Karine Faure

Electrochromatography in cyclic olefin copolymer microchips: a step towards field portable analysis.

In order to develop a portable and disposable instrument for on-site analysis of neutral compounds, a lauryl methacrylate monolith has been synthesized into a cyclic olefin copolymer microdevice for reversed-phase electrochromatography purposes. This monolith was tested in capillary to evaluate electrochromatographic performances in terms of electroosmotic flow (EOF) mobility, retention and efficiency prior to its transfer into the microfluidic device. The produced monolithic bed exhibited a good run-to-run repeatability, column-to-column reproducibility and batch-to-batch reproducibility, with relative standard deviation (RSD) values below 9% for EOF mobility, retention factors and heights of theoretical plate. The electrochromatographic performances of the monolith were optimized by reducing irradiation time. Photopolymerization time of 10 min was found to be the best process in order to obtain a robust, retentive and efficient system. The on-chip performances of this monolith were evaluated in detail for the reversed-phase electrochromatographic separation of polycyclic aromatic hydrocarbons, with plate heights reaching down to 15 μm when working at optimal velocity. Aiming at the maximum simplification of instrumental fabrication and operation, a direct injection from a 2 μL droplet was compared with more conventional dynamic injection process.

New “one-step” method for the simultaneous synthesis and anchoring of organic monolith inside COC microchip channels

A new method for monolith synthesis and anchoring inside cyclic olefin copolymer (COC) microchannels in a single step is proposed. It is shown that type I photoinitiators, typically used in a polymerization mixture to generate free radicals during monolith synthesis, can simultaneously act as type II photoinitiators and react with the plastic surface through hydrogen abstraction. This mechanism is used to photograft poly(ethylene glycol) methacrylate (PEGMA) on COC surfaces. Contact angle measurements were used to observe the changes in surface hydrophilicity when increasing initiator concentration and irradiation duration. The ability of type I photoinitiators to synthesize and anchor a monolith inside COC microchannels in a single step was proved through SEM observations. Different concentrations of photoinitiators were tried. Finally, electrochromatographic separations of polycyclic aromatic hydrocarbons were realized to illustrate the beneficial effect of anchoring on chromatographic performances. The versatility of the method was demonstrated with two widely used photoinitiators: benzoin methyl ether (BME) and azobisisobutyronitrile (AIBN).

Carnosol purification. Scaling-up centrifugal partition chromatography separations

This paper illustrates the application of a recently proposed protocol allowing the scale-up prediction on hydrostatic countercurrent chromatography columns (centrifugal partition chromatographs or CPC). A commercial extract of rosemary (Rosmarinus officinalis L.) was used as the starting material containing 0.48% of carnosol, an active pharmaceutical ingredient with great potential. After a rapid method development on a small-scale 35-mL CPC instrument that allowed for the determination of the solvent system and maximum sample concentration and volume, the purification was transferred on two larger instruments using the free space between peaks method. The method takes into account the technical limitations of the larger instruments, such as pressure and/or maximum centrifugal field, and allows, by simply running an analytical-sized injection on the large scale rotor, to give an accurate prediction of the maximum sample load and best throughput. The 0.27g of rosemary extract maximum load on a 35-mL CPC was transferred as a 1.9g load on a 254-mL medium size CPC and 9g load on a 812-mL CPC. The maximum process efficiency of 3.1mg of carnosol per hour obtained on the small 35-mL column was transferred on the 254-mL CPC giving 8.3mg/h and, on the larger 812-mL column 49.4mg of carnosol could be obtained per hour. If the scaling-up in CPC instruments is not directly homothetic, it can be highly predictable through a few simple experiments.

Preparative two dimensional separations involving liquid–liquid chromatography☆

The increasing performances of analytical techniques, especially two-dimensional liquid chromatography hyphenated with highly sensitive detectors, allow discovery of new targeted compounds in highly complex samples, whether biomarkers in environmental effluents, natural metabolomes in natural products or unknown impurities in synthetic chemical process. While structural identification can usually be achieved through mass spectrometry and databases, it can be useful to confirm the potential structure via NMR analysis and/or to obtain standard reference material for quantitative purposes, incentive for the production of μg to mg of new target compounds. Hence, preparative chromatography, which was initially run for large-scale production, is now facing new challenges, with small volumes of highly complex samples to deal with. Unfortunately, one dimensional chromatographic methods exhibit limited performances when targets are minor compounds in a complex matrix. Moreover, for process intensification or limited amount of sample, it is now a priority to be able to isolate multiple components with the largest yield possible using as few purification steps as possible. With these specifications in mind, a comprehensive multidimensional chromatographic method for preparative purposes is becoming appealing.

Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC

AbstractPuwainaphycins are a recently described group of β-amino fatty acid cyclic lipopeptides of cyanobacterial origin that possess interesting biological activities. Therefore, the development of an efficient method for their isolation from natural sources is necessary. Following the consecutive adsorption of the crude extract on Amberlite XAD-16 and XAD-7 resins, countercurrent chromatography (CCC) was applied to separate seven puwainaphycin variants from a soil cyanobacterium (Cylindrospermum alatosporum CCALA 988). The resin-enriched extract was first fractionated by CCC into fractions I and II with use of the n-hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system at a flow rate of 2xa0mLxa0min−1 and a rotational speed of 1400xa0rpm. The CCC separation of fraction I, with use of the ethyl acetate–ethanol–water (5:1:5, v/v/v) system, afforded compounds 1 and 2. The CCC separation of fraction II, with use of the n-hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system, afforded compounds 3–7. In both cases, the lower phases were used as mobile phases at a flow rate of 1xa0mLxa0min−1 with a rotational speed of 1400xa0rpm and a temperature of 28xa0°C. The CCC target fractions obtained were repurified by semipreparative high-performance liquid chromatography (HPLC), leading to compounds 1–7 with purities of 95xa0%, 95xa0%, 99xa0%, 99xa0%, 95xa0%, 99xa0%, and 90xa0% respectively, as determined by HPLC–electrospray ionization high-resolution mass spectrometry (ESI-HRMS). The chemical identity of the isolated puwainaphycins (compounds 1–7) was confirmed by ESI-HRMS and NMR analyses. Three new puwainaphycin variants (compounds 1, 2, and 5) are reported for the first time. This study provides a new approach for the isolation of puwainaphycins from cyanobacterial biomass.n Graphical AbstractSeparation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC. Compounds 1 (12-hydroxy-4-methyl-Ahtea-Puw-F), 2 (11-chloro-4-methyl-Ahdoa-Puw-F), 3 (4-methyl-Ahdoa-Puw-F), 4 (4-methyl-Ahdoa-Puw-G), 5 (12-chloro-4-methyl-Ahtea-Puw-F), 6 (4-methyl-Ahtea-Puw-F) and 7 (4-methyl-Ahtea-Puw-G). Ahtea: 3-amino-2-hydroxy tetradecanoic acid. Ahdoa: 3-amino-2-hydroxy dodecanoic acid

Two-dimensional multi-heart cutting centrifugal partition chromatography–liquid chromatography for the preparative isolation of antioxidants from Edelweiss plant

The Edelweiss plant has been recognized as a very valuable source of anti-aging principles due to its composition of antioxidants compounds: leontopodic acid A and 3,5-dicaffeoylquinic acid. In this work, off-line multi-heart cutting CPC-LC separation was set up at industrial scale in order to isolate and produce new high quality reference material of these two antioxidants from Edelweiss. For this purpose, CPC and HPLC methods were developed and optimized at laboratory scale and a comprehensive CPCxHPLC analysis of the crude extract was established. Thereby, the CPC method led to a first separation of the target compounds according to their partition coefficient in the solvent system and the HPLC method was performed on the recovered fractions to lead to a second separation. A 2D CPCxHPLC plot was established in order to know the fractions to select at the industrial scale. Then, the CPC and HPLC methods were transferred at industrial scale and the multi-heart cutting CPC-LC was performed in off-line mode. Using CPC with methyl ter-butyl ether-water 1:1 (v/v) solvent system and LC with Denali C18 column, 2g of crude extract sample were injected and leontopodic acid A and 3,5-dicaffeoylquinic acid were recovered with purity over 97%. The compounds were identified by MS and NMR.

Functionalization of cyclic olefin copolymer substrates with polyethylene glycol diacrylate for the in situ synthesis of immobilized nanoparticles

Nanoparticles as novel stationary phase could enhance chromatographic performance (efficiency and resolution). However, their implementation in columns is rather challenging. The methodological approach we propose is an in situ synthesis of such nanoparticles through photopolymerization of a miniemulsion containing hexyl acrylate monomers. While the miniemulsion composition was previously optimized, resulting in highly spherical monodisperse nanoparticles with a mean size inferior to 200xa0nm, the immobilization of such particles is not straightforward. The strategy consists in carrying out the miniemulsion polymerization in the very close vicinity of a reactive surface, namely cyclic olefin copolymer (COC), from which covalent bond can grow thanks to hydrogen abstraction. While the hydrophobic nature of COC appears to disturb the stability of the miniemulsion, a more polar surface allows simultaneous polymerization and anchoring of nanoparticles on the surface. Surface functionalization of COC with different monomers was tested in this work and polyethylene glycol dimethacrylate photografting was found to provide an adequate surface modification. It appears however that the extent of surface modification and especially the amount of initiator that is applied on the COC surface have consequences on both the spherical aspect and the number of resulting nanoparticles. An optimization of the PEGDA surface modification by central composite design in regards to PEGDA and BME amounts and irradiation time is proposed. SEM images after miniemulsion photopolymerization for different conditions of PEGDA photografting showed the impact of photografting on nanoparticle shape.

