Characterisation of poly(N-isopropylacrylamide) by asymmetrical flow field-flow fractionation, dynamic light scattering, and size exclusion chromatography

Asymmetrical flow field-flow fractionation (AsFIFFF) was used to determine the hydrodynamic particle sizes, molar masses, and phase transition behaviour of various poly(N-isopropylacrylamide) (PNIPAM) samples synthesised by reversible addition--fragmentation chain transfer (RAFT) and conventional free radical polymerisation processes. The results were compared with corresponding data obtained by dynamic light scattering (DLS) and size exclusion chromatography (SEC). Agreement between the three methods was good except at higher molar masses, where the molar mass averages obtained by SEC were much lower than those obtained by AsFIFFF and light scattering. The aggregation of the polymers, which are thermally sensitive, was studied by DLS and AsFIFFF at various temperatures. In deionised water there was an abrupt change in the particle size due to phase separation at approximately equal to 32-35 degrees C. The critical temperatures determined by AsFIFFF were 3-5 degrees C higher than those obtained by DLS.     

Characteristics of the particle size distribution of hydrolyzed starch, as determined by dynamic light scattering

The unimodal distribution of hydrodynamic radii is determined by DLS measurements of starch molecule sizes. The starch was hydrolyzed in 0.3 M hydrochloric acid for 3–9 h. The ratio of amylose to amylopectin is shown to remain constant.     

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.     

Carboxymethylation of corn starch and characterization using asymmetrical flow field-flow fractionation coupled with multiangle light scattering

Asymmetrical flow field-flow fractionation (AsFlFFF) was coupled online with multiangle light scattering (MALS) to study the changes in the molecular weight and the size distribution of the corn starch during carboxymethylation. A corn starch was derivatized with sodium chloroacetate in alcoholic medium under alkaline condition to produce carboxymethyl starches (CMS) having various degrees of substitution (DS). The change in thermal characteristics and granule structure of the native corn starch and CMS were compared using Thermogravimetric analysis and scanning electron microscope. The ionic strength of the carrier liquid (water with 0.02% NaN₃) was optimized by adding 50 mM NaNO₃ to minimize the interactions among the starch molecules and between the starch molecules and the AsFlFFF membrane. A field-programmed AsFlFFF allowed determination of the molecular weight distribution (MWD) of starches within about 25 min. It was found that carboxymethylation of starch results in reduction in the molecular weight due to molecular degradation by the alkaline treatment. The weight-average molecular weight (M_w) was reduced down to about 4.4 × 10⁵ from about 7.2 × 10⁶ when DS was 0.14. It seems AsFlFFF coupled with MALS (AsFlFFF/MALS) is a useful tool for monitoring the changes taking place in the molecular weight and the size of starch during derivatization.     

Characterization of sodium hyaluronate blends using frit inlet asymmetrical flow field-flow fractionation and multiangle light scattering

We characterized ultrahigh molecular weight sodium hyaluronate (NaHA) and blended pharmaceutical products containing NaHA using flow field-flow fractionation and multiangle light scattering–differential refractive index (FlFFF-MALS-DRI). NaHA is a water-soluble polysaccharide with a range of molecular weights (MW; 10⁵~10⁸ Da) that is found in body fluids and tissues. NaHA is also used commercially in pharmaceutical and cosmetic applications. We used a frit inlet asymmetrical FlFFF channel to separate aqueous polymers according to their hydrodynamic size, and we used on-line measurements of light scattering to obtain the MW distribution (MWD) as well as structural information about NaHA in aqueous solution. In this study, we investigated NaHA and anti-adhesive blend mixtures of NaHA (a commercial NaHA blend mixture containing sodium carboxymethyl cellulose and a new blend with hydroxyethyl starch (HES)) to determine the molecular weight distribution MWD of NaHA and the blend mixtures and to obtain structural information about these compounds in aqueous solution. We also examined the characteristics of NaHA–HES–polylactic-co-glycolic acid film products exposed to gamma radiation for sterilization purposes.     

