Analytica Chimica Acta (v.963, #C)

Combining ANOVA-PCA with POCHEMON to analyse micro-organism development in a polymicrobial environment by Brigitte P. Geurts; Anne H. Neerincx; Samuel Bertrand; Manja A.A.P. Leemans; Geert J. Postma; Jean-Luc Wolfender; Simona M. Cristescu; Lutgarde M.C. Buydens; Jeroen J. Jansen (1-16).
Revealing the biochemistry associated to micro-organismal interspecies interactions is highly relevant for many purposes. Each pathogen has a characteristic metabolic fingerprint that allows identification based on their unique multivariate biochemistry. When pathogen species come into mutual contact, their co-culture will display a chemistry that may be attributed both to mixing of the characteristic chemistries of the mono-cultures and to competition between the pathogens. Therefore, investigating pathogen development in a polymicrobial environment requires dedicated chemometric methods to untangle and focus upon these sources of variation. The multivariate data analysis method Projected Orthogonalised Chemical Encounter Monitoring (POCHEMON) is dedicated to highlight metabolites characteristic for the interaction of two micro-organisms in co-culture. However, this approach is currently limited to a single time-point, while development of polymicrobial interactions may be highly dynamic. A well-known multivariate implementation of Analysis of Variance (ANOVA) uses Principal Component Analysis (ANOVA-PCA). This allows the overall dynamics to be separated from the pathogen-specific chemistry to analyse the contributions of both aspects separately. For this reason, we propose to integrate ANOVA-PCA with the POCHEMON approach to disentangle the pathogen dynamics and the specific biochemistry in interspecies interactions. Two complementary case studies show great potential for both liquid and gas chromatography - mass spectrometry to reveal novel information on chemistry specific to interspecies interaction during pathogen development.Display Omitted
Keywords: ANOVA-PCA; POCHEMON; Micro-organism; Co-culture; Interspecies interaction;

In this work, biomimetic polydopamine nanospheres (PDANSs) were easily prepared and were firstly exploited as an electrochemiluminescence (ECL) nanoquencher for the development of a sandwich type immunosensor. The PDANSs with abundant active functional groups can facilely label the detection antibody and show remarkable quenching effect towards the ECL of the tris-(2,2’-bipyridine)ruthenium (Ru(bpy)3 2+). The amino-modified multiwall carbon nanotubes/Nafion (MWCNTs-NH2@N) composite-film was adopted as a matrix to incorporate the luminophor Ru(bpy)3 2+ and immobilize the capture antibody. The prominent decrease of ECL signal intensity was obtained on account of the unique quenching ability of the labeled PDANSs. The quenching mechanism is believed that the excited states of Ru(bpy)3 2+ can be annihilated by quinone units in PDANSs via energy transfer. The ECL quenching efficiency was logarithmically related to the concentration of the prostate specific antigen (PSA) in the range from 0.1 pg mL−1 to 20 ng mL−1 with a detection limit of 35 fg mL−1. Furthermore, the proposed ECL immunosensor presented good stability, repeatability and selectivity.Display Omitted
Keywords: Electrochemiluminescence; Ru(bpy)3 2+; Polydopamine nanospheres; Quenching;

An ionic liquid-(IL) based micellar extraction combined with microcrystalline cellulose- (MCC) assisted dispersive micro solid-phase extraction method was developed to extract phenolic compounds from propolis. A total of 20 target compounds were identified by ultra-high- performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. The main extraction parameters were optimized and included the ultrasonic power, ultrasonic time, sample pH, type of IL, the concentration of [C12mim]Br, extraction time, concentration of MCC, type of sorbent and type of elution solvents. Under the optimum conditions, the proposed method exhibited good linearities (r2 ≥ 0.999) for all plant phenolic compounds with the lower limits of detection in the range of 0.21–0.41 ng/mL. The recoveries ranged from 82.74% to 97.88% for pinocembrin, chrysin and galangin. Compared with conventional solvent extraction, the present method was simpler and more efficient and required less organic solvent and a shorter extraction time. Finally, the methodology was successfully used for the extraction and enrichment of phenolic compounds in propolis.Display Omitted
Keywords: Dispersive micro solid-phase extraction; Ionic liquid; Microcrystalline cellulose; Propolis; Quadrupole time-of-flight tandem mass spectrometry; Ultra-high- performance liquid chromatography;

