Karl-Gustav Wahlund

Fast molecular mass and size characterization of polysaccharides using asymmetrical flow field-flow fractionation-multiangle light scattering

Asymmetrical flow field-flow fractionation (asymmetrical flow FFF), connected on-line to multi-angle light scattering detection (MALS) was shown here to be an efficient method for size characterization of pullulan standards and dextrans ranging from 20 000 up to 2 000 000 in molecular mass. The characterization of molecular mass and the molecular mass distribution of these polysaccharides is often complex and may require different methods. Using asymmetrical flow FFF-MALS, information was obtained not only about molecular mass and molecular mass distribution but also about hydrodynamic size as well as radius of gyration and conformation. The analysis time was very short, often below 5 min. It was shown that the pullulan standards have a narrow molecular mass distribution compared to the more polydisperse dextrans. Obtained molecular masses and distributions were in good agreement with data from the manufacturer. The dextrans, especially at high molecular mass, were found to have a more compact structure than the pullulans in both water and 0.1 M NaCl.

Flow field-flow fractionation: Critical overview

This overview regards some critical issues in performing flow field-flow fractionation (flow FFF, FlFFF, AF4, HF-FlFFF, HF5). It includes the channel thickness, void time, channel-flow parabolic profile, channel-flow velocity gradient, uniformity of the cross-flow, sample injection time, relaxation/focusing time, width of sample starting zone, retention level, theoretical and experimental zone broadening, hydrodynamic threshold immobilisation/re-mobilisation, sample loss and adsorption, membrane fouling, sample mass overloading, problems with symmetrical channels, non-spherical sample particles, and method development. Good method development practice (GMDP) and good fractogram practice (GFP) is suggested.

Miniaturised on-line solid-phase extraction for enhancement of concentration sensitivity in capillary electrophoresis

Abstract Two of the most important issues facing the technology of capillary electrophoresis (CE) are the limited concentration sensitivity and the applicability to biological samples. An enrichment technique using miniaturised on-line solid-phase extraction did enhance the concentration sensitivity by a factor of 7000 using UV-absorbance detection. The cationic drug terbutaline was used as a model compound. The extractor consisted of a short length (1–3 mm) of a capillary (200 μm I.D.) packed with C18 alkyl-diol silica (dp 12 μm). This capillary was connected to a 50 μm I.D. separation capillary and glass-fibre filters retained the sorbent. Preparation and performance of the enrichment capillary is discussed and described in detail. The on-line enrichment procedure includes washing, wetting, conditioning, sorption, washing, filling and desorption. Then follows CE separation. The separation efficiency was excellent as 250 000 plates were obtained in a 58 cm enrichment capillary (550 000 plates m−1). The concentration limit of detection for terbutaline in aqueous solution with on-line enrichment was 0.6 nM (10 min×140 kPa pressure injection) compared to 4.4 μM (1 s×3.4 kPa pressure injection) without enrichment. The enrichment procedure was successfully adapted to high efficiency separation of terbutaline enantiomers using cyclodextrin as chiral selector. It was possible to enrich terbutaline from directly injected spiked plasma as well.

Retention behaviour of amylopectins in asymmetrical flow field-flow fractionation studied by multi-angle light scattering detection

Asymmetrical flow field-flow fractionation (FFF) with multi-angle light scattering (MALS) detection was applied for the fractionation of amylopectins from four different sources. Samples originated from genetically modified potatoes and waxy maize. Amylopectins were dissolved in a 1 mol l(-1) sodium hydroxide solution or water. With an injected mass of 0.2 microg, well below overloading conditions, a decrease of the apparent hydrodynamic radius with increasing inlet flow-rate was observed. Moreover, a decrease of the radius of gyration with increasing elution volume was recorded by the MALS detector. Steric/hyperlayer effects are a feasible explanation for this behaviour. The observed radius of gyration at the steric inversion point was in the order of 0.3 microm, which is smaller than the theoretically calculated inversion point. Apparently, the amylopectin behave as macromolecules with a larger hydrodynamic radius than expected on basis of their radius of gyration and are subjected to significant lift forces. The results were confirmed by four fractionations with varying flow-rates but constant ratio of cross to outlet-flow. In contrast to the normal mode operation, the retention of the amylopectins depended strongly on the applied flow-rates and was close to that of a much smaller 10 kDa dextran. Apparent molar masses in the order of between 10(7) and 10(9) g mol(-1) were obtained. The results are contrasted with enzymatically degraded and oxidised starch samples that were fractionated in the normal mode.

Effects of temperature, carrier composition and sample load in asymmetrical flow field-flow fractionation

Abstract The use of asymmetrical flow field-flow fractionation for accurate sample characterization requires good control of side effects which can adversely influence sample retention. Some of these factors are identified and means to avoid or minimize them are given. The behaviour of a model protein and virus was investigated. Bad reproducibility of the retention times and peak deformations were traced to incomplete temperature control and adsorption to the accumulation wall membrane. Good thermostatic control gave constant retention times because it eliminated temperature variations caused by friction heat produced in the carrier stream. Rinsing of the channel removed adsorbed species. The mechanism involved in the adsorption is unknown but the pH of the carrier liquid appears to have an influence. Exceeding the sample load limit leads to a complex behaviour of peak deformation. This behaviour as well as the load limit is governed by the choice of buffer and ionic strength of the carrier liquid.