Asymmetric flow field flow fractionation of aqueous C60 nanoparticles with size determination by dynamic light scattering and quantification by liquid chromatography atmospheric pressure photo-ionization mass spectrometry

A size separation method was developed for aqueous C₆₀ fullerene aggregates (aqu/C₆₀) using asymmetric flow field flow fractionation (AF4) coupled to a dynamic light scattering detector in flow through mode. Surfactants, which are commonly used in AF4, were avoided as they may alter suspension characteristics. Aqu/C₆₀ aggregates generated by sonication in deionized water ranged in size from 80 to 260 nm in hydrodynamic diameter (D_h) as determined by DLS in flow through mode, which was corroborated by analysis of fractions by DLS in batch mode and by TEM. The mass of C₆₀ in each fraction was determined by LC–APPI–MS. Only 5.2 ± 6.7% of the total aqu/C₆₀ mass had D_h less than 80 nm, while 58 ± 32% of the total aqu/C₆₀ mass had D_h between 80 and 150 nm and 14 ± 9.2% of the total aqu/C₆₀ were between 150 and 260 nm in D_h. With the optimal fractionation parameters, 77 ± 5.8% of the aqu/C₆₀ mass eluted from the AF4 channel, indicating deposition on the AF4 membrane had occurred during fractionation; use of alternative membranes did not reduce deposition. Channel flow splitting increased detector response although channel split ratios greater than 80% of the channel flow led to decreased detector response. This is the first report on the use of AF4 for fractionating a colloidal suspension of aqu/C₆₀.     

Contribution of flow field flow fractionation with on line static and dynamic light scattering to the study of hydrosoluble polyelectrolyte complexes

Water soluble polyelectrolyte complexes (PECs) formed between polyaspartate (anionic polymer) and poly(trimethylammonium propyl methacrylamide chloride) (cationic polymer) were studied by flow field flow fractionation with on-line coupling multi-angle laser light scattering-quasi elastic light scattering-differential refractive index determination (F4/MALLS/QELS/DRI). The separation technique permits to characterize polydisperse PECs. The molar mass of the polycation (PC) influences the stiffness of the PECs and the proportion between single PECs (i.e. nPA/1PC) and multiple PECs (i.e. nPA/n’PC). High ionic strength with NaCl (>0.1 M) tends to break the multiple PECs while CaCl₂ destroys PECs and leads to the formation of complexes polyaspartate/Ca²⁺. The studied PECs can be used as inhibitors to the calcite formation in the drilling fluids.     

Optimisation of asymmetrical flow field flow fractionation for environmental nanoparticles separation

The fractionation of natural nanoparticles by Asymmetrical Flow Field Flow Fractionation (As-Fl-FFF) was optimised by considering the following operating conditions: ionic strength, surfactant concentration and crossflow rate. The method performances such as fractionation recovery and fractionation efficiency were evaluated on a stable solution of colloidal-size natural inorganic particles. The online multi-detection by ultraviolet/visible spectrophotometer (UV) and multi-angle laser light scattering (MALLS) provided the monitoring of the sample during the separation and the evaluation of the fractionation efficiency. The lowest ionic strength and surfactant concentrations (i.e. 10(-3)molL(-1) NH(4)NO(3) and 3x10(-4)molL(-1) SDS) allowed to obtain the highest sample recovery and lowest loss of the largest particles. The crossflow rate was investigated in order to avoid significant membrane-sample interaction. The applicability of the fractionation in optimised conditions was evaluated on a natural soil leachate, which was filtrated with different filter cut-offs. Filtration efficiency was stressed by the decrease of the large unfractionated particle influence in the void volume. For the first time, robust operating conditions were proposed to well size-fractionate and characterize soil nanoparticles within a single multi-detection analysis.     

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.     

Macromolecular size of polyelectrolytes containing ammonium and sulfonic acid groups, as determined by light scattering

