Discussion.

A recent article in Journal of Chromatography A 1561 (2018) 67-75 authored by J.-L. Wang, E.

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.

Comments on the separation efficiency of asymmetrical flow field-flow fractionation in channels of constant channel and crossflow velocities leading to constant separation efficiency.

It is shown theoretically that a claim in the literature about the overall separation efficiency of asymmetrical flow FFF channels being improved by geometries that permit a uniform channel flow velocity throughout the channel length is untrue.

Size separations of starch of different botanical origin studied by asymmetrical-flow field-flow fractionation and multiangle light scattering.

Asymmetrical-flow field-flow fractionation combined with multiangle light scattering and refractive index detection has been revealed to be a powerful tool for starch characterization. It is based on size separation according to the hydrodynamic diameter of the starch components. Starch from a wide range of different botanical sources were studied, including normal starch and high-amylose and high-amylopectin starch.

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).

Complex analytical approach to characterization of the influence of carbon dioxide concentration on carbohydrate composition in Norway spruce needles.

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.

Competitive adsorption of a polydisperse polymer during emulsification: experiments and modeling.

In this paper we study the selective adsorption of a high molar mass polymer, OSA-starch, at the cyclohexane/water interface during emulsification. This was made possible through the use of AsFlFFF-MALS-RI which enables us to characterize the size and molar mass of polydisperse ultrahigh molar mass polymers. The results show that the high molar mass components in the molar mass distribution of the polymer were selectively adsorbed.

NMR, cloud-point measurements and enzymatic depolymerization: complementary tools to investigate substituent patterns in modified celluloses.

The substituent patterns of some chemically modified celluloses were characterized as a function of their size distribution, using size-exclusion chromatography coupled to both nuclear magnetic resonance spectroscopy (NMR) and cloud-point measurements. Intact and enzymatically hydrolyzed methyl cellulose (MC) was fractionated according to size, and the level of substitution of the fractions was measured off-line using NMR. Clouding behavior was also measured as a function of size.

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.

On-line capillary based quantitative analysis of a heterocyclic amine in human urine.

A high through-put miniaturised separation-quantification method for the heterocyclic aromatic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in urine was developed. The limit of detection was of 0.65 fmol (0.

Mechanical degradation and changes in conformation of hydrophobically modified starch.

In this paper, we study the mechanical degradation and changes in conformation of a branched ultrahigh molar mass biomacromolecule, hydrophobically modified starch, as caused by high-pressure homogenization. The characterization was performed with asymmetrical flow field-flow fractionation (AsFlFFF) with multiangle light scattering (MALS) and refractive index detection. The starch which had been chemically modified with octenyl succinate anhydride (OSA) proved to be very large and polydisperse.

Size characterization of green fluorescent protein inclusion bodies in E. coli using asymmetrical flow field-flow fractionation-multi-angle light scattering.

The goal of this study was to investigate the applicability of asymmetrical flow field-flow fractionation-multi-angle light scattering (AsFlFFF-MALS) for size analysis of green fluorescent protein inclusion bodies (GFPIBs). The size distributions of GFPIBs prepared by various culture conditions were determined. For GFPIBs prepared at 37 degrees C the peak maximum hydrodynamic diameter (d(H)) first increased and then decreased with the increase of the induction times in the presence of 0.

Improved chemical analysis of cellulose ethers using dialkylamine derivatization and mass spectrometry.

Oligosaccharides of hydroxypropylmethyl cellulose, hydroxypropyl cellulose, and methyl cellulose were investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The cellulose ether oligosaccharides were produced either by enzymatic depolymerization utilizing the purified family 5 endoglucanase from Bacillus agaradhaerens or by partial acidic depolymerization. To lower the limit of detection in MALDI-MS three dilakylamines, dimethyl-, diethyl-, and dipropylamine were studied as reagents for reductive amination of the oligosaccharides.

Derivatization using dimethylamine for tandem mass spectrometric structure analysis of enzymatically and acidically depolymerized methyl cellulose.