Papain-functionalized gold nanoparticles as heterogeneous biocatalyst for bioanalysis and biopharmaceuticals analysis by Siyao Liu; Markus Höldrich; Adrian Sievers-Engler; Jeannie Horak; Michael Lämmerhofer (33-43).
Surface-modified gold nanoparticles (GNPs) were synthesized via layer-by-layer process with alternating cationic polyallylamine and anionic poly(acrylic acid) polyelectrolyte layers leading to a highly hydrophilic biocompatible shell supporting colloidal stability. Afterwards, papain was covalently immobilized on the modified GNPs via amide coupling between the amino groups on papain and the terminal carboxylic groups of the modified GNPs by using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysulfosuccinimide sodium as coupling agents. The resultant papain-functionalized gold nanoparticles were characterized by surface plasmon resonance, dynamic light scattering and zeta potential measurements. The new technology resonant mass measurement was applied for determining the average number of papain molecules immobilized per GNP by measurement of the single nanoparticle buoyant mass in the range of femtograms. The activity of the immobilized enzyme was estimated by determination of the kinetic parameters (K m , V max and k cat ) with the standard chromogenic substrate N α -benzoyl-dl-arginine-4-nitroanilide hydrochloride. It was found that K m of immobilized and free enzyme are in the same order of magnitude. On contrary, turnover numbers k cat were significantly higher for GNP-conjugated papain. Further, the gold nanobiocatalyst was applied for digestion of polyclonal human immunoglobulin G to yield protein fragments. The resultant fragment mixture was further analyzed by high-performance liquid chromatography-microelectrospray ionization-quadrupole-time-of-flight mass spectrometry, which demonstrated the applicability of the bioreactor based on papain functionalized GNPs. The immobilized papain not only has higher catalytic activity and better stability, but also can be easily isolated from the reaction medium by straightforward centrifugation steps for reuse.Display Omitted
Keywords: Immobilized papain; Gold nanoparticle; Resonant mass measurement; Sample preparation; Biopharmaceuticals (biologicals) analytics; Immobilized enzyme reactor;

Selective solid phase extraction of lanthanides from tap and river waters with ion imprinted polymers by Manel Moussa; Massamba Mbacké Ndiaye; Thomas Pinta; Valérie Pichon; Thomas Vercouter; Nathalie Delaunay (44-52).
For the first time, an ion imprinted polymer (IIP) able to selectively extract simultaneously all the lanthanide ions was successfully synthesized in acetonitrile using Nd3+ as a template ion, methacrylic acid as a complexing monomer, and ethylene glycol dimethacrylate as a cross-linker. A non-imprinted polymer (NIP) was synthesized under the same conditions as those of the IIP, but in the absence of the template ion. After the removal of the template ions, grounding and sieving, the IIP particles were packed in solid phase extraction (SPE) cartridges. The selectivity of the IIP was evaluated by comparing its behavior with the one of the NIP. Each SPE step (percolation, washing, and elution) was optimized in order to find the best compromise between the selectivity and the extraction recoveries. Using the optimized SPE conditions, the extraction recoveries of eight lanthanide ions representative of the lanthanide family were higher than 77% with an average value of 83% with the IIP, whereas, in the case of the NIP, they ranged between 14 and 36% and they were below 3% for the interfering ions from alkali, transition, and post-transition metal families with the IIP. A first evaluation of the reproducibility of the SPE profiles was carried out by performing statistical tests on the data obtained with several cartridges filled with particles obtained from two different IIP and NIP syntheses. Promising results were obtained. The specific capacity, i. e. the adsorption capacity of Nd3+ ions by the specific cavities of the imprinted polymer, was about 9 mg of Nd3+ per gram of IIP (60 μmol g−1), which is more than enough for the extraction of the lanthanide ions at trace levels. The breakthrough volume was about 1 mL per mg of IIP, leading to an enrichment factor of 15, which allows not only to selectively extract the lanthanides but also to concentrate them. Finally, the imprinted polymer was successfully used to selectively extract lanthanides from tap and river waters spiked at 1 μg L−1.Display Omitted
Keywords: Ion imprinted polymer; Lanthanides; Solid-phase extraction (SPE); Tap water; River water;

