## Editorial Board

Publication date: 19 January 2018 Source:Journal of Chromatography A, Volume 1533

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

## Detection and confirmation of α-cobratoxin in equine plasma by solid-phase extraction and liquid chromatography coupled to mass spectrometry

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Fuyu Guan, Youwen You, Xiaoqing Li, Mary A. Robinson
α-Cobratoxin (CTX) is a large peptide (71 amino acids) with strong analgesic effect and may be misused in sports such as horse racing. To prevent such misuse, a sensitive method is required for detection and confirmation of the toxin in equine samples. CTX was extracted from equine plasma using an optimized mixed-mode solid-phase extraction (SPE) procedure. Extracted CTX was reduced with dithiothreitol and alkylated with iodoacetamide, and then was digested by trypsin at 56°C for 30min. The digest was analysed by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), and tryptic peptides T2 (3CFITPDITSK12) and T4 (24TWCDAFCSIR33) were monitored for detection and confirmation of CTX. The limit of detection (LOD) was 0.05ng/mL for CTX in plasma, and the limit of confirmation (LOC) 0.2ng/mL. Unlike small peptides consisting of the 20 canonical amino acids, CTX was stable in equine plasma at ambient temperature for at least 24h. The developed analytical method was successfully applied to analysis of incurred plasma samples; CTX was detected in plasma collected 15min through 36h post subcutaneous administration of CTX (2.0mg dose) to a research horse, and confirmed 30min through 24h. Additionally, an approach named “reliable targeted SEQUEST search” has been proposed for assessing the specificity of T2 at product ion spectrum level for confirmation of CTX. T2 is uniquely specific for CTX, as evaluated with this approach and BLAST search. Furthermore, the effect of dimethyl sulfoxide (DMSO) as a mobile phase additive on electrospray (ESI) response of T2 and T4, background noise level and signal to noise ratio (S/N) was examined; DMSO increased signal intensity of T2 and T4 by a factor of less than 2. It is the first report that DMSO raised background noise level and did not improve S/N for the peptides, to the authors’ knowledge. The developed analytical method may be applicable for analysis of CTX in plasma from other species such as greyhound dogs or even human beings.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Fuyu Guan, Youwen You, Xiaoqing Li, Mary A. Robinson

α-Cobratoxin (CTX) is a large peptide (71 amino acids) with strong analgesic effect and may be misused in sports such as horse racing. To prevent such misuse, a sensitive method is required for detection and confirmation of the toxin in equine samples. CTX was extracted from equine plasma using an optimized mixed-mode solid-phase extraction (SPE) procedure. Extracted CTX was reduced with dithiothreitol and alkylated with iodoacetamide, and then was digested by trypsin at 56°C for 30min. The digest was analysed by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), and tryptic peptides T2 (3CFITPDITSK12) and T4 (24TWCDAFCSIR33) were monitored for detection and confirmation of CTX. The limit of detection (LOD) was 0.05ng/mL for CTX in plasma, and the limit of confirmation (LOC) 0.2ng/mL. Unlike small peptides consisting of the 20 canonical amino acids, CTX was stable in equine plasma at ambient temperature for at least 24h. The developed analytical method was successfully applied to analysis of incurred plasma samples; CTX was detected in plasma collected 15min through 36h post subcutaneous administration of CTX (2.0mg dose) to a research horse, and confirmed 30min through 24h. Additionally, an approach named “reliable targeted SEQUEST search” has been proposed for assessing the specificity of T2 at product ion spectrum level for confirmation of CTX. T2 is uniquely specific for CTX, as evaluated with this approach and BLAST search. Furthermore, the effect of dimethyl sulfoxide (DMSO) as a mobile phase additive on electrospray (ESI) response of T2 and T4, background noise level and signal to noise ratio (S/N) was examined; DMSO increased signal intensity of T2 and T4 by a factor of less than 2. It is the first report that DMSO raised background noise level and did not improve S/N for the peptides, to the authors’ knowledge. The developed analytical method may be applicable for analysis of CTX in plasma from other species such as greyhound dogs or even human beings.

## Determination of heavy polycyclic aromatic hydrocarbons by non-aqueous reversed phase liquid chromatography: Application and limitation in refining streams

Publication date: 19 January 2018 Source:Journal of Chromatography A, Volume 1533 Author(s): Saroj K. Panda, Hendrik Muller, Thunayyan A. Al-Qunaysi, Omer R. Koseoglu The heavy polycyclic aromatic hydrocarbons (HPAHs) cause det…

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Saroj K. Panda, Hendrik Muller, Thunayyan A. Al-Qunaysi, Omer R. Koseoglu

The heavy polycyclic aromatic hydrocarbons (HPAHs) cause detrimental effects to hydrocracker operations by deactivating the catalysts and depositing in the downstream of the reactor/ exchangers. Therefore, it is essential to continuously monitor the accumulation of HPAHs in a hydrocracker unit. To accurately measure the concentration of HPAHs, the development of a fast and reliable analytical method is inevitable. In this work, an analytical method based on non-aqueous reversed phase chromatography in combination with high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was developed. As a first step, five different types of stationary phases were evaluated for the separation of HPAHs in non-aqueous mode and the best suited phase was further used for the fractionation of HPAHs in a fractionator bottom sample obtained from a refinery hydrocracker unit. The eight major fractions or peaks obtained from the separation were further characterized by UV spectroscopy and FT-ICR MS and the compounds in the fractions were tentatively confirmed as benzoperylene, coronene, methylcoronene, naphthenocoronene, benzocoronene, dibenzoperylene, naphthocoronene and ovalene. The developed liquid chromatography method can be easily adapted in a refinery laboratory for the quantitation of HPAHs in hydrocracking products. The method was further tested to check the interference of sulfur aromatics and/or large alkylated aromatic hydrocarbons on the determination of HPAHs in hydrocracking products.

