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Highlights: [•] SFC speed-resolution properties of sub-2μm particles are given under adiabatic conditions. [•] The effect of the organic modifier content on column performance was theoretically studied. [•] At low pressure drop, increasing methanol content decreases efficiency and analysis speed. [•] At high pressure drop, increasing methanol content does not affect the maximum efficiency. [•] At high pressure drop, increasing methanol content minimizes the loss of analysis speed. [ABSTRACT FROM AUTHOR]

Highlights: [•] The concept of Poppe plot is extended and used to compare column performance in SFC and HPLC. [•] The columns were assumed to be radially homogeneous and to operate under adiabatic conditions. [•] The dispersion process in adjacent column slices is assumed to be independent. [•] For axial column non-uniformity, the linear addition rule applies to the time variance only. [•] In RPLC and SFC, columns of core–shell particles outperform those of fully porous particles. [Copyright &y& Elsevier]

Abstract: The sources of band broadening in fast gradient elution chromatography include the contributions of the tubings placed upstream the column, the isocratic migration of the sample that takes place in the column until the moment when the front of the gradient catches up with it, dispersion and mass transfer resistance during band migration under gradient elution to the column outlet, and dispersion in the tubings downstream the column to the detector. The measurements of all these contributions is meticulously described, analyzed, and applied to assess the kinetic performance of monolithic columns of the second generation (KGaA Merck) in gradient elution, at the highest possible speed at which they can be operated (t 0=25s at P =200bar). These results are compared to those provided by several 4.6mm × 100mm columns packed with sub-3μm core–shell particles (Kinetex, Phenomenex and Halo, Advanced Material Technologies) and by a 2.1mm × 100mm column packed with fully porous 1.7μm BEH particles (Waters). A RPLC checkout sample (Agilent) containing nine alkanophenones (molecular weights between 120 and 204g/mol) was used for this purpose. The mobile phase was a mixture of acetonitrile and water and the instrument, an optimized version of the 1290 Infinity HPLC system (Agilent). The results show that the overall peak capacity provided by this second generation of monolithic columns is nearly 30% smaller than that provided by commercial columns packed with sub-3 μm core–shell particles and 20% smaller than that provided by a narrow-bore column packed with sub-2μm fully porous particles. After isolating the sole band broadening contribution due to the gradient migration of the analytes along the columns, the BEH column proved to provide the highest resolution power (apparent average plate height, of 2μm), followed by the 2.6μm Kinetex particles (3.0μm), the Halo particles (3.2μm), and the second generation of 4.6mm I.D. monolithic columns (6.5μm). This work demonstrates that analysts have an urgent need for very high pressure liquid chromatographs having smaller dwell and extra-column volumes that those of the currently available instruments. The gradient elution peak capacity provided by the modern, narrow-diameter columns, which are short and highly efficient (plate heights of 3–4μm), is barely half what the column alone could produce if it were fitted to a perfect instrument. The other half of the peak capacity is just wasted by the instrument. [Copyright &y& Elsevier]

Abstract: The gradient elution performance of narrow-bore 2.3mm×50mm (N733) and wider bore 3.2mm×50mm (N648 and N655) prototype silica monolithic columns was investigated and compared to the performance of commercially available columns packed with sub-2μm fully porous particles (2.1mm×50mm, 1.7μm BEH-C18, Waters) and sub-3μm superficially porous particles (2.1mm×50mm, 2.7μm Halo-ES-Peptide-C18 (AMT), 1.7 and 2.6μm Kinetex-C18, Phenomenex). Results show that the two wide monolithic columns show peak capacities similar to the one measured for the Kinetex column. In contrast, the narrow-bore monolithic column delivers a lower performance (−30%) than the BEH, the Halo and the Kinetex columns. This work stresses out the importance of reducing the extra-column band broadening contribution of HPLC instruments when short 2.1mm I.D. columns are used. The part of the instrument contribution originating downstream the column is important for all compounds; the one originating upstream the column is significant only for weakly retained compounds. [Copyright &y& Elsevier]