Development of asymmetrical flow field-flow fractionation-multi angle laser light scattering analysis for molecular mass characterization of cationic potato amylopectin

The goal of this study is to investigate the applicability of asymmetrical flow field-flow fractionation (AsFlFFF)-multi angle laser light scattering (MALLS), and to develop a method for analysis of cationic potato amylopectin (CPAP) having ultrahigh molecular mass (UHMr). Use of the aqueous carrier having low salt content (3 mM NaN3) resulted in a distortion in AsFlFFF fractograms of CPAP with a general pattern of a sharp rise at the beginning of the elution followed by a long tailing, probably due to combination of attractive and repulsive charge interactions (attractive interaction between CPAP molecules and the channel membrane, and repulsion among cationic CPAP molecules). As the cross flow-rate (Fc) increases, the tailing tends to increase, and the repeatability of the AsFlFFF retention data tends to decrease, which is an indication of the presence of the charge interactions. The tailing gradually decreased, and the repeatability of the AsFIFFF retention data increased, as the salt content of the carrier increased. The distortion of the fractogram finally disappeared at Fc of about 0.2 ml/min and the channel flow-rate (F(out)) of about 1 ml/min with the aqueous carrier having the salt content of 40 mM (3 mM NaN3 +37 mM NaNO3). The weight-average molecular mass (Mw) and the z-average radius of gyration ((rg),) determined by MALLS were 5.2 x 10(7) and 34 x 10(1) nm, respectively. With the flow-rate ratio, Fc/F(out) kept constant, the degree of the charge interactions (and thus the distortion of fractogram) seems to increase with the cross flow-rate (Fc) and with the sample injection mass. AsFIFFF-MALLS was applied for determination of molecular mass distributions (MrDs) and the sizes of CPAPs prepared by various cooking procedures.

Analysis of film coating thickness and surface area of pharmaceutical pellets using fluorescence microscopy and image analysis

A method is presented which enables geometrical characterisation of pharmaceutical pellets and their film coating. It provides a high level of details on the single pellet level. Image analysis was used to determine the coating thickness (h) applied on the pellets and the surface area (A) of the pellet cores. Different definitions of A and h are evaluated. Hierarchical analysis of variance was used to resolve different sources contributing to the total variance. The variance within pellets and the variance between pellets were found as significant sources of variation. Special emphasis was put on evaluation of A/h due to its influence on the release rate of an active drug substance from the pellet core. The pellet images were thus used to predict variations in the release rate using a mathematical model as a link between the image data and the release rate. General aspects of image analysis are discussed. The method would be useful in calibration of near infrared spectra to h in process analytical chemistry.

Effects of flow-rates and sample concentration on the molar mass characterisation of modified celluloses using asymmetrical flow field-flow fractionation–multi-angle light scattering

Abstract Modified celluloses are an important group of polymers used in many applications, such as in the food and drug industry. Thus, their physicochemical properties are of considerable interest and need to be characterised carefully. In this study, asymmetrical flow field-flow fractionation (asymmetrical flow FFF) connected on-line to a multi-angle light scattering (MALS) detector was used to study the molar mass and molar mass distribution of three different hydroxypropylmethylcelluloses (HPMCs). The influence of the flow-rates and the sample concentration on the results obtained was found to be significant, emphasising the importance of optimising the experimental conditions so as to obtain reliable information about the polymer system. With the use of appropriate conditions, flow FFF–MALS was found to be a suitable method for the characterisation of these complex samples. The weight-average molar mass ranged from 132 000 g/mol to 309 000 g/mol. The z-average radius of gyration was found to be high relative to the molar mass, ranging from 58 nm to 73 nm, suggesting an expanded structure. This was also confirmed by double logarithmic plots of the molar mass versus the radius of gyration, the slope being approximately 0.7 for the two high molar mass samples.

Time-minimized determination of ribosome and tRNA levels in bacterial cells using flow field-flow fractionation.

The evaluation of the translation capacity of cells that produce recombinant proteins can be made by monitoring their ribosomal composition. In a previous use of asymmetrical flow field-flow fractionation (AsFlFFF) for this purpose the overall analysis time was more than 1 h and 40 min, based on a standard protocol for cell harvest, washing, cell disruption, and the final 8-min AsFlFFF determination of ribosome and subunits. In the present work the overall analysis time was reduced to 16 min. The washing step was deleted and a time-consuming freeze-thaw cycle. Cell disruption was obtained by a time-minimized lysozyme and detergent treatment for 1.5 min, respectively. The ribosomal material was finally fractionated and quantified in only 6 min, without previous centrifugation, using AsFlFFF. The great time reduction will enable the future use of AsFlFFF at-line to a growing cell cultivation, continuously monitoring the change in ribosomal composition or in other applications requiring high sample throughput. To demonstrate the high efficiency of the method the ribosome and tRNA composition in an Escherichia coli cultivation was monitored every half an hour, giving 18 measurements across the complete growth curve, a frequency of data enough to make decisions about induction or termination of the cultivation.

Macromolecular geometries determined with field-flow fractionation and their impact on the overlap concentration.

In this paper we aim to understand the size/conformation relationship in waxy barley starch, a polydisperse and ultrahigh molar mass biomacromolecule. Characterizations are performed with asymmetrical flow field-flow fractionation (AsFlFFF). Furthermore, we study the effect of homogenization on the molar mass, rms radius (r rms) and hydrodynamic radius (r h). For the untreated sample, the macromolecules are elongated objects with low apparent density. As a result of homogenization, molar mass, and r rms decrease, while r h remains unaffected. The process also induces an increase, and scaling with size, of apparent density as well as changes in conformation, represented qualitatively by r rms/ r h. Finally, results from AsFlFFF are compared with viscosimetry and discussed in terms of concentration and close-packing in relation to macromolecular shape and conformation. Hence, the results show that AsFlFFF and our novel methodology enable the determination of several physical properties with high relevance for the solution behavior of polydisperse macromolecules.