## Adsorption behavior of optical brightening agent on microfibrillated cellulose studied through inverse liquid chromatography: The need to correct for axial dispersion effect

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Sonia Serroukh, Patrick Huber, Abdelaziz Lallam
Inverse liquid chromatography is a technique for studying solid/liquid interaction and most specifically for the determination of solute adsorption isotherm. For the first time, the adsorption behaviour of microfibrillated cellulose was assessed using inverse liquid chromatography. We showed that microfibrillated cellulose could adsorb 17 mg/g of tetrasulfonated optical brightening agent in typical papermaking conditions. The adsorbed amount of hexasulfonated optical brightening agent was lower (7 mg/g).The packing of the column with microfibrillated cellulose caused important axial dispersion (Da = 5e-7 m²/s). Simulation of transport phenomena in the column showed that neglecting axial dispersion in the analysis of the chromatogram caused significant error (8%) in the determination of maximum adsorbed amount. We showed that conventional chromatogram analysis technique such as elution by characteristic point could not be used to fit our data. Using a bi-Langmuir isotherm model improved the fitting, but did not take into account axial dispersion, thus provided adsorption parameters which may have no physical significance. Using an inverse method with a single Langmuir isotherm, and fitting the transport equation to the chromatogram was shown to provide a satisfactory fitting to the chromatogram data. In general, the inverse method could be recommended to analyse inverse liquid chromatography data for column packing with significant axial dispersion (Da  &gt; 1e-7 m²/s).

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Sonia Serroukh, Patrick Huber, Abdelaziz Lallam

Inverse liquid chromatography is a technique for studying solid/liquid interaction and most specifically for the determination of solute adsorption isotherm. For the first time, the adsorption behaviour of microfibrillated cellulose was assessed using inverse liquid chromatography. We showed that microfibrillated cellulose could adsorb 17 mg/g of tetrasulfonated optical brightening agent in typical papermaking conditions. The adsorbed amount of hexasulfonated optical brightening agent was lower (7 mg/g). The packing of the column with microfibrillated cellulose caused important axial dispersion ($D a$ = 5e-7 m²/s). Simulation of transport phenomena in the column showed that neglecting axial dispersion in the analysis of the chromatogram caused significant error (8%) in the determination of maximum adsorbed amount. We showed that conventional chromatogram analysis technique such as elution by characteristic point could not be used to fit our data. Using a bi-Langmuir isotherm model improved the fitting, but did not take into account axial dispersion, thus provided adsorption parameters which may have no physical significance. Using an inverse method with a single Langmuir isotherm, and fitting the transport equation to the chromatogram was shown to provide a satisfactory fitting to the chromatogram data. In general, the inverse method could be recommended to analyse inverse liquid chromatography data for column packing with significant axial dispersion ($D a$  &gt; 1e-7 m²/s).

## High-resolution separation of neodymium and dysprosium ions utilizing extractant-impregnated graft-type particles

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Shoichiro Uchiyama, Takaaki Sasaki, Ryo Ishihara, Kunio Fujiwara, Takanobu Sugo, Daisuke Umeno, Kyoichi Saito
An efficient method for rare metal recovery from environmental water and urban mines is in high demand. Toward rapid and high-resolution rare metal ion separation, a novel bis(2-ethylhexyl) phosphate (HDEHP)-impregnated graft-type particle as a filler for a chromatography column is proposed. To achieve rapid and high-resolution separation, a convection-flow-aided elution mode is required. The combination of 35 μm non-porous particles and a polymer-brush-rich particle structure minimizes the distance from metal ion binding sites to the convection flow in the column, resulting in minimized diffusional mass transfer resistance and the convection-flow-aided elution mode. The HDEHP-impregnated graft-type non-porous-particle-packed cartridge developed in this study exhibited a higher separation performance for model rare metals, neodymium (III) and dysprosium (III) ions, and a narrower peak at a higher linear velocity, than those of previous HDEHP-impregnated fiber-packed and commercially available Lewatit® VP OC 1026-packed cartridges.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Shoichiro Uchiyama, Takaaki Sasaki, Ryo Ishihara, Kunio Fujiwara, Takanobu Sugo, Daisuke Umeno, Kyoichi Saito

An efficient method for rare metal recovery from environmental water and urban mines is in high demand. Toward rapid and high-resolution rare metal ion separation, a novel bis(2-ethylhexyl) phosphate (HDEHP)-impregnated graft-type particle as a filler for a chromatography column is proposed. To achieve rapid and high-resolution separation, a convection-flow-aided elution mode is required. The combination of 35 μm non-porous particles and a polymer-brush-rich particle structure minimizes the distance from metal ion binding sites to the convection flow in the column, resulting in minimized diffusional mass transfer resistance and the convection-flow-aided elution mode. The HDEHP-impregnated graft-type non-porous-particle-packed cartridge developed in this study exhibited a higher separation performance for model rare metals, neodymium (III) and dysprosium (III) ions, and a narrower peak at a higher linear velocity, than those of previous HDEHP-impregnated fiber-packed and commercially available Lewatit® VP OC 1026-packed cartridges.

## Metal-organic framework-derived three-dimensional porous graphitic octahedron carbon cages-encapsulated copper nanoparticles hybrids as highly efficient enrichment material for simultaneous determination of four fluoroquinolones

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533
Author(s): Yang Wang, Yao Tong, Xu Xu, Lei Zhang
A unique 3D porous Cu@graphitic octahedron carbon cages were constructed by rapid room-temperature synthesis of a Cu-based metal–organic framework (MOF) with further pyrolysis in N2, which exhibited good enrichment ability for four fluoroquinolones (FQs) due to their superior chemical affinities to the target analytes. Applied Cu@graphitic octahedron carbon cages as adsorbent, a dispersive solid phase extraction (DSPE) method combined with HPLC was developed for detecting four FQs in real samples. Various parameters affecting residues FQs extraction efficiency were inquired in more detail. Under optimal conditions, the extraction recoveries of four FQs in chicken muscle, fish tissue, seawater and river water samples were in the range of 81.3∼104.3% and the RSDs (n = 5) were less than 5.2%. This method was successfully used to the determination of FQs in real samples.

