkg−1, respectively. The accuracy of the method was calculated as percent error, which was less than 10%. The relative standard deviation in reproducibility conditions ranged between 2% and 14%. [Copyright &y& Elsevier]

Abstract: Branching has a strong influence on the processability and properties of polymers. However, the accurate characterization of branched polymers is genuinely difficult. Branched molecules of a certain molecular weight exhibit the same hydrodynamic volumes as linear molecules of substantially lower weights. Therefore, separation by size-exclusion chromatography (SEC), will result in the co-elution of molecules with different molecular weights and branching characteristics. Chromatographic separation of the polymer molecules in sub-μm channels, known as molecular-topology fractionation (MTF), may provide a better separation based on topological differences among sample molecules. MTF elution volumes depend on both the topology and molar mass. Therefore co-elution of branched molecules with linear molecules of lower molar mass may also occur in this separation. Because SEC and MTF exhibit significantly different selectivity, the best and clearest separations can be achieved by combining the two techniques in a comprehensive two-dimensional (MTF×SEC) separation system. In this work such a system has been used to demonstrate branching-selective separations of star branched polymers and of randomly long-chain-branched polymers. Star-shaped polymers were separated from linear polymers above a column-dependent molecular weight or size. [Copyright &y& Elsevier]

Abstract: One- and two-dimensional separation methods have been developed for the analysis of lignosulphonates and sulphonated kraft lignins. The evaluated sulphonated lignins are all used as dispersants in agrochemical formulations, where some give rise to physical instabilities of formulations. It is of interest to identify the properties of the sulphonated lignins that determine the formulation characteristics. Tetrapentylammonium bromide has been used as an ion-pair reagent in a gradient-elution reversed-phase liquid-chromatographic (IP-RPLC) method, as well as in aqueous size-exclusion chromatography (SEC). Clear differences in the size distribution were observed between different batches of sulphonated lignins. The RPLC and SEC methods were combined in a comprehensive two-dimensional liquid chromatography system. The retention times in the two dimensions were highly correlated. Therefore, the full potential of comprehensive two-dimensional liquid chromatography was not yet realized. However, the results did reveal that retention in IP-RPLC was not determined by the degree of sulphonation of similar-size molecules. Rather, molecules were separated according to size and the degree of sulphonation appears to be approximately constant. The information obtained in this study represents a significant step towards meaningful correlations between the requirements of surfactants within an agrochemical formulation and structural parameters, such as the size and the degree of sulphonation of lignin oligomers. [Copyright &y& Elsevier]

Abstract: Accelerated membrane-assisted clean-up (AMAC) is a new technique to purify lipid-rich extracts of biota samples. It combines a separation of organic analytes and lipid-like matrix components using a semi-permeable membrane with the technical options provided by a Dionex ASE device which was developed for pressurized liquid extraction (PLE; Dionex trade name ASE for accelerated solvent extraction). The ASE device automates the procedure. It offers the possibility to increase the permeation of the analytes through the membrane via application of higher temperatures and automatic solvent exchange to ensure steep gradients across the membrane. The optimal operating parameters of AMAC were determined in this study. Satisfactory recoveries of over 70% for most target compounds, as well as excellent removal of lipid-like matrix components were achieved using 80μm thick low-density polyethylene (LD-PE) membranes, at a temperature and pressure of 40°C and 3.45MPa, respectively, over 16 static cycles lasting 10min each. The recoveries obtained were similar to those achieved using gel permeation chromatography (GPC), however AMAC showed a higher matrix removal capacity. AMAC removed up to 4.5g of lipids in a single run, which was more than 98% of the original matrix content. Additionally, a 13-fold lower solvent consumption, compared with GPC, was achieved. AMAC separates analytes and matrix compounds mainly by size, making this method a beneficial tool for non-target or effect analysis. AMAC is especially applicable for processing large amounts of sample as a result of the high matrix removal capacity. [Copyright &y& Elsevier]