An automatic on-line preconcentration and detection system for analysis of trace dissolved manganese (Mn) in estuarine and coastal waters was established, using preconcentration with IDA chelating resin and detection with flame atomic fluorescence spectrometer (FAFS). The rinse (pre-eluent) solution was optimized, for removing the interference ions while retaining the target element. It was found that the interference ions affected the chelating efficiency of Mn, causing variation of the detection blank and sensitivity. This effect varied when sample volume presented as preconcentration time changed. The influence at preconcentration times of 120 s, 30 s and 10 s were carefully investigated and reported. Ten folds of the foreign trace metals Zn, Cu, Ni, Fe, and Al did not show obvious interference on Mn preconcentration and detection. The method detection limit was 0.9 nmol L-1 (n = 7, preconcentration time 120 s). The linear detection range could be adjusted with designed preconcentration time. In addition to high precision and accuracy, the proposed analytical system had the advantages of high integration, and required normal site preparation, low energy supply and simple auxiliary equipment, which was appropriate for in-field operation. Compared with other common in-field applied molecular spectrometry instruments, the inherent high selectivity and multi-element applicability of FAFS highlighted the superiority and potential of the proposed analytical system. It was successfully applied to in-field vehicle-board determination of dissolved Mn in coastal waters around Xiamen, Fujian, China, and it was also used to analyze natural water samples collected from the Jiulongjiang Estuary, Fujian, China.Display Omitted
Keywords: In-field; Trace dissolved manganese; IDA chelating resin; Flame atomic fluorescence spectrometer; Estuarine and coastal waters;

The Amplex Red/horseradish peroxidase (AR/HRP) assay for H2O2 is one of the most sensitive and simple approaches for H2O2 quantification, which is effected by measuring the highly-fluorescent resorufin formed from oxidation of AR by the oxidizing intermediates generated by reaction of HRP and H2O2. The direct reactions of S(−II) with both H2O2 and resorufin are too slow to be of relevance on analytical timescales, however, the reaction between S(−II) and the HRP/H2O2 oxidizing intermediates is rapid enough to compete with the desired reaction of these oxidizing intermediates with AR, suppressing formation of the resorufin analyte. As this mode of interference can be considered simply a competition between the AR reagent and S(−II) for the intermediate oxidizing species, a simple equation is derived in this work enabling one to correct for this interference and obtain a good estimate of the true H2O2 concentration after measuring the apparent H2O2 concentration and the S(−II) concentration. This mode of interference is general to any compound that can act as a HRP substrate even if not directly reactive with H2O2. As such, the approach described is widely applicable to many potential reducing interferents and opens up the use of the AR/HRP assay to a much wider range of conditions, as well as demonstrating the utility of explicitly considering the mechanism of the analytical process.Display Omitted
Keywords: Hydrogen peroxide; Amplex Red; Horseradish peroxidase; Sulfide;