Publication date: 19 January 2018
Source:Journal of Chromatography A, Volume 1533

Author(s): Yang Wang, Yao Tong, Xu Xu, Lei Zhang

A unique 3D porous Cu@graphitic octahedron carbon cages were constructed by rapid room-temperature synthesis of a Cu-based metal–organic framework (MOF) with further pyrolysis in N2, which exhibited good enrichment ability for four fluoroquinolones (FQs) due to their superior chemical affinities to the target analytes. Applied Cu@graphitic octahedron carbon cages as adsorbent, a dispersive solid phase extraction (DSPE) method combined with HPLC was developed for detecting four FQs in real samples. Various parameters affecting residues FQs extraction efficiency were inquired in more detail. Under optimal conditions, the extraction recoveries of four FQs in chicken muscle, fish tissue, seawater and river water samples were in the range of 81.3∼104.3% and the RSDs (n = 5) were less than 5.2%. This method was successfully used to the determination of FQs in real samples.

## High-resolution dynamic computer simulation of electrophoresis using a multiphysics software platform

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532
Author(s): Saara Mikkonen, Henrik Ekström, Wolfgang Thormann
The modeling and simulation software COMSOL Multiphysics® was recently extended with an electrophoretic transport interface. Its performance was investigated by comparison to results obtained using the 1D dynamic electrophoresis simulators GENTRANS and SIMUL5. Simulations of zone electrophoresis, isotachophoresis, isoelectric focusing and of an oscillating electrolyte system were performed. Smooth profiles were essentially identical indicating that the COMSOL electrophoretic transport interface is able to reproduce results of the 1D simulators. Differences in the way the respective numerical schemes handle steep concentration gradients and associated instabilities were observed. The COMSOL electrophoretic transport interface is expected to be useful as a general model for simulations in 1D, 2D or 3D geometries, as well as for simulations combining electrophoresis with other physical phenomena.

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532

Author(s): Saara Mikkonen, Henrik Ekström, Wolfgang Thormann

The modeling and simulation software COMSOL Multiphysics® was recently extended with an electrophoretic transport interface. Its performance was investigated by comparison to results obtained using the 1D dynamic electrophoresis simulators GENTRANS and SIMUL5. Simulations of zone electrophoresis, isotachophoresis, isoelectric focusing and of an oscillating electrolyte system were performed. Smooth profiles were essentially identical indicating that the COMSOL electrophoretic transport interface is able to reproduce results of the 1D simulators. Differences in the way the respective numerical schemes handle steep concentration gradients and associated instabilities were observed. The COMSOL electrophoretic transport interface is expected to be useful as a general model for simulations in 1D, 2D or 3D geometries, as well as for simulations combining electrophoresis with other physical phenomena.

## Differentiation of modern and ancient varieties of common wheat by quantitative capillary electrophoretic profile of phenolic acids

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532
Author(s): Roberto Gotti, Elisa Amadesi, Jessica Fiori, Sara Bosi, Valeria Bregola, Ilaria Marotti, Giovanni Dinelli
Phenolic compounds have received great attention among the health promoting phytochemicals in common wheat (Triticum aestivum L.), mainly because of their strong antioxidant properties. In the present study a simple Capillary Zone Electrophoresis (CZE) method with UV detection was optimized and validated for the quantitation of six of the most important phenolic acids in whole grain i.e., sinapic, ferulic, syringic, p-coumaric, vanillic and p-hydroxybenzoic acid. The separation was achieved in a running buffer composed of sodium phosphate solution (50 mM) in water/methanol 80:20 (v/v) at pH 6.0 and using a fused-silica capillary at the temperature of 30 °C under application of 27 kV. By means of diode array detector, and made possible by the favorable characteristic UV spectra, the quantitation of the solutes was carried out at 200, 220 and 300 nm, in the complex matrices represented by the soluble and bound fractions of wheat flours. The validation parameters of the method i.e., linearity, sensitivity, precision, accuracy and robustness were in line with those obtained by consolidated separation techniques applied for the same purposes (e.g., HPLC-UV), with a significant advantage in term of analysis time (less than 12 min). Ten varieties of soft wheat (five modern Italian and five old Italian genotypes) were analysed and the data were subjected to Principal Components Analysis (PCA). Interestingly, significant differences of the quantitative phenolic acids profile were observed between the modern and the ancient genotypes, with the latter showing higher amount of the main represented phenolic acids.

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532

Author(s): Roberto Gotti, Elisa Amadesi, Jessica Fiori, Sara Bosi, Valeria Bregola, Ilaria Marotti, Giovanni Dinelli

Phenolic compounds have received great attention among the health promoting phytochemicals in common wheat (Triticum aestivum L.), mainly because of their strong antioxidant properties. In the present study a simple Capillary Zone Electrophoresis (CZE) method with UV detection was optimized and validated for the quantitation of six of the most important phenolic acids in whole grain i.e., sinapic, ferulic, syringic, p-coumaric, vanillic and p-hydroxybenzoic acid. The separation was achieved in a running buffer composed of sodium phosphate solution (50 mM) in water/methanol 80:20 (v/v) at pH 6.0 and using a fused-silica capillary at the temperature of 30 °C under application of 27 kV. By means of diode array detector, and made possible by the favorable characteristic UV spectra, the quantitation of the solutes was carried out at 200, 220 and 300 nm, in the complex matrices represented by the soluble and bound fractions of wheat flours. The validation parameters of the method i.e., linearity, sensitivity, precision, accuracy and robustness were in line with those obtained by consolidated separation techniques applied for the same purposes (e.g., HPLC-UV), with a significant advantage in term of analysis time (less than 12 min). Ten varieties of soft wheat (five modern Italian and five old Italian genotypes) were analysed and the data were subjected to Principal Components Analysis (PCA). Interestingly, significant differences of the quantitative phenolic acids profile were observed between the modern and the ancient genotypes, with the latter showing higher amount of the main represented phenolic acids.