Abstract: Native and n-alkyl-bonded (n-octadecyl) monolithic silica rods with mesopores in the range between 10 and 25nm and macropores in the range between 1.8 and 6.0μm were examined by mercury intrusion/extrusion, inverse size exclusion chromatography (ISEC) and nitrogen sorption. Our results reveal very good agreement for the mesopore size distribution obtained from nitrogen adsorption (in combination with an advanced NLDFT analysis) and ISEC. Our studies highlight the importance of mercury porosimetry for the assessment of the macropore size distribution and show that mercury porosimetry is the only method which allows obtaining a combined and comprehensive structural characterization of macroporous/mesoporous silica monoliths. Our data clearly confirm that mercury porosimetry hysteresis and entrapment have different origin, and indicate the intrinsic nature of mercury porosimetry hysteresis in these silica monoliths. Within this context some silica monoliths show the remarkable result of no entrapment of mercury after extrusion from the mesopore system (i.e. for the first intrusion/extrusion cycle). The results of a systematic study of the mercury intrusion/extrusion behavior into native silica monoliths and monoliths with bonded n-alkyl groups reveals that the macro (through) pore structure, which controls the mass transfer to and from the mesopores, here mainly controls the entrapment behavior. Our data suggest that mercury intrusion/extrusion porosimetry does not only allow to obtain a comprehensive pore structure analysis, but can also serve as a tool to estimate the mass transport properties of silica monoliths to be employed in liquid-phase separation processes. [Copyright &y& Elsevier]

Abstract: A new method for N,N′-ethylenebisstearamide (EBS) analysis was developed and validated in normal phase-HP liquid chromatography (NP-HPLC) with diol column at 50°C with 100% CHCl3 at 1mLmin−1 and evaporative light scattering detection with elution time at 3.0min. EBS solubility was the best at 0.80gL−1 in CHCl3/methanol 90:10. The molecular structure of commercial samples of EBS was determined by GC–MS which ascertained that the main structure is C18/C18 at approximately 45%. The remaining part was constituted by molecules with different alkyl chain length. The HPLC quantification method was proved linear (r =0.9983), accurate (99.6%) and precise (1.95%). Limit of quantification (LOQ) and limit of detection (LOD) were equal to 2.0 and 0.8μgmL−1, respectively. The suitability of this method was assessed with a dissolution/precipitation extraction procedure of EBS from ethylene vinyl acetate (EVA) polymer which showed that other additives and polymer do not interfere with EBS analysis. The intra-day and day-to-day precisions of extraction method were equal to 9.1% and 9.9%, respectively. [Copyright &y& Elsevier]

Abstract: A novel organic carbon detector (OCD) for size exclusion chromatography (SEC), and its application to the characterisation of aquatic natural organic matter (NOM) in natural and treated potable water samples, is described. The instrument uses a conventional UV-persulfate oxidation technique to convert organic carbon to CO2. The novelty of the technique is detection of the evolved CO2 using a sensitive Fourier transform infrared (FTIR) spectroscopy ‘lightpipe’ detector originally designed for detection of analytes after gas chromatographic separation. With the exception of the lightpipe, the OCD system was constructed using simple, inexpensive, readily available components. The system was designed to minimise deadvolume, allowing for use of smaller sample sizes and smaller columns, substantially shortening analysis time, while maintaining chromatographic integrity through the OCD system. Downscaling resulted in some loss of separation but it was shown that this was caused by the lower separation efficiency of the smaller capacity column, rather than from sample dispersion within the OCD system. [Copyright &y& Elsevier]

Abstract: We report on the size-exclusion chromatography (SEC) operated at high column temperature to reduce the analysis time. The column temperature was raised beyond the normal boiling point of the eluent and a sufficient column backpressure was applied to prevent the mobile phase from boiling by inserting a narrow bore tubing between the separation column and the detector. The narrow bore tubing also functions to cool the effluent down to the room temperature before it reaches the detector. Therefore, normal SEC detectors can be used without any modification. It was confirmed that the SEC analysis time could be shortened significantly by the high-temperature operation without serious deterioration in the resolution. [Copyright &y& Elsevier]