Simultaneous determination of ethanol's four types of non-oxidative metabolites in human whole blood by liquid chromatography tandem mass spectrometry by Xinyu Zhang; Feng Zheng; Zebin Lin; Sys Stybe Johansen; Tianfang Yu; Yuming Liu; Zhibin Huang; Jiaolun Li; Jie Yan; Yulan Rao (68-75).
The importance of ethanol non-oxidative metabolites as the specific biomarkers of alcohol consumption in clinical and forensic settings is increasingly acknowledged. Simultaneous determination of these metabolites can provide a wealth of information like drinking habit and history, but it was difficult to achieve because of their wide range of polarity. This work describes development and validation of a simple liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for 4 types of ethanol non-oxidative metabolites (ethyl glucuronide, ethyl sulfate, fatty acid ethyl esters and phosphatidylethanols) in 50 μL of human whole blood. Pretreatment method, column and MS conditions were optimized. For the first time, the four types of ethanol non-oxidative metabolites with enormous discrepancies of property were simultaneously extracted and analyzed in one run within 40 min. The limits of detections (LODs) were among 0.1–10 ng/mL, and good linearity was obtained. Deviations in precision and accuracy were all lower than 15% at three QC levels. This method was then applied to two forensic samples, resulting in information on drinking habits and drinking time which were very useful for the interpretation of the blood alcohol results.Display Omitted
Keywords: Ethanol; Non-oxidative metabolites; Human whole blood; LC-MS/MS;

Systematic assessment of surfactants for matrix-assisted laser desorption/ionization mass spectrometry imaging by Bijay Banstola; Eulalie T. Grodner; Fan Cao; Fabrizio Donnarumma; Kermit K. Murray (76-82).
A systematic method for evaluation of MALDI profiling and imaging was developed and applied to the use of three surfactants, sodium dodecyl sulfate (SDS), Triton X-100, and Tween 20, on rat brain tissue. For profiling studies, mass spectra were acquired from regular arrays of spots with manually deposited surfactant and matrix. The studies recorded the total number of peaks in the mass spectra from 2 to 20 kDa and compared the number of peaks and peak intensities with and without surfactant. It was found that SDS decreases the total number of peaks at all concentrations but does lead to an increase in the number of peaks below 5 kDa. Triton X-100 at 0.05% concentration yielded the highest number of peaks and highest number of new peaks, with the best results above 5 kDa. Correlation of the increase in signal with the estimated hydrophobicity suggests that Triton X-100 improves mass spectrometry quality through an increase in the intensity of hydrophobic protein peaks. Tween 20 provided good performance at 0.05% concentration across all mass ranges. For imaging studies, multiple images were obtained and the integrated intensity ratio for images obtained with and without surfactant was compared for 10 selected peaks. It was found that SDS tends to degrade imaging performance whereas Triton X-100 and Tween 20 improved performance compared to no surfactant, especially above 7 kDa.Display Omitted
Keywords: Mass spectrometry; MALDI imaging; Surfactant;

Microfluidic immunosensor based on mesoporous silica platform and CMK-3/poly-acrylamide-co-methacrylate of dihydrolipoic acid modified gold electrode for cancer biomarker detection by Matías Regiart; Martin A. Fernández-Baldo; Jhonny Villarroel-Rocha; Germán A. Messina; Franco A. Bertolino; Karim Sapag; Aaron T. Timperman; Julio Raba (83-92).
We report a hybrid glass-poly (dimethylsiloxane) microfluidic immunosensor for epidermal growth factor receptor (EGFR) determination, based on the covalent immobilization of anti-EGFR antibody (anti-EGFR) on amino-functionalized mesoporous silica (AMS) retained in the central channel of a microfluidic device. The synthetized AMS was characterized by N2 adsorption-desorption isotherm, scanning electron microscopy (SEM), energy dispersive spectrometry (EDS) and infrared spectroscopy. The cancer biomarker was quantified in human serum samples by a direct sandwich immunoassay measuring through a horseradish peroxidase-conjugated anti-EGFR. The enzymatic product was detected at −100 mV by amperometry on a sputtering gold electrode, modified with an ordered mesoporous carbon (CMK-3) in a matrix of poly-acrylamide-co-methacrylate of dihydrolipoic acid (poly(AC-co-MDHLA)) through in situ copolymerization. CMK-3/poly(AC-co-MDHLA)/gold was characterized by cyclic voltammetry, EDS and SEM. The measured current was directly proportional to the level of EGFR in human serum samples. The linear range was from 0.01 ng mL−1 to 50 ng mL−1. The detection limit was 3.03 pg mL−1, and the within- and between-assay coefficients of variation were below 5.20%. The microfluidic immunosensor is a very promising device for the diagnosis of several kinds of epithelial origin carcinomas.Display Omitted
Keywords: EGFR; Cancer biomarker; Cancer diagnosis; Mesoporous silica; Microfluidic immunosensor; Nanostructured electrode;