## Broad range chemical profiling of natural deep eutectic solvent extracts using a high performance thin layer chromatography–based method

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532
Author(s): Xiaojie Liu, Samantha Ahlgren, Henrie A.A.J. Korthout, Luis F. Salomé-Abarca, Lina M. Bayona, Robert Verpoorte, Young Hae Choi

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532

Author(s): Xiaojie Liu, Samantha Ahlgren, Henrie A.A.J. Korthout, Luis F. Salomé-Abarca, Lina M. Bayona, Robert Verpoorte, Young Hae Choi

## Detection of estrogen active compounds in hops by planar yeast estrogen screen

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532
Author(s): Dinah Schick, Wolfgang Schwack
Hops used in the brewing process of beer for flavoring are known to contain estrogen active compounds (EAC) and to be the source of EAC in beer. The recently developed planar yeast estrogen screen (pYES) with the substrate resorufin-β-d-galactopyranoside (RGP) successfully was applied for the detection of EAC in ethanolic extracts of hops pellet samples. The only pYES positive compound was identified as the hop flavanone prenylnaringenin (PN) by thin-layer chromatography–mass spectrometry. The heat-induced formation of estrogen active PN from the inactive hop flavonoid desmethylxanthohumol was confirmed by simulation of wort boiling, extraction of both the hops’ remainder and the supernatant water, and subsequent investigation of the extracts by pYES. By means of the dose-response curve of PN of a hops’ remainder extract, the estradiol equivalent concentration (EEQ) and thus the estradiol equivalent amount (EEA) of PN in the hops’ remainder after simulation of the wort boiling was determined to 39 μg/L and 52 μg/kg, respectively.

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532

Author(s): Dinah Schick, Wolfgang Schwack

Hops used in the brewing process of beer for flavoring are known to contain estrogen active compounds (EAC) and to be the source of EAC in beer. The recently developed planar yeast estrogen screen (pYES) with the substrate resorufin-β-d-galactopyranoside (RGP) successfully was applied for the detection of EAC in ethanolic extracts of hops pellet samples. The only pYES positive compound was identified as the hop flavanone prenylnaringenin (PN) by thin-layer chromatography–mass spectrometry. The heat-induced formation of estrogen active PN from the inactive hop flavonoid desmethylxanthohumol was confirmed by simulation of wort boiling, extraction of both the hops’ remainder and the supernatant water, and subsequent investigation of the extracts by pYES. By means of the dose-response curve of PN of a hops’ remainder extract, the estradiol equivalent concentration (EEQ) and thus the estradiol equivalent amount (EEA) of PN in the hops’ remainder after simulation of the wort boiling was determined to 39 μg/L and 52 μg/kg, respectively.

## A study on the onset of turbulent conditions with supercritical fluid chromatography mobile-phases

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532
Author(s): Abhijit Tarafder
Following a recent publication [1], the topic of turbulent flow in SFC has generated both interest and questions. Liquid-like density, coupled with significantly low viscosity of CO2-based mobile-phases may result in high Reynolds number (Re) – higher than what represents laminar flow conditions, reaching the so-called turbulent regions. Although such turbulent flows can form only in the connecting tubings, thus not directly affecting the chromatographic process, it is important to know under many situations, whether the flow inside the tubing is laminar or turbulent.In this report a comprehensive guideline to identify the possibilities of turbulent flow conditions is provided through a series of charts. Flow properties depend on state conditions (composition, pressure and temperature) and also on the tubing material and geometry. Here guidelines to detect the onset of turbulent conditions is provided for cylindrical stainless-steel tubings of different internal diameters (i.d.) under a wide range of SFC mobile-phase conditions.

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532

Author(s): Abhijit Tarafder

Following a recent publication [1], the topic of turbulent flow in SFC has generated both interest and questions. Liquid-like density, coupled with significantly low viscosity of CO2-based mobile-phases may result in high Reynolds number (Re) – higher than what represents laminar flow conditions, reaching the so-called turbulent regions. Although such turbulent flows can form only in the connecting tubings, thus not directly affecting the chromatographic process, it is important to know under many situations, whether the flow inside the tubing is laminar or turbulent. In this report a comprehensive guideline to identify the possibilities of turbulent flow conditions is provided through a series of charts. Flow properties depend on state conditions (composition, pressure and temperature) and also on the tubing material and geometry. Here guidelines to detect the onset of turbulent conditions is provided for cylindrical stainless-steel tubings of different internal diameters (i.d.) under a wide range of SFC mobile-phase conditions.

## Characterization of charged polymer self-assemblies by multidetector thermal field-flow fractionation in aqueous mobile phases

Publication date: 12 January 2018 Source:Journal of Chromatography A, Volume 1532 Author(s): Guilaume Greyling, Harald Pasch Charged block copolymer self-assemblies, such as charged micelles, have attracted much attention as versat…

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532

Author(s): Guilaume Greyling, Harald Pasch

Charged block copolymer self-assemblies, such as charged micelles, have attracted much attention as versatile drug delivery systems due to their readily tunable characteristics such as size and surface charge. However, current column-based analytical techniques are not suitable to fractionate and comprehensively characterize charged micelles in terms of size, molar mass, chemical composition and morphology. Multidetector thermal field-flow fractionation (ThFFF) is shown to be a unique characterization platform that can be used to characterize charged micelles in terms of size, molar mass, chemical composition and morphology in aqueous mobile phases with various ionic strengths and pH. This is demonstrated by the characterization of poly(methacrylic acid)-b-poly(methyl methacrylate) self-assemblies in high pH buffers as well as the characterization of cationic poly(2-vinyl pyridine)-b-polystyrene and poly(4-vinyl pyridine)-b-polystyrene self-assemblies in low pH buffers. Moreover, it is shown that ThFFF is capable of separating charged micelles according to the corona composition. These investigations prove convincingly that ThFFF is broadly applicable to the comprehensive characterization of amphiphilic self-assemblies even when aqueous mobile phases are used.