The macromolecular dimensions of fractionated polyelectrolytes poly(4-vinylpyridine) quaternized (PVPyQ), poly[(3-methacryloylamino) propyltrimethyl ammonium chloride] (PMPTA), poly(diallyl dimethyl ammonium chloride) (PDDA), and poly[(3-methacryloylamino) propyltrimethyl ammonium chloride-co-2-acrylamido-2-methyl-1-propanesulfonic acid] P(MPTA-co-APSA), were determined by light scattering, using aqueous electrolyte as solvent. PVPyQ, PMPTA, and P(MPTA-co-APSA) of various comonomer compositions were synthesized by radical polymerization. All polymers were fractionated by membrane ultrafiltration. Light scattering measurements on dialyzed solutions give good Zimm plots from which the average molecular weight, , mean squared radius of gyration, 〈s²〉, and second virial coefficient, A₂, were determined. No significant selective sorption of the electrolyte by the polyelectrolytes was detected. Positive A₂ values are obtained in all cases, corresponding to good solvent conditions, however, the interpenetration function, Ψ, has low values (0.01-0.10), showing that the excluded volume effect is relatively low. In PDDA, the dependence of 〈s²〉 on suggests that the coil of this polyelectrolyte is contracted by branching. In the ammonium-sulfonic copolymers, the comonomer stoichiometric feed 1:1 yields an insoluble copolymer (possible complex formation between amino and sulfonic groups). The soluble copolymers (with unbalanced comonomer compositions) have dimensions similar to the homopolymers, and not appreciably dependent on composition; hence no contraction due to complex formation is detected in these copolymers.     

Determination of spherulite size distribution by small-angle light scattering

The theoretical backgrounds of the method of spherulite size distribution determination, on the basis of the small-angle light scattering technique, are presented. Special corrections of experimental data are introduced to achieve great accuracy of the determination of scattered light intensity distribution. Experiments have been done on low-density polyethylene samples obtained in various crystallization conditions. For these samples, the stepwise distributions of spherulite size have been determined.     

Field programming in frit inlet asymmetrical flow field-flow fractionation/multiangle light scattering: application to sodium hyaluronate

The capability of field-programmed separation in frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF) has been examined for separating a high molecular weight sodium hyaluronate (NaHA) by varying the field programming parameters. Experiments were performed with on-line coupling of the field programming FI-AFlFFF and multiangle light scattering (MALS) detection. Sample relaxation, a pre-requisite step to establish equilibrium states of sample materials prior to the beginning of separation in most forms of FFF techniques, is obtained by hydrodynamically in FI-AFlFFF without stopping the migration flow. Thus, the procedures of sample injection -- hydrodynamic relaxation -- separation in FI-AFlFFF are continuously achieved without halting the sample migration. In this study, field programming in FI-AFlFFF was investigated for the separation of NaHA, water-soluble polysaccharides, by examining the influence of field decay pattern, initial field strength condition, and ionic strength of carrier solution on the successful separation of a degraded NaHA sample. Results were compared with molecular weight calculations of eluting materials among different field programming conditions from multiangle light scattering (MALS) signals. It was found that when the field programming was utilized in FI-AFlFFF, a proper selection of initial cross-flow rate, the field decay pattern, and an appropriate control of final field strength needed to be carefully selected in achieving a successful separation of a broad molecular weight water-soluble polymer sample.     

Protein diffusion coefficients determined by macroscopic-gradient Rayleigh interferometry and dynamic light scattering

Dynamic light scattering (DLS) is extensively used for measuring macromolecule diffusion coefficients. Contrary to classical techniques based on macroscopic concentration gradients, DLS probes microscopic fluctuations in concentration. DLS accuracy and its concordance with macroscopic-gradient techniques remains an outstanding important issue. We measured lysozyme diffusion coefficients in aqueous salt using both DLS and Rayleigh interferometry, a highly accurate macroscopic-gradient technique. The precision of our results is unprecedented. We find that our DLS values were systematically 2% higher than interferometry values. We believe that our interferometric measurements have produced the most accurate diffusion data ever reported for a protein, providing a new standard for quality control of DLS measurements. Furthermore, by interferometry, we have determined the whole diffusion coefficient matrix required for rigorously describing lysozyme-salt coupled diffusion. For the first time, we experimentally demonstrate that DLS does not provide the protein diffusion coefficient but one eigenvalue of the diffusion coefficient matrix.     

Latex particle size distribution by dynamic light scattering: novel data processing for multiangle measurements

Multiangle dynamic light scattering (DLS) provides a better estimate of particle size distributions (PSD) than single-angle DLS. However, multiangle data treatment requires appropriate weighting of each autocorrelation measurement prior to calculation of the PSD. The weighting coefficients may be directly obtained from (i). the autocorrelation baselines or (ii). independent measurement of the average light intensity by elastic light scattering. However, the propagation of errors associated with such procedures may intolerably corrupt the PSD estimate. In this work, an alternative recursive least-squares calculation is proposed that estimates the weighting coefficients on the basis of the complete autocorrelation measurement. The method was validated through a numerical example that simulates the analysis of a polystyrene latex with a bimodal PSD and with "measurements" taken at 10 detection angles. The ill-conditioned nature of the problem determines that the "true" PSD cannot be recovered, even in the absence of errors. A sensitivity analysis was carried out to determine the effect of errors in the weighting coefficients on the PSD recoveries.     