Paper-polymer composite devices with minimal fluorescence background by Chang-Ming Wang; Chong-You Chen; Wei-Ssu Liao (93-98).
Polymer film incorporated paper-based devices show advantages in simplicity and rugged backing. However, their applications are restricted by the high fluorescence background interference of conventional laminating pouches. Herein, we report a straightforward approach for minimal fluorescence background device fabrication, in which filter paper was shaped and laminated in between two biaxially oriented polypropylene (OPP) and polyvinyl butyral (PVB) composite films. This composite film provides mechanical strength for enhanced device durability, protection from environmental contamination, and prevents reagent degradation. This approach was tested by the determination of copper ions with a fluorescent probe, while the detection of glucose was used to illustrate the improved device durability. Our results show that lamination by the polymer composite lengthens device lifetime, while allowing for fluorescence detection methods combination with greatly reduced fluorescent background widely present in commercially available lamination pouches. By the combination of rapid device prototyping with low cost materials, we believe that this composite design would further expand the potential of paper-based devices.Display Omitted
Keywords: Paper-polymer composite; Fluorescence; Sensing device; Microfluidics;

External cavity-quantum cascade laser (EC-QCL) spectroscopy for protein analysis in bovine milk by Julia Kuligowski; Andreas Schwaighofer; Mirta Raquel Alcaráz; Guillermo Quintás; Helmut Mayer; Máximo Vento; Bernhard Lendl (99-105).
The analytical determination of bovine milk proteins is important in food and non-food industrial applications and yet, rather labour-intensive wet-chemical, low-throughput methods have been employed since decades. This work proposes the use of external cavity-quantum cascade laser (EC-QCL) spectroscopy for the simultaneous quantification of the most abundant bovine milk proteins and the total protein content based on the chemical information contained in mid-infrared (IR) spectral features of the amide I band. Mid-IR spectra of protein standard mixtures were used for building partial least squares (PLS) regression models. Protein concentrations in commercial bovine milk samples were calculated after chemometric compensation of the matrix contribution employing science-based calibration (SBC) without sample pre-processing. The use of EC-QCL spectroscopy together with advanced multivariate data analysis allowed the determination of casein, α-lactalbumin, β-lactoglobulin and total protein content within several minutes.Display Omitted
Keywords: External cavity-quantum cascade laser (EC-QCL) spectroscopy; Mid-infrared (IR); Protein analysis; Milk analysis; Science-based calibration (SBC);

A new colorimetric immunoassay method was developed for the rapid and sensitive detection of a tumor biomarker of carcinoembryonic antigen (CEA) by combination of a magnetic bead (MB)-based sandwich immunoassay and a copper chromogenic reaction. The magnetic immunoassay platform was constructed through the covalent immobilization of the capture antibody on the surface of carboxylated magnetic beads. After immuno-recognition of CEA, signal antibody-functionalized copper oxide nanoparticle (CuO NP) probes were applied for sandwich immunoreaction to form an immunocomplex. The CuO NP labels quantitatively captured onto the immunocomplex were then dissolved in acid solution to release high-content copper ions. Based on the coordination of these ions with the newly synthesized chromogenic agent of 1,2-diphenyl-2-(2-(pyridin-2-yl)hydrazono)ethanone, a red complex was produced for the colorimetric signal readout, resulting in the successful construction of a sensitive immunoassay method for CEA detection. Under the optimum conditions, this method showed a wide linear range over three orders of magnitude and a low detection limit of 26 pg/mL. Besides, this method showed excellent performance with low cost, rapid and convenient operation as well as satisfactory reproducibility, stability and accuracy, thus providing great potentials for practical applications.Display Omitted
Keywords: Immunoassay; Tumor biomarkers; Colorimetric analysis; Magnetic beads; Copper oxide;