## Aqueous size-exclusion chromatography of polyelectrolytes on reversed-phase and hydrophilic interaction chromatography columns

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532
Author(s): Anna M. Caltabiano, Joe P. Foley, André M. Striegel
The size-exclusion separation of a water-soluble polyelectrolyte polymer, sodium polystyrene sulfonate (NaPSS), was demonstrated on common reversed-phase (C18, C4, phenyl, and cyano) and hydrophilic interaction chromatography (HILIC) columns. The effect of common solvents – acetonitrile (ACN), tetrahydrofuran (THF), and methanol (MeOH), used as mobile phase modifiers – on the elution of NaPSS and the effect of column temperature (within a relatively narrow range corresponding to typical chromatographic conditions, i.e., 10 °C–60 °C) on the partition coefficient, KSEC, were also investigated. Non-size-exclusion chromatography (non-SEC) effects can be minimized by the addition of an electrolyte and an organic modifier to the mobile phase, and by increasing the column temperature (e.g., to 50 °C or 60 °C). Strong solvents such as THF and ACN are more successful in the reduction of such effects than is the weaker solvent MeOH. The best performance is seen on medium polarity and polar stationary phases, such as cyanopropyl- and diol-modified silica (HILIC), where the elution of the NaPSS polyelectrolyte is by a near-ideal SEC mechanism. Hydrophobic stationary phases, such as C18, C4, and phenyl, require a higher concentration of a strong solvent modifier (THF) in the mobile phase to reduce non-SEC interactions of the solute with the stationary phase.

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532

Author(s): Anna M. Caltabiano, Joe P. Foley, André M. Striegel

The size-exclusion separation of a water-soluble polyelectrolyte polymer, sodium polystyrene sulfonate (NaPSS), was demonstrated on common reversed-phase (C18, C4, phenyl, and cyano) and hydrophilic interaction chromatography (HILIC) columns. The effect of common solvents – acetonitrile (ACN), tetrahydrofuran (THF), and methanol (MeOH), used as mobile phase modifiers – on the elution of NaPSS and the effect of column temperature (within a relatively narrow range corresponding to typical chromatographic conditions, i.e., 10 °C–60 °C) on the partition coefficient, $K S E C$, were also investigated. Non-size-exclusion chromatography (non-SEC) effects can be minimized by the addition of an electrolyte and an organic modifier to the mobile phase, and by increasing the column temperature (e.g., to 50 °C or 60 °C). Strong solvents such as THF and ACN are more successful in the reduction of such effects than is the weaker solvent MeOH. The best performance is seen on medium polarity and polar stationary phases, such as cyanopropyl- and diol-modified silica (HILIC), where the elution of the NaPSS polyelectrolyte is by a near-ideal SEC mechanism. Hydrophobic stationary phases, such as C18, C4, and phenyl, require a higher concentration of a strong solvent modifier (THF) in the mobile phase to reduce non-SEC interactions of the solute with the stationary phase.

## Modelling ordered packed beds of spheres: The importance of bed orientation and the influence of tortuosity on dispersion

Publication date: 12 January 2018 Source:Journal of Chromatography A, Volume 1532 Author(s): Fabian Dolamore, Conan Fee, Simone Dimartino Ordered packing has previously been considered for porous media applications in the industr…

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532

Author(s): Fabian Dolamore, Conan Fee, Simone Dimartino

Ordered packing has previously been considered for porous media applications in the industrial and analytical worlds, with implementation constrained only by the lack of feasible fabrication methods. Additive manufacturing now provides the answer to this limitation, which leads to the novel domain of customized ordered packing and a variety of optimized geometries. In this work, the chromatographic behaviour of ordered configurations of particles was described using computational fluid dynamics methods based on the Lattice Boltzmann Model. The model was first validated by matching van Deemter trends for ordered and random packings shown in previous research. The influence of rotations of the ordered configurations was then considered, indicating that orientational changes with respect to the main flow axis can strongly affect minimum plate height. In particular, it is demonstrated that targeted rotations of ordered packings can reduce axial dispersion while improving transverse dispersion, thus improving chromatographic performance. This principle is clearly shown in a strong linear correlation between tortuosity and plate height, offering an additional parameter to enable a priori control of the performance of ordered packings. Furthermore, rotation of the packing does not change porosity or surface area and has a relatively small effect on permeability. Thus, highly permeable packings with poor dispersion can be improved in terms of chromatographic impedance by simple rotation of the packing orientation. This work further demonstrates the advantages of ordered packings over randomly packed beds, and introduces new perspectives on the development of chromatographic structures with improved performance.

## Simultaneous determination of borate, chloride and molybdate in pyrohydrolysis distillates of plant and soil samples by ion chromatography

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532
Author(s): Vivekchandra Guruprasad Mishra, Mrinal Kanti Das, Dipti Jayesh Shah, Subbiah Jeyakumar, Bhupendra Singh Tomar, Karanam Lakshminarayana Ramakumar
Determination of concentrations of micronutrients in plant samples is important in order to assess the growth and quality of plants. An ion chromatography (IC) method was developed for the simultaneous determination of B, Cl and Mo (micronutrients present in their anionic form in aqueous samples) using a gradient elution with d-mannitol and NaOH. Despite their different chemical nature, these elements could be separated from the matrix by employing pyrohydrolysis. IC was employed for their sequential determination from single aliquot injection into the IC column. It was observed that the optimised procedures developed earlier in our laboratory for the separation of B and halogens using d-Mannitol-NaOH or bicarbonate eluents could not be extended to B-Mo-Cl separation. The concentration levels of d-mannitol in the eluent required for separation of boron impeded the conductivity detection of Mo and the peak sensitivity was seen to be critically dependent on d-mannitol concentration in the eluent. In addition, d-mannitol in NaOH eluent altered the retention times of analytes (B, F, Cl, NO3, SO42−and MoO42−) which were not observed in the case of bicarbonate eluent. The current study deals with the investigation on the influence of d-mannitol on the molybdate as well as its role on the retention time. Formation of Mo-mannitol complex at different pH conditions and de-protonation of mannitol were correlated to the observed effects. Based on the observations, a gradient elution method was proposed for the simultaneous separation and determination of B, Cl and Mo in the pyrohydrolysis distillates of plant samples. The method was calibrated for B (0.05–1 mg/L), Cl (0.1–10 mg/L) and Mo (0.5–10 mg/L) and the linear regression coefficients obtained were 0.9992, 0.9998 and 0.997 respectively. The limit of detection (LOD) for B, Cl and Mo was calculated to be 19, 23 and 96 μg/L, respectively. The developed IC method after pyrohydrolytic separation of B, Cl and Mo was successfully applied for the analysis of real samples.