Structure analysis of partially depolymerized methyl cellulose was performed by nanoelectrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) and by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). Dimethylamine (DMA) was used for the first time as a reducing end derivatization reagent for oligosaccharides. This is an attractive reagent since it could be easily removed from the reaction mixture.

Improved matrix-assisted laser desorption/ionisation sample preparation of a partially depolymerised cellulose derivative by continuous spray deposition and interfacing with size-exclusion chromatography.

Continuous spray deposition (CSD) of aqueous solutions of partially depolymerised methyl cellulose was found to improve matrix-assisted laser desorption/ionisation (MALDI) sample preparation. One feature was that the sensitivity in MALDI time-of-flight mass spectrometry increased up to an order of magnitude compared with the standard sample preparation method. Another feature was that CSD provided targets for MALDI with homogeneously distributed analyte.

Size and structure characterization of ethylhydroxyethyl cellulose by the combination of field-flow fractionation with other techniques. Investigation of ultralarge components.

Ethylhydroxyethyl cellulose (EHEC) of three different viscosity classes (EHEC I, II, and III) was analyzed by programmed cross-flow asymmetrical flow field-flow fractionation coupled to multiangle light scattering and refractive index detectors to determine their size and molar mass distribution. Two size populations were detected in the two lower viscosity classes, EHEC I and II, one high molar mass and one ultrahigh molar mass (UHM). The two covered molar masses from 10(4) up to 10(9) g X mol(-1).

Accuracy in multiangle light scattering measurements for molar mass and radius estimations. Model calculations and experiments.

Multiangle light scattering (MALS) is a well-established technique used to determine the size of macromolecules and particles. In this study, different extrapolation procedures used in MALS were investigated with regard to accuracy and robustness in the obtained molar mass and rms radius. Three different mathematical transformations of the light scattering function referred to as the Debye, Zimm, and Berry methods for constructing the Debye plot were investigated for two idealized polymer shapes, homogeneous spheres and random coils, with radii from 25 to 250 nm.

Enhanced ribosome and tRNA contents in Escherichia coli expressing a truncated Vitreoscilla hemoglobin mutant analyzed by flow field-flow fractionation.

The ribosome and tRNA levels of Escherichia coli cells, transformed with a native or mutated Vitreoscilla hemoglobin genes (vhb), were investigated using asymmetrical flow field-flow fractionation (AFFFF). Mutagenesis of rhb by error-prone PCR was carried out to alter the growth behavior of microaerobically cultivated native VHb-expressing E. coli.

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.

Mass overloading in the flow field-flow fractionation channel studied by the behaviour of the ultra-large wheat protein glutenin.

Flow field-flow fractionation (FFF) has previously been used in successful fractionation and characterisation of the ultra-large wheat protein glutenin. The many parameters, which may influence the retention behaviour, especially when analysing extremely high-molecular-mass samples such as glutenin, are here reported. Size determination from the sample retention time, using FFF theory, will as a result have a very low accuracy.

Sample preparation effects in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry of partially depolymerised methyl cellulose.

Methyl cellulose (MC) was partially depolymerised and the oligomers thus obtained were studied by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). The depolymerisation was either enzymatic or acidic. Fractions of enzymatically depolymerised MC were collected from size-exclusion chromatography and subjected to a sample preparation investigation.

Sample preparation effects in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry of partially depolymerised carboxymethyl cellulose.

Sample preparation effects in matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) of partially depolymerised carboxymethyl cellulose (CMC) have been investigated. The depolymerisation was either enzymatic or acidic. Fractions of enzymatically depolymerised CMC were collected from size-exclusion chromatography (SEC) and further investigated by MALDI-TOFMS.

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.

Size separation of supermicrometer particles in asymmetrical flow field-flow fractionation. Flow conditions for rapid elution.

The performance of lift-hyperlayer asymmetrical flow field-flow fractionation using rapid elution conditions was tested through the separation of standard polystyrene latex particles of diameters from 2 to 20 microm. Optimization of flowrates was studied not only in order to obtain efficient and rapid separation, but also to work under conditions of various shape and steepness of the axial flow velocity gradient. Using extreme flow conditions, the five widely spaced particle sizes, 20.