Highly sensitive colorimetric immunosensor for influenza virus H5N1 based on enzyme-encapsulated liposome by Cuiying Lin; Yajuan Guo; Mengmeng Zhao; Mi Sun; Fang Luo; Longhua Guo; Bin Qiu; Zhenyu Lin; Guonan Chen (112-118).
Development of simple but sensitive biosensor for influenza detection is highly important in immediate and effective clinical treatment. In this study, a sensitive colorimetric immunosensor which combines the advantages of high selectivity of immunoassay and simplicity of colorimetric detection has been developed to detect influenza virus H5N1 based on enzyme-encapsulated liposome. Biotin-tagged liposome encapsulated with large amount of horseradish peroxidase (HRP) was firstly synthesized. In the presence of H5N1, H5N1 co-bound with the capture antibody and the biotinylated detection antibody to form sandwich immunocomplex. Subsequently, the HRP-encapsulated liposome was introduced to conjugate with the detection antibody through biotin-avidin-biotin linkage. Upon the addition of substrate (mixture of 3,3′,5,5′-tetramethylbenzidine (TMB) and H2O2), the liposome was directly lysed to release large amount of HRP by TMB. The released HRP catalyzed the H2O2-mediated oxidation of TMB, resulting in color change of the system, which was observed by naked eyes or UV–vis spectra. The result showed that the absorption intensity enhanced with the increase of H5N1 concentration ranging from 0.1 to 4.0 ng/mL, and the detection limit was calculated to be 0.04 ng/mL. The sensitivity of the proposed biosensor is much higher than that of conventional enzyme-linked immunosorbent assay method. The proposed immunosensor is relatively simple, low-cost, sensitive, and selective without using any sophisticated instruments, therefore it may have a promising prospect for detecting targets in clinical medicine, food safety analysis, and environmental monitoring.Display Omitted
Keywords: Liposome; Signal amplification; Immunoassay; Colorimetric; H5N1;

“Turn-off” fluorescent sensor for highly sensitive and specific simultaneous recognition of 29 famous green teas based on quantum dots combined with chemometrics by Li Liu; Yao Fan; Haiyan Fu; Feng Chen; Chuang Ni; Jinxing Wang; Qiaobo Yin; Qingling Mu; Tianming Yang; Yuanbin She (119-128).
Fluorescent “turn-off” sensors based on water-soluble quantum dots (QDs) have drawn increasing attention owing to their unique properties such as high fluorescence quantum yields, chemical stability and low toxicity. In this work, a novel method based on the fluorescence “turn-off” model with water-soluble CdTe QDs as the fluorescent probes for differentiation of 29 different famous green teas is established. The fluorescence of the QDs can be quenched in different degrees in light of positions and intensities of the fluorescent peaks for the green teas. Subsequently, with aid of classic partial least square discriminant analysis (PLSDA), all the green teas can be discriminated with high sensitivity, specificity and a satisfactory recognition rate of 100% for training set and 98.3% for prediction set, respectively. Especially, the “turn-off” fluorescence PLSDA model based on second-order derivatives (2nd der) with reduced least complexity (LVs = 3) was the most effective one for modeling. Most importantly, we further demonstrated the established “turn-off” fluorescent sensor mode has several significant advantages and appealing properties over the conventional fluorescent method for large-class-number classification (LCNC) of green teas. This work is, to the best of our knowledge, the first report on the rapid and effective identification of so many kinds of famous green teas based on the “turn-off” model of QDs combined with chemometrics, which also implies other potential applications on complex LCNC classification system with weak fluorescence or even without fluorescence to achieve higher detective response and specificity.Display Omitted
Keywords: Quantum dots; “Turn-off”; Green teas; Chemometrics;