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532

Author(s): Vivekchandra Guruprasad Mishra, Mrinal Kanti Das, Dipti Jayesh Shah, Subbiah Jeyakumar, Bhupendra Singh Tomar, Karanam Lakshminarayana Ramakumar

Determination of concentrations of micronutrients in plant samples is important in order to assess the growth and quality of plants. An ion chromatography (IC) method was developed for the simultaneous determination of B, Cl and Mo (micronutrients present in their anionic form in aqueous samples) using a gradient elution with d-mannitol and NaOH. Despite their different chemical nature, these elements could be separated from the matrix by employing pyrohydrolysis. IC was employed for their sequential determination from single aliquot injection into the IC column. It was observed that the optimised procedures developed earlier in our laboratory for the separation of B and halogens using d-Mannitol-NaOH or bicarbonate eluents could not be extended to B-Mo-Cl separation. The concentration levels of d-mannitol in the eluent required for separation of boron impeded the conductivity detection of Mo and the peak sensitivity was seen to be critically dependent on d-mannitol concentration in the eluent. In addition, d-mannitol in NaOH eluent altered the retention times of analytes (B, F, Cl, NO3 , SO4 2−and MoO4 2−) which were not observed in the case of bicarbonate eluent. The current study deals with the investigation on the influence of d-mannitol on the molybdate as well as its role on the retention time. Formation of Mo-mannitol complex at different pH conditions and de-protonation of mannitol were correlated to the observed effects. Based on the observations, a gradient elution method was proposed for the simultaneous separation and determination of B, Cl and Mo in the pyrohydrolysis distillates of plant samples. The method was calibrated for B (0.05–1 mg/L), Cl (0.1–10 mg/L) and Mo (0.5–10 mg/L) and the linear regression coefficients obtained were 0.9992, 0.9998 and 0.997 respectively. The limit of detection (LOD) for B, Cl and Mo was calculated to be 19, 23 and 96 μg/L, respectively. The developed IC method after pyrohydrolytic separation of B, Cl and Mo was successfully applied for the analysis of real samples.

## Improved separation by at-column dilution in preparative hydrophilic interaction chromatography

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532
Author(s): Geoffrey Jaffuel, Laura Chappuis, Davy Guillarme, Ted C.J. Turlings, Gaétan Glauser
We evaluated at-column dilution (ACD) as a possible approach to reduce peak distortion caused by the injection of strong solvents in preparative hydrophilic interaction chromatography (HILIC). Three model compounds, namely uridine, cytidine and guanosine were selected and injected either in conventional or ACD conditions, using various proportions of water and acetonitrile in the sample diluent. Plate number and peak capacity were systematically investigated under both isocratic and gradient elution conditions, respectively. In isocratic conditions, ACD was found to provide higher plate numbers with sample diluent containing more than 40% water, but lower plate numbers with injections of less than 40% water, in comparison to a conventional injection system at the preparative scale. These contrasting results were attributed to the fact that i) efficiency was fundamentally reduced in ACD, due to the low make-up flow rate that was used to flush the injection loop, but ii) the trend was reversed for highly aqueous injection thanks to the capacity of ACD to maintain similar efficiency regardless of the injection solvent. On the other hand, peak capacity was comparable between ACD and conventional systems in gradient elution for injections containing low amounts of water. However, ACD became increasingly advantageous as the proportion of water in the injection solvent and/or the injection volume increased. This was confirmed by the separation of a plant root aqueous extract. Altogether, our findings suggest that ACD is beneficial both in isocratic and gradient modes when strongly aqueous solutions are injected, offering an attractive approach to increase loadability, while preserving peak shapes in preparative HILIC.

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532

Author(s): Geoffrey Jaffuel, Laura Chappuis, Davy Guillarme, Ted C.J. Turlings, Gaétan Glauser

We evaluated at-column dilution (ACD) as a possible approach to reduce peak distortion caused by the injection of strong solvents in preparative hydrophilic interaction chromatography (HILIC). Three model compounds, namely uridine, cytidine and guanosine were selected and injected either in conventional or ACD conditions, using various proportions of water and acetonitrile in the sample diluent. Plate number and peak capacity were systematically investigated under both isocratic and gradient elution conditions, respectively. In isocratic conditions, ACD was found to provide higher plate numbers with sample diluent containing more than 40% water, but lower plate numbers with injections of less than 40% water, in comparison to a conventional injection system at the preparative scale. These contrasting results were attributed to the fact that i) efficiency was fundamentally reduced in ACD, due to the low make-up flow rate that was used to flush the injection loop, but ii) the trend was reversed for highly aqueous injection thanks to the capacity of ACD to maintain similar efficiency regardless of the injection solvent. On the other hand, peak capacity was comparable between ACD and conventional systems in gradient elution for injections containing low amounts of water. However, ACD became increasingly advantageous as the proportion of water in the injection solvent and/or the injection volume increased. This was confirmed by the separation of a plant root aqueous extract. Altogether, our findings suggest that ACD is beneficial both in isocratic and gradient modes when strongly aqueous solutions are injected, offering an attractive approach to increase loadability, while preserving peak shapes in preparative HILIC.