Characterization of branched ultrahigh molar mass polymers by asymmetrical flow field-flow fractionation and size exclusion chromatography

The molar mass distribution (MMD) of synthetic polymers is frequently analyzed by size exclusion chromatography (SEC) coupled to multi angle light scattering (MALS) detection. For ultrahigh molar mass (UHM) or branched polymers this method is not sufficient, because shear degradation and abnormal elution effects falsify the calculated molar mass distribution and information on branching. High temperatures above 130 °C have to be applied for dissolution and separation of semi-crystalline materials like polyolefins which requires special hardware setups. Asymmetrical flow field-flow fractionation (AF4) offers the possibility to overcome some of the main problems of SEC due to the absence of an obstructing porous stationary phase. The SEC-separation mainly depends on the pore size distribution of the used column set. The analyte molecules can enter the pores of the stationary phase in dependence on their hydrodynamic volume. The archived separation is a result of the retention time of the analyte species inside SEC-column which depends on the accessibility of the pores, the residence time inside the pores and the diffusion ability of the analyte molecules. The elution order in SEC is typically from low to high hydrodynamic volume. On the contrary AF4 separates according to the diffusion coefficient of the analyte molecules as long as the chosen conditions support the normal FFF-separation mechanism. The separation takes place in an empty channel and is caused by a cross-flow field perpendicular to the solvent flow. The analyte molecules will arrange in different channel heights depending on the diffusion coefficients. The parabolic-shaped flow profile inside the channel leads to different elution velocities. The species with low hydrodynamic volume will elute first while the species with high hydrodynamic volume elute later. The AF4 can be performed at ambient or high temperature (AT-/HT-AF4). We have analyzed one low molar mass polyethylene sample and a number of narrow distributed polystyrene standards as reference materials with known structure by AT/HT-SEC and AT/HT-AF4. Low density polyethylenes as well as polypropylene and polybutadiene, containing high degrees of branching and high molar masses, have been analyzed with both methods. As in SEC the relationship between the radius of gyration (R(g)) or the molar mass and the elution volume is curved up towards high elution volumes, a correct calculation of the MMD and the molar mass average or branching ratio is not possible using the data from the SEC measurements. In contrast to SEC, AF4 allows the precise determination of the MMD, the molar mass averages as well as the degree of branching because the molar mass vs. elution volume curve and the conformation plot is not falsified in this technique. In addition, higher molar masses can be detected using HT-AF4 due to the absence of significant shear degradation in the channel. As a result the average molar masses obtained from AF4 are higher compared to SEC. The analysis time in AF4 is comparable to that of SEC but the adjustable cross-flow program allows the user to influence the separation efficiency which is not possible in SEC without a costly change of the whole column combination.     

Size fractionation and characterization of natural colloids by flow-field flow fractionation coupled to multi-angle laser light scattering

Flow-field flow fractionation (FlFFF) coupled to multi-angle laser light scattering (MALLS) was evaluated for size and shape determination of standard spherical and arbitrarily shaped natural colloids. Different fitting methods for light scattering data retrieved from MALLS were evaluated to determine the particle size of spherical standards and natural colloids. In addition, FlFFF was optimized for best fractionation in connection to MALLS, minimal colloids-membrane interaction, and minimal sample losses. FlFFF, calibrated with standard particles, was used to determine hydrodynamic diameter, or radius (D(h) or R(h)), of the fractionated colloids, whereas the MALLS was used to determine root mean square radius of gyration (R(g)) for fractionated colloids. Combining both results, by calculating the R(g)/R(h) ratio, allows an estimation of colloid deviation from the shape of homogeneous sphere. Accordingly, this study demonstrates that, FlFFF-MALLS is a valuable technique for characterizing heterogeneous and arbitrarily shaped natural colloidal particles in terms of size and shape. To check the usefulness of FlFFF-MALLS in natural colloid studies, the technique was used to investigate the sedimentation behavior of extracted soil colloidal particles. Results illustrate that, in a silty till sample, carbonates function as cement between the colloidal particles, and consequently, change their sedimentation behavior. On the other hand, carbonate dissolution generates a more homogeneous colloidal sample.     