Colorimetric immunoassays have been attracting more attention for use in practical applications, especially in point−of−care diagnostics. In comparison with a single color immunoassay, the dose−dependent multicolor strategy greatly improves the detection resolution and accuracy of visual inspection. In the current study, a concentration−dependent multicolor conversion strategy was developed based on gold nanoparticle (AuNP)−mediated copper deposition for signal amplification and Prussian blue for color generation. Under optimal conditions, a dose−dependent multicolor from yellow through green to blue were successfully achieved, which was easier to be differentiated from each other by the naked eyes. With rabbit IgG and prostate specific antigen (PSA) as model analytes, semi−quantitative evaluations were demonstrated in lab buffer and serum by direct readout with the naked eyes. Quantitative detections were also accomplished by measurement of absorbance of Prussian blue with a common UV–Vis spectrophotometer. A limit of detection (LOD) down to sub−picogram per milliliter was determined. In addition, this newly developed colorimetric assay method can be easily adapted for the detection of other biomolecules by simply changing the recognition pairs.Display Omitted
Keywords: Gold nanoparticle (AuNP); Copper deposition; Signal amplification; Colorimetric immunoassay; Multicolor conversion; Cancer diagnosis;

A methacrylate based monolith, containing the innovative zwitterionic monomer (3-allyl-1-imidazol)propane sulfonate, was prepared in 100 μm I.D. silica capillaries by UV initiated photo-polymerization. Composition of the porogen, i.e. a mixture of 1-propanol, 1,4 butanediol and water, was of great importance to obtain a homogeneous monolith with satisfactory permeability and good electrochromatographic performance. Morphology of the stationary phase was studied in Scanning Electron Microscopy and IR experiments, which revealed a good attachment to the capillary wall, flowthrough-pores in the range of 0.5–2 μm, and a continuous monolithic structure. The developed material was well suited for the analysis of six common phenolic acids (salicylic, cinnamic, syringic, rosmarinic, caffeic and chlorogenic acid) by CEC. Their separation was possible in less than 8 min with a mobile phase comprising a 12 mM aqueous ammonium acetate solution with pH 8.5 and acetonitrile, at an applied voltage of - 20 kV. The developed method was validated (R2 ≥ 0.995; LOD ≤ 3.9 μg mL−1, except for salicylic acid; recovery rates from 94 to 104%) and successfully used for the determination of phenolic acids in Coffea arabica samples. All of them contained cinnamic, syringic and caffeic acid, however only in unroasted coffee beans chlorogenic acid (0.06%) was found. The quantitative results were in good agreement to reported literature data.Display Omitted
Keywords: CEC; Zwitterionic stationary phase; Natural products; Coffee;

In this study, multi-acrylate based dipentaerythritol penta-/hexa-acrylate (DPEPA) was exploited for fabrication of highly cross-linked hybrid monolithic column by copolymerization with polyhedral oligomeric silsesquioxane methacryl substituted (POSS-MA) via a “one-pot” method. The new DPEPA-POSS hybrid monolithic column was respectively characterized by Fourier transform infrared spectrometry, thermogravimetric analysis, scanning electron microscopy and nitrogen adsorption/desorption measurement. When it was used for the separation of amides, thioureas and positional isomers of phenols, ultrahigh column efficiency separation (up to 511,000 N m−1) was achieved with excellent selectivity. Moreover, intact protein standards could be efficiently separated with minimum tailing peaks, outperforming the commercially available silica-based C8 column. Furthermore, successful separation of complex egg white proteins and expressed BARD1 BRCT domains protein sample was also achieved with good chromatographic performance. In the future work, the DPEPA-POSS hybrid monolithic column will be further exploited and applied in capillary electrochromatography as well as the top-down based proteome research.Display Omitted
Keywords: Ultrahigh efficiency separation; Proteins; Highly cross-linked; Hybrid monolithic column; Dipentaerythritol penta-/hexa-acrylate; Polyhedral oligomeric silsesquioxane methacryl substituted;