## Co-elution phenomena in polymer mixtures studied by asymmetric flow field-flow fractionation

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532
Author(s): Claudia Zielke, Catalina Fuentes, Lennart Piculell, Lars Nilsson
Most polymers generally have complex characteristics. Analysis and understanding of these characteristics is crucial as they, for instance, influence functionality. Separation and analysis of samples of polymers, biopolymers in particular, is challenging since they often display broad distributions in size, structure and molar mass (M) and/or a tendency to form aggregates. Only few analytical techniques are suitable for the task. AF4-MALS-dRI is highly suited for the task, but the analysis can nevertheless be especially challenging for heterogeneous mixtures of polymers that exhibit wide size distributions or aggregation. For such systems, systematic and thorough method development is clearly a requirement. This is the purpose of the present work, where we approach the problem of heterogeneous polymer samples systematically by analyzing mixtures of two different polymers which are also characterized individually.An often observed phenomenon in AF4 of samples with a high polydispersity is a downturn in M vs. elution time, especially common at high retention. This result is often dismissed as an artifact attributed to various errors in detection and data processing.In this work, we utilize AF4-MALS-dRI to separate and analyze binary mixtures of the well-known polysaccharides pullulan and glycogen, or pullulan and poly(ethylene oxide), respectively, in solution. The results show that an observed downturn – or even an upturn – in M can be a correct result, caused by inherent properties of the analyzed polymers.

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532

Author(s): Claudia Zielke, Catalina Fuentes, Lennart Piculell, Lars Nilsson

Most polymers generally have complex characteristics. Analysis and understanding of these characteristics is crucial as they, for instance, influence functionality. Separation and analysis of samples of polymers, biopolymers in particular, is challenging since they often display broad distributions in size, structure and molar mass (M) and/or a tendency to form aggregates. Only few analytical techniques are suitable for the task. AF4-MALS-dRI is highly suited for the task, but the analysis can nevertheless be especially challenging for heterogeneous mixtures of polymers that exhibit wide size distributions or aggregation. For such systems, systematic and thorough method development is clearly a requirement. This is the purpose of the present work, where we approach the problem of heterogeneous polymer samples systematically by analyzing mixtures of two different polymers which are also characterized individually. An often observed phenomenon in AF4 of samples with a high polydispersity is a downturn in M vs. elution time, especially common at high retention. This result is often dismissed as an artifact attributed to various errors in detection and data processing. In this work, we utilize AF4-MALS-dRI to separate and analyze binary mixtures of the well-known polysaccharides pullulan and glycogen, or pullulan and poly(ethylene oxide), respectively, in solution. The results show that an observed downturn – or even an upturn – in M can be a correct result, caused by inherent properties of the analyzed polymers.

## Methodologies to determine b-term coefficients revisited

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532
Author(s): Huiying Song, Donatela Sadriaj, Gert Desmet, Deirdre Cabooter
The accuracy of the longitudinal diffusion term (b-term) plays a vital role in the study of mass transfer mechanisms in high performance liquid chromatography (HPLC). In this study, three commonly used methodologies (peak parking; fitting of an experimental van Deemter curve; and the so-called dynamic method) for the determination of the b-term constant were investigated in detail. The three methods were compared based on their mutual agreement, the intra- and inter-day variation of the obtained values and the time required to measure them. Whereas the dynamic method was found to be plagued by impractically long waiting times and concomitant baseline variations compromising accurate measurements of the band broadening, the two other methods lead to very similar b-values, i.e., well within the 1% RSD inter-day variation typically marking both methods in the present study.The best way to study the agreement of the peak parking and plate height fitting method is in a plot of h.ν versus ν, providing a much better zoom on the b-term region of the van Deemter curve than the customarily employed h versus ν-curve and hence allowing to identify any anomalous measurement values (usually related to measurements with a long experimentation time). Verifying the mutual agreement between both methods is proposed here as an additional accuracy check of the obtained data.

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532

Author(s): Huiying Song, Donatela Sadriaj, Gert Desmet, Deirdre Cabooter

The accuracy of the longitudinal diffusion term (b-term) plays a vital role in the study of mass transfer mechanisms in high performance liquid chromatography (HPLC). In this study, three commonly used methodologies (peak parking; fitting of an experimental van Deemter curve; and the so-called dynamic method) for the determination of the b-term constant were investigated in detail. The three methods were compared based on their mutual agreement, the intra- and inter-day variation of the obtained values and the time required to measure them. Whereas the dynamic method was found to be plagued by impractically long waiting times and concomitant baseline variations compromising accurate measurements of the band broadening, the two other methods lead to very similar b-values, i.e., well within the 1% RSD inter-day variation typically marking both methods in the present study. The best way to study the agreement of the peak parking and plate height fitting method is in a plot of h.ν versus ν, providing a much better zoom on the b-term region of the van Deemter curve than the customarily employed h versus ν-curve and hence allowing to identify any anomalous measurement values (usually related to measurements with a long experimentation time). Verifying the mutual agreement between both methods is proposed here as an additional accuracy check of the obtained data.

## A capillary zone electrophoresis method for detection of Apolipoprotein C-III glycoforms and other related artifactually modified species

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532
Author(s): Coralie Ruel, Marco Morani, Arnaud Bruneel, Christophe Junot, Myriam Taverna, François Fenaille, Nguyet Thuy Tran
ApolipoproteinC-III (ApoC-III) is a human plasma glycoprotein whose O-glycosylation can be altered as a result of congenital disorders of glycosylation (CDG). ApoC-III exhibits three major glycoforms whose relative quantification is of utmost importance for the diagnosis of CDG patients. Considering the very close structures of these glycoforms and their tendency to adsorb on the capillary, a thorough optimization of capillary electrophoresis (CE) parameters including preconditioning and in-between rinsing procedures was required to efficiently separate all the ApoC-III glycoforms. Permanent coatings did not contribute to high resolution separations. A fast and reliable method based on a bare-silica capillary combining the effect of urea and diamine additives allowed to separate up to six different ApoC-III forms. We demonstrated by a combination of MALDI-TOF mass spectrometry (MS) analyses and CE of intact and neuraminidase-treated samples that this method well resolved glycoforms differing not only by their sialylation degree but also by carbamylation state, an undesired chemical modification of primary amines. This method allowed to demonstrate the carbamylation of ApoC-III glycoforms for the first time. Our CZE method proved robust and accurate with excellent intermediate precision regarding migration times (RSDs &lt; 0.7%) while RSDs for peak areas were less than 5%. Finally, the quality of three distinct batches of commercial ApoC-III obtained from different suppliers was assessed and compared. Quite similar but highly structurally heterogeneous ApoC-III profiles were observed for these samples.