Abstract: New hyphenated technique for the extraction and determination of isoflavones in sea and freshwater algae and cyanobacteria was developed. The method consists of sonication sample pretreatment, extraction by supercritical CO2 modified by 3% (v/v) of MeOH/H2O mixture (9:1, v/v) at 35MPa and 40°C for 60min, fast chromatography analysis by the means of Agilent 1200 Series Rapid Resolution and MS/MS determination. Agilent 1200 Series RRLC was used with Zorbax SB-CN chromatographic column (100mm×2.1mm, particle size 3.5μm), 3μl injection volume, mobile phase consisting of 0.2% (v/v) acetic acid in water (solvent A) and acetonitrile (solvent B) and used with linear gradient (30% B at 0min, from 0min to 3min up to 50% B, from 3 to 6min up to 80% B and from 6 to 10min down to 30% B). The flow-rate was 0.4mL/min, column oven temperature 35°C. MS detector Agilent Technologies 6460 Triple quadrupole LC/MS with Agilent Jet Stream was used in a negative ESI mode under following conditions: gas temperature 350°C, gas flow 13L/min, nebulizer gas pressure 50psi, sheath gas temperature 400°C, sheath gas flow 12L/min, capillary voltage was 4kV. Samples were analysed in the multiple reaction monitoring (MRM) mode. Eight isoflavone compounds were found for the first time in seven real samples of sea algae and in three control samples of freshwater algae and cyanobacteria. Usual optimisation study of extraction parameters was performed. Pressure and temperature optima for algae matrix are different from those obtained sooner for other matrices for most of the analytes, but the results of modifier optimisation study are in good accordance with those obtained sooner for spiked samples and red clover matrix. It seems that matrix has very small or no effect on the modifier selection. Two different approaches of sonication pretreatment were tested: sonication bath and the thorn instrument. In longer extraction time experiments, thorn sonication was more efficient and recovery of following supercritical fluid extraction was higher. [Copyright &y& Elsevier]

Abstract: A novel fast and sensitive method has been developed for the specific simultaneous determination of polysorbate 20 (Tween 20) and unbound polyethylene-glycol (PEG) from liquid formulations in the presence of proteins and excipients. The quantitative determination is based on a fast liquid chromatographic (HPLC) separation and condensation nucleation light scattering detection (CNLSD or NQAD™). The method uses a Kinetex core–shell column (100mm×3mm, 2.6μm) and methanol–water–trifluoroacetic acid mobile phase. The rapid HPLC-CNLSD method presented here is suitable for quantifying polysorbate 20 in the range of 10–60μg/ml and unbound PEG in the range of 2–40μg/ml in protein solutions within good manufacturing practices (GMP) of the pharmaceutical industry. [Copyright &y& Elsevier]

Abstract: Macroporous cellulose beads (MCB) used as anion exchangers were successfully prepared from cellulose solution in ionic liquid by double emulsification followed by cross-linking and modification with diethylaminoethyl. The pore structure and properties of the MCB were investigated and the results were compared with homogeneous cellulose beads (HCB). The MCB in size of about 71μm is characterized by two sets of pores, i.e., diffusion pores (10–20nm) and macropores (800–2000nm), determined by mercury porosimeter. In addition, the bed permeability and effective porosity for BSA of MCB-packed column are 58% and 25% higher than those of HCB-packed column, respectively. The adsorption properties of MCB were evaluated, and compared with HCB and commercial absorbent (Sepharose 6 Fast Flow, CSFF). It is found that the pore diffusivity of BSA in MCB is over 7.9 times higher than HCB, and 6.7 times higher than CSFF, respectively. While the equilibrium adsorption capacity (q m ) of BSA on MCB is obviously lower than that on HCB and CSFF, the dynamic binding capacity (DBC) on MCB at 10% breakthrough reaches 47.7mg/mL, higher than HCB (40.3mg/mL) and CSFF (46.2mg/mL) at flow rate of 360cm/h. In addition, the MCB-packed column showed better column efficiency over the HCB packed one. Therefore, we demonstrated that the MCB possessed more advantages than other ones, like HCB and CSFF, and was expected as an ideal material for fast chromatography. [Copyright &y& Elsevier]

Chromatographic behavior of a series of N-methylated tetra and octapeptides on a reversed-phase sorbent was studied considering the information obtained on these compounds by NMR spectroscopy. The modified tetrapeptides were derived from GFFY-NH2, GFFF-NH2 and GFFH-NH2 primary structures by N-methylation at various peptide bond positions. Similarly, the N-methylated octapeptides were based on TPK(Pac)T C-terminally elongated forms of GFFY and GFFF. It was found that many studied N-methylated peptides provide broad peaks as a consequence of cis/trans isomerism of the R1CON(CH3)R2 peptide bond. The extent of the peak spreading depends on the following important factors: the nature of the surrounding amino acid residues, the location of the modified peptide bond within the