Programmed cross flow asymmetrical flow field-flow fractionation for the size separation of pullulans and hydroxypropyl cellulose

Different functions for the programming of the cross flow in asymmetrical flow field-flow fractionation were studied with the aim to find the flow conditions most suitable for the molar mass distribution analysis of high molecular weight polysaccharides. A mixture of four differently sized pullulans covering the molar mass range 5.8 x 10(3)-1.6 x 10(6) g mol(-1) were used as a model sample. Two types of programs were studied, linear and exponential decays, both with and without initial periods of a constant cross flow. For comparison, nonprogrammed runs, i.e. using constant cross flow, were studied. It was found that exponentially decaying cross flow gave the most uniform molar mass selectivity across the fractogram. The programmed cross flow was applied to the molar mass distribution analysis of a technical quality of hydroxypropyl cellulose.     

Polyelectrolyte complexes of poly(methacryloxyethyl trimethylammonium chloride) and poly(ethylene oxide)-block-poly(sodium methacrylate) studied by asymmetrical flow field-flow fractionation and dynamic light scattering

Polyelectrolyte complex (PEC) formation between cationic poly(methacryloxyethyl trimethylammonium chloride) (PMOTAC) and anionic poly(ethylene oxide)-block-poly(sodium methacrylate) (PEO-b-PMANa) was studied by asymmetrical flow field-flow fractionation and dynamic light scattering. The influence of ionic strength and mixing ratios of the charged units of the polyelectrolytes on the complex formation was evaluated. The diffusion coefficients and the hydrodynamic diameter distributions of the free and complexed polyelectrolytes were measured. In the absence of salt, the weight averaged hydrodynamic diameters were 48 and 28 nm for PMOTAC and PEO-b-PMANa, respectively. In the presence of salt, the particles were smaller, with weight averaged hydrodynamic diameters of 44-45 and 8-10 nm, respectively. In salt-free solution, at 1:1 mixing ratio of the charged monomer units of PMOTAC and PEO-b-PMANa, polydisperse particles with diameters of 2000-4000 nm were formed. In the presence of 20, 80, and 160 mM of sodium chloride, the 1:1 complexes were relatively monodisperse particles with weight averaged hydrodynamic diameters of 93, 124, and 120 nm, respectively.     

Particle size analysis by sedimentation field flow fractionation. Performance and application

Recently commercial equipment using sedimentation field flow fractionation (SFFF) has become available for analysis of particulate materials in the sub-micron range. This paper describes the DuPont instrument and discusses its performance. A particular study is described on the comparison of the SFFF technique with that of quasi-elastic light scattering (QELS). The paper concludes that the instrument is capable of measuring particle size distributions with high resolution and precision, provided that no particles above the upper size limit — about 1 μm — are present.     

Depolymerization study of sodium hyaluronate by flow field-flow fractionation/multiangle light scattering

Thermal depolymerization of ultrahigh-molecular-weight (UHMW) sodium hyaluronate (NaHA) was studied systematically by using frit-inlet asymmetrical flow field-flow fractionation/multiangle light scattering/differential refractive index (FI-AFlFFF/MALS/DRI). FI-AFlFFF was utilized for the size separation of NaHA samples which had been thermally degraded for varied treatment times, followed by light-scattering detection to determine MW and structural information of degraded NaHA products. Analysis of NaHA products showed time-dependent depolymerization of raw molecules into smaller-MW components, as well as unfolding of compact structures of UHMW NaHA. To determine whether the observed decrease in MW of sodium hyaluronate originated from the chain degradation of UHMW molecules or from dissociation of entangled complex particles that may have been formed by intermolecular association, narrow size fractions (1 × 10⁷–6 × 10⁷ and >6 × 10⁷ MW) of NaHA molecules were collected during FlFFF separation and followed by thermal treatment. Subsequent FI-AFlFFF/MALS analysis of collected fractions after thermal treatment suggested that the ultrahigh-MW region (>10⁷ Da) of NaHA is likely to result from supermolecular structures formed by aggregation of large molecules.