Design, synthesis and application of a new class of stimuli-responsive separation materials by Roshanak Sepehrifar; Reinhard I. Boysen; Basil Danylec; Yuanzhong Yang; Kei Saito; Milton T.W. Hearn (153-163).
A new class of efficient stationary phase has been investigated for use in the liquid chromatographic separation of low molecular weight analytes and high molecular weight biomolecules, based on the application of immobilised stimuli-responsive polymers (SRPs). To this end, two polymeric units, namely poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) and poly(acrylic acid) (PAA) were tethered to a triazine core. The derived poly(2-dimethyl-aminoethyl methacrylate)-block-poly(acrylic acid) (PDMAEMA-b-PAA), as a diblock co-polymer, was then immobilised onto the surface of porous silica particles. The performance of this microparticulate adsorbent was evaluated under various temperature, ionic strength and/or pH conditions in packed columns in a high-performance liquid chromatography (HPLC) format. Baseline separations of a variety of low molecular weight analytes were achieved at different temperatures with this SRP-based adsorbent using 10 mM sodium phosphate buffer, pH 6.0, as the mobile phase. Moreover, when the ionic strength of the mobile phase was increased to 40 mM sodium phosphate buffer, pH 6.0, similar temperature changes resulted in further increases in resolution for the hydrophobic analytes. In addition, changes in the pH of the mobile phase from pH 6.0 to pH 8.0 led to significant changes in selectivity of the analytes, including reversal in their elution orders. Upon increasing the temperature, the retention times of all analytes decreased but without loss of resolution. These findings can be attributed to the consequence of the immobilised copolymer undergoing a phase transition at its lower critical solution temperature (LCST), which leads to changes in its solvated structure, including how the electrostatic, hydrophilic and hydrophobic regions/domains of the copolymer are exposed to the bulk mobile phase. Thermodynamic data were indicative of a temperature-related re-organisation of the structure of the immobilised PDMAEMA-b-PAA stationary phase with exothermic binding of the analytes occurring at temperatures below the lower critical solution temperature (LCST). In this manner; changes in the system temperature could directly be used to manipulate the adsorption and desorption behaviour of these analytes with this stimuli-responsive, polymer-modified porous silica stationary phase. Additional studies with several proteins further documented the versatility of these stimuli-responsive separation materials. The results indicated that these separations could be tuned by variation of the temperature with fully aqueous mobile phases at specific ionic strength and pH values, without the need to use an organic solvent as a component in the mobile phase.Display Omitted
Keywords: Stimuli-responsive polymers; Diblock copolymer; Polyelectrolyte; Tuneable selectivity; Liquid chromatography;

Quinine bonded to superficially porous particles for high-efficiency and ultrafast liquid and supercritical fluid chromatography by Darshan C. Patel; Zachary S. Breitbach; JeongJae Yu; Kate A. Nguyen; Daniel W. Armstrong (164-174).
Two new anion-exchange columns were prepared by bonding tert-butyl carbamoylated quinine to 2.7 μm superficially porous particle (SPP) silica to create chiral stationary phases for high-efficiency and ultrafast chromatography. Performance and retention parameters of these new columns are compared with an analogous 5 μm fully porous particle (FPP) based Chiralpak QNAX column and a 3–4 fold increase in efficiency was observed. Ultrafast separations ranging from 12 s down to sub-second are shown using 2.7 μm SPPs bonded via hydrosilation to the selector. Potential benefits of 2.7 μm SPP based columns for increased LC-MS compatibility were investigated. A van Deemter plot comparison showed 2.7 μm SPP based columns provided a lower reduced plate height and a higher optimal linear velocity compared to the 5 μm FPP based column. With geometry-independent kinetic plots, 2.7 μm SPP and 5 μm FPP based columns were assessed for their kinetic performance and the maximal number of plates each column can generate in a given analysis time. The 2.7 μm SPP based column showed remarkable performance improvements in speed and efficiency as indicated by the kinetic plots.Display Omitted
Keywords: Chiral separations; Superficially porous particles; High-efficiency chiral stationary phase; Ultra-high performance liquid chromatography (UHPLC); Sub/supercritical fluid chromatography (SFC); Kinetic plots;