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532

Author(s): Coralie Ruel, Marco Morani, Arnaud Bruneel, Christophe Junot, Myriam Taverna, François Fenaille, Nguyet Thuy Tran

ApolipoproteinC-III (ApoC-III) is a human plasma glycoprotein whose O-glycosylation can be altered as a result of congenital disorders of glycosylation (CDG). ApoC-III exhibits three major glycoforms whose relative quantification is of utmost importance for the diagnosis of CDG patients. Considering the very close structures of these glycoforms and their tendency to adsorb on the capillary, a thorough optimization of capillary electrophoresis (CE) parameters including preconditioning and in-between rinsing procedures was required to efficiently separate all the ApoC-III glycoforms. Permanent coatings did not contribute to high resolution separations. A fast and reliable method based on a bare-silica capillary combining the effect of urea and diamine additives allowed to separate up to six different ApoC-III forms. We demonstrated by a combination of MALDI-TOF mass spectrometry (MS) analyses and CE of intact and neuraminidase-treated samples that this method well resolved glycoforms differing not only by their sialylation degree but also by carbamylation state, an undesired chemical modification of primary amines. This method allowed to demonstrate the carbamylation of ApoC-III glycoforms for the first time. Our CZE method proved robust and accurate with excellent intermediate precision regarding migration times (RSDs &lt; 0.7%) while RSDs for peak areas were less than 5%. Finally, the quality of three distinct batches of commercial ApoC-III obtained from different suppliers was assessed and compared. Quite similar but highly structurally heterogeneous ApoC-III profiles were observed for these samples.

## Performance comparison of chiral separation materials derived from N-cyclohexylcarbonyl and N-hexanoyl chitosans

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532
Author(s): Sheng Tang, Jian-De Liu, Wei Chen, Shao-Hua Huang, Juan Zhang, Zheng-Wu Bai
Chitosan bis(phenylcarbamate)-(N-cyclohexylformamide)s and chitosan bis(phenylcarbamate)-(N-hexanamide)s were synthesized as chiral selectors for enantiomeric separation. Since two types of substituents with different structures were, respectively, introduced onto the 2-position and the 3-/6-positions of the glucose skeleton in the chitosans through a “heterogeneous” modification pathway, the enantioseparation performances of the chiral selectors could be improved. Influence of the type and position of the substituents on chiral recognition and enantioseparation abilities was studied in detail, and the structural dependence on enantioseparation performance was particularly demonstrated. It was found that methyl- and chloro-substituted chitosan bis(phenylcarbamate)-(N-hexanamide)s possessed comparable enantioseparation performances, whereas chloro-substituted chitosan bis(phenylcarbamate)-(N-cyclohexylformamide)s exhibited much more powerful chiral recognition and enantioseparation abilities than the methyl-substituted ones. Among all the prepared chiral selectors, those with the combination of the cyclohexyl group at the 2-position of the glucose skeleton in the chitosan derivatives and the chlorophenyl group at the 3-/6-positions seemed to be more preferable for enantiomeric separation. As a result, the chitosan bis(3,4-dichlorophenylcarbamate)-(N-cyclohexylformamide) possessed the best enantioseparation performance. The solvent tolerability of the prepared chiral selectors was also investigated in the present study. Compared with the classical coated-type chiral separation materials derived from cellulose/amylose derivatives, the N-cyclohexylcarbonyl and N-hexanoyl chitosans based chiral stationary phases were observed to possess more favorable solvent tolerability, thus possibly widening their applications for various practical enantioseparations.

Publication date: 12 January 2018
Source:Journal of Chromatography A, Volume 1532

Author(s): Sheng Tang, Jian-De Liu, Wei Chen, Shao-Hua Huang, Juan Zhang, Zheng-Wu Bai

Chitosan bis(phenylcarbamate)-(N-cyclohexylformamide)s and chitosan bis(phenylcarbamate)-(N-hexanamide)s were synthesized as chiral selectors for enantiomeric separation. Since two types of substituents with different structures were, respectively, introduced onto the 2-position and the 3-/6-positions of the glucose skeleton in the chitosans through a “heterogeneous” modification pathway, the enantioseparation performances of the chiral selectors could be improved. Influence of the type and position of the substituents on chiral recognition and enantioseparation abilities was studied in detail, and the structural dependence on enantioseparation performance was particularly demonstrated. It was found that methyl- and chloro-substituted chitosan bis(phenylcarbamate)-(N-hexanamide)s possessed comparable enantioseparation performances, whereas chloro-substituted chitosan bis(phenylcarbamate)-(N-cyclohexylformamide)s exhibited much more powerful chiral recognition and enantioseparation abilities than the methyl-substituted ones. Among all the prepared chiral selectors, those with the combination of the cyclohexyl group at the 2-position of the glucose skeleton in the chitosan derivatives and the chlorophenyl group at the 3-/6-positions seemed to be more preferable for enantiomeric separation. As a result, the chitosan bis(3,4-dichlorophenylcarbamate)-(N-cyclohexylformamide) possessed the best enantioseparation performance. The solvent tolerability of the prepared chiral selectors was also investigated in the present study. Compared with the classical coated-type chiral separation materials derived from cellulose/amylose derivatives, the N-cyclohexylcarbonyl and N-hexanoyl chitosans based chiral stationary phases were observed to possess more favorable solvent tolerability, thus possibly widening their applications for various practical enantioseparations.