In-situ protein determination to monitor contamination in a centrifugal partition chromatograph

Centrifugal partition chromatography (CPC) works with biphasic liquid systems including aqueous two-phase systems. Metallic rotors are able to retain an aqueous stationary phase able to purify proteins. But the adhesion of proteins to solid surface may pose a cross-contamination risk during downstream processes. So it is of utmost importance to ensure the cleanliness of the equipment and detect possible protein contamination in a timely manner. Thereby, a direct method that allows the determination of the effective presence of proteins and the extent of contamination in the metallic CPC rotors was developed. This in-situ method is derived from the Amino Density Estimation by Colorimetric Assay (ADECA) which is based on the affinity of a dye, Coomassie Brillant Blue (CBB), with protonated N+ groups of the proteins. In this paper, the ADECA method was developed dynamically, on a 25xa0mL stainless-steel rotor with various extents of protein contaminations using bovine serum albumin (BSA) as a fouling model. The eluted CBB dye was quantified and found to respond linearly to BSA contamination up to 70xa0mg injected. Limits of detection and quantification were recorded as 0.9xa0mg and 3.1xa0mg, respectively. While the non-specific interactions between the dye and the rotor cannot currently be neglected, this method allows for in situ determination of proteins contamination and should contribute to the development of CPC as a separation tool in protein purification processes.

Effect of operating parameters on a centrifugal partition chromatography separation

Centrifugal partition chromatography (CPC) is the branch of countercurrent chromatography (CCC) that works with single axis hydrostatic columns with rotary seals. The hydrodynamic of the liquid stationary phase-liquid mobile phase equilibrium in the CPC chambers has been studied theoretically and with specially designed CPC columns. In this work, we selected a simple analytical separation (no loading study) of three test solutes, coccine red, coumarin and carvone, with a commonly used heptane/ethyl acetate/methanol/water 1:1:1:1v/v biphasic liquid system and two different rotors: a commercially available 30-mL CPC instrument and a 80-mL prototype rotor designed for productivity. We fully studied this separation in many possible practical operating conditions of the two rotors, aiming at a generic column characterization. The rotor rotation was varied between 1000 and 2800rpm, the aqueous mobile phase flow rate was varied between 1 and 22mL/min with the 30-mL rotor and 10 and 55mL/min with the 80-mL rotor, the upper limits being mechanical constraints and some liquid stationary phase remaining in the rotor. The variations of Sf, the volume ratio of stationary phase in the rotor, were studied versus mobile phase flow rate and rotor rotation speed. A maximum mobile phase linear velocity was found to depend on the centrifugal field for the 30-mL rotor. This maximum velocity was not observed with the 80-mL rotor. Studying the changes in coumarin and carvone peak efficiencies, it is established that the number of cells required to make one theoretical plate, i.e. one chromatographic exchange, is minimized at maximal rotation speed and, to a lesser extent, at high mobile phase flow rate (or linear velocity). Considering the throughput, there is evidence of an optimal flow rate depending on the rotor rotation that is not necessarily the highest possible.

Application of HPCCC Combined with Polymeric Resins and HPLC for the Separation of Cyclic Lipopeptides Muscotoxins A–C and Their Antimicrobial Activity

Muscotoxins are cyanobacterial cyclic lipopeptides with potential applications in biomedicine and biotechnology. In this study, Desmonostoc muscorum CCALA125 strain extracts were enriched by polymeric resin treatment, and subjected to HPCCC affording three cyclic lipopeptides (1–3), which were further repurified by semi-preparative HPLC, affording 1, 2, and 3, with a purity of 86%, 92%, and 90%, respectively. The chemical identities of 2–3 were determined as muscotoxins A and B, respectively, by comparison with previously reported ESI-HRMS/MS data, whereas 1 was determined as a novel muscotoxin variant (muscotoxin C) using NMR and ESI-HRMS/MS data. Owing to the high yield (50 mg), compound 2 was broadly screened for its antimicrobial potential exhibiting a strong antifungal activity against Alternaria alternata, Monographella cucumerina, and Aspergillus fumigatus, with minimum inhibitory concentration (MIC) values of 0.58, 2.34, and 2.34 µg/mL; respectively, and weak antibacterial activity against Bacillus subtilis with a MIC value of 37.5 µg/mL. Compounds 1 and 3 were tested only against the plant pathogenic fungus Sclerotinia sclerotiorum due to their low yield, displaying a moderate antifungal activity. The developed chromatographic method proved to be an efficient tool for obtaining muscotoxins with potent antifungal properties.