Abstract: Two methods for extracting organochlorine (OCs) and organophosphorus (OPs) pesticides from animal liver have been developed. The determination was carried out by gas chromatography with electron impact ionization tandem mass spectrometry (GC–(EI-)MS/MS) using a triple quadrupole (QqQ) analyzer. First, a liquid–solid extraction performed with a high-speed homogenizer (Polytron) using ethyl acetate as solvent, and a subsequent clean-up by gel permeation chromatography (GPC) was applied, determining 34 pesticides. Secondly, a matrix solid phase dispersion (MSPD) extraction with octadecylsilyl (C18) sorbent combined with a Florisil clean-up and ethyl acetate elution was performed, analyzing 25 compounds. These methodologies have been tested and compared in the sample pre-treatment due to the fatty nature of the matrix. The GPC method was finally selected and validated, yielding recoveries in the range 70–115%, with precision values expressed as relative standard deviation (RSD) lower or equal to 20%, at the spiking levels of 25 and 50μgkg−1, and limits of quantification (LOQs) lower than the maximum residue levels (MRLs) set by the European Union in animal products, except for isofenphos. Linearity was also studied ranging between 5 and 300μgkg−1 for most of pesticides. This method was applied to the analysis of real liver samples of chicken, pork and lamb. [Copyright &y& Elsevier]

Abstract: Fulvic acids from an Australian floodplain river and billabong were isolated using DEAE and DAX-8 resins, and characterised with the use of size exclusion chromatography and solid-state CP-MAS 13C NMR spectroscopy. Differences between the two resin isolates were evident. Fulvic acids isolated using DEAE-cellulose had higher apparent M n and M w values, while the DAX-8 resin showed a slight preference for aliphatic components. Fulvic acids from the river and billabong had the same functional groups present, however, the river fulvic acids had higher apparent M n (number average molecular weight) and M w values (weight average molecular weight), and were more polydisperse than the billabong fulvic acid. There were no significant changes in the characteristics of the fulvic acid isolated from the river at four sampling times: summer, autumn, winter and spring. In contrast, fulvic acids isolated from a billabong displayed seasonal variation in molecular weights. This work emphasizes the importance in ecological studies of isolation procedure for the operationally defined fulvic acids. [Copyright &y& Elsevier]

Abstract: Water-soluble non-structural carbohydrates (NSC) in the needles of Norway spruce Picea abies [L.] Karst have been studied by using a combination of several separation techniques, having various detectors, with mass spectrometry. The intent was to find a suitable methodology that enables the characterization and determination of NSC, covering a wide range of molar masses, and being suitable to assess how NCS are influenced by both external conditions, e.g. different carbon dioxide (CO2) concentrations, light intensity, and by internal conditions such as the needle age. The techniques were liquid–liquid extraction, high performance liquid chromatography (HPLC), size exclusion chromatography (SEC), asymmetrical flow field-flow fractionation (AsFlFFF), electrospray ionization mass spectrometry (ESI-MS), and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). NSC were extracted by a methanol/chloroform/water mixture into the water-rich phase. Application of AsFlFFF and SEC, using refractive index (RI) and multi-angle light scattering (MALS) detectors to the water-rich extracts resulted in three or four main fractions covering molar masses from 103 to 106 g/mol. Individual fractions collected from SEC were directly subjected to both MALDI and ESI-MS analysis in order to identify NSC. MALDI mass spectra confirmed the presence of hexose oligomers in individual fractions while ESI-MS was used for evaluation of low mass NSC. HPLC–RI was used for quantification of NSC and predominant carbohydrates were found to be fructose, glucose, and sucrose. The changes in their content during seasonal course were studied in detail. HPLC coupled to ESI-MS enabled the identification of low concentration NSC like raffinose that occurred in the needles of autumn samplings. An influence of the increased CO2 concentration on sucrose and glucose accumulation was observed and it was found that the light intensity as well as the needle age has significant influence on the sucrose content. [Copyright &y& Elsevier]

Abstract: Liquid chromatography (LC) coupled on line with UV/visible diode array detector (DAD) and cold vapour generation atomic fluorescence spectrometry (CVGAFS) has been developed for the speciation, determination and characterization of phytochelatins (PCs). The method is based on a bidimensional approach, e.g. on the analysis of synthetic PC solutions (apo-PCs and Cd2+-complexed PCs) (i) by size exclusion chromatography coupled to UV diode array detector (SEC–DAD); (ii) by the derivatization of PC –SH groups in SEC fractions by p-hydroxymercurybenzoate (PHMB) and the indirect detection of PC-PHMB complexes by reversed phase liquid chromatography coupled to atomic fluorescence detector (RPLC–CVGAFS). MALDI-TOF/MS (matrix assisted laser desorption ionization time of flight mass spectrometry) analysis of underivatized synthetic PC samples was performed in order have a qualitative information of their composition. Quantitative analysis of synthetic PC solutions has been performed on the basis of peak area of PC-PHMB complexes of the mercury specific chromatogram and calibration curve of standard solution of glutathione (GSH) complexed to PHMB (GS-PHMB). The limit of quantitation (LOQ) in terms of GS-PHMB complex was 90nM (CV 5%) with an injection volume of 35μL, corresponding to 3.2pmol (0.97ng) of GSH. The method has been applied to analysis of extracts of cell cultures from Phaeodactylum tricornutum grown in Cd-containing nutrient solutions, analysed by SEC–DAD–CVGAFS and RPLC–DAD–CVGAFS. [Copyright &y& Elsevier]

Abstract: Statistical properties of the band-broadening function calculated from the equilibrium and kinetic theories of Giddings and Eyring are compared with real liquid-chromatography elution curves of low-molecular-weight substances and with light-scattering elution curves of a high-molecular-weight polystyrene standard injected at several concentrations. The curves obtained by liquid chromatography agree in a large span of elution times with the theoretical ones and can be described by one value of the plate-height. The size exclusion chromatography (SEC) elution curves seem to approach the predicted statistical properties with decreasing concentration of injected polymer but, in general, a reliable prediction of band-broadening (BB) functions in SEC of polymers, especially of non-symmetric BB functions near the exclusion limit, seems not possible at this point. The relation of the theory with the characteristic-function approach developed by Dondi is also discussed. [Copyright &y& Elsevier]

Abstract: A new method called promoter trapping was developed to purify promoter-protein complex using the c-jun promoter (−200+81) as a model, which was shown to have significant promoter activity. Polymerase chain reaction (PCR), lambda exonuclease digestion combined with (AC)5-Sepharose DNA affinity chromatography were used to produce c-jun promoter with a (GT)5 tail at each 3′ end. The intact promoter and different length pieces with one or two (GT)5 tails had almost the same capacity to bind with (AC)5-Sepharose. In solution, tailed c-jun promoter (60nM) and competitor poly dI:dC (30ng/μl) was incubated with crude HEK293 nuclear extract to form a large protein-promoter complex, and the complex was then trapped by (AC)5-Sepharose by centrifugation or on a column. Compared with a popular alternative method, called here the immobilized promoter method, the products of promoter trapping were purer. The preinitiation complex purified by promoter trapping had the expected components including RNA polymerase II, TATA-box binding protein (TBP), TFIIF subunit RAP74, and transcription factor SP1, and transcribed RNA in vitro. Thus, the promoter trapping approach provides a useful tool for the purification and investigation of transcription complexes. [Copyright &y& Elsevier]

Abstract: Reversible dimer formation in size exclusion chromatography (SEC) can cause peak splitting, merging, tailing, and fronting. Such behavior can be predicted by the association rate and the dissociation rate relative to the convection rate. Slow association and dissociation result in separated monomer and dimer peaks. Fast association and slow dissociation result in one single dimer peak. Slow association and fast dissociation result in one single monomer peak. Intermediate association and dissociation result in a merged, broad peak with either fronting when monomers dominate or tailing when dimers dominate. A diagram based on the two relative rates is generated to predict general peak shape and retention behavior in SEC. [Copyright &y& Elsevier]

Abstract: The suitability of three different retention models to predict the retention times of poly(ethylene glycol)s (PEGs) in gradient and isocratic chromatography was investigated. The models investigated were the linear (LSSM) and the quadratic solvent strength model (QSSM). In addition, a model describing the retention behaviour of polymers was extended to account for gradient elution (PM). It was found that all models are suited to properly predict gradient retention volumes provided the extraction of the analyte specific parameters is performed from gradient experiments as well. The LSSM and QSSM on principle cannot describe retention behaviour under critical or SEC conditions. Since the PM is designed to cover all three modes of polymer chromatography, it is therefore superior to the other models. However, the determination of the analyte specific parameters, which are needed to calibrate the retention behaviour, strongly depend on the suitable selection of initial experiments. A useful strategy for a purposeful selection of these calibration experiments is proposed. [Copyright &y& Elsevier]

Abstract: 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×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. [Copyright &y& Elsevier]

Abstract: The effect of deuterated solvents in size-exclusion chromatography (SEC) was studied by comparing intrinsic viscosity measurements, SEC calibration curves, and column efficiency using water-soluble polymers. For aqueous SEC, the use of deuterium oxide slightly increases the SEC elution volume. To verify that adsorption onto the packing was absent, data from exclusion experiments were compared at 35 and 50°C. Our results indicate that adsorption is not occurring for pullulan or polyethylene glycol (PEG)/poly(ethylene oxide) (PEO); for the latter, however, the elution volume increased using both D2O and H2O, indicative of slight hydrodynamic volume contraction of PEG/PEO at higher temperatures. A moderate increase in band broadening (moderate decrease in column efficiency) was observed using D2O. Finally, the effects of chloroform versus deuterated chloroform were evaluated, but no hydrodynamic volume changes were observed. [Copyright &y& Elsevier]

Abstract: The feasibility of different clean-up procedures was studied for the determination of polycyclic aromatic hydrocarbons (PAHs) in biota samples such as oysters, mussels and fish liver. In this sense, once the samples were extracted – essentially with acetone and in a microwave system – and before they could be analysed by gas chromatography–mass spectrometry (GC–MS), three different approaches were studied for the clean-up step: solid phase extraction (SPE), microwave-assisted saponification (MAS) and gel permeation chromatography (GPC). The main aim of this work was to maximise the recoveries of PAHs and to minimise the presence of interfering compounds in the last extract. In the case of SPE, Florisil® cartridges of 1, 2 and 5g, and silica cartridges of 5g were studied. In that case, and with oysters and mussels, microwave-assisted extraction and 5g Florisil® cartridges provided good results. In addition, the concentrations obtained for Standard Reference Material (SRM) NIST 2977 (mussel tissue) were in good agreement with the certified values. In the case of microwave-assisted saponification, the extracts were not as clean as those obtained with 5g Florisil® and this fact lead to overestimate the concentration of the heaviest PAHs. Finally, the cleanest extracts were obtained by GPC. The method was successfully applied to mussels, oysters and hake liver, and the results obtained for NIST 2977 (mussel tissue) were within the confidence interval of the certified reference material for most of the certified analytes. [Copyright &y& Elsevier]

Abstract: The modified universal calibration of gel permeation chromatography (GPC) has been applied in the case of native proteins. Plotting log([η]M/Φ) versus elution volume, instead of log[η]M versus elution volume used till now, we obtain unique curves with different proteins and non-proteonic polymers ([η]: intrinsic viscosity, M: molecular mass, Φ: Flory''s parameter). The values of Flory''s parameter Φ are calculated for each protein using an indirect method based on GPC. [Copyright &y& Elsevier]

Abstract: A comprehensive two-dimensional liquid chromatography system was constructed on the combination of size exclusion chromatography (SEC) and reverse phase liquid chromatography (RPLC). The first dimension used a SEC column with 300mm×8mm i.d., packed with Toyopearl HW-40S. The column was eluted with 0.05mol/l Tris–HCl (pH 6.9) at a flow rate of 0.4ml/min. The second dimension used a RPLC column with 100mm×4.6mm i.d., which was operated in gradient form at a flow rate of 1.0ml/min. An automatic switching valve was used to collect the effluent of the SEC column every 3min, and the effluent was automatically injected into the RPLC column. Mass spectrometer was used for peak identification. This system was used to separate Qingkailing injection, a traditional Chinese medicine (TCM). The result showed that the total peak capacity of this system could reach 1134 and the qualitative analysis of seven chemical components of the Qingkailing injection was accomplished by this system. The results show that comprehensive two-dimensional liquid chromatography system is of great importance and high value in the separation of complex TCM. [Copyright &y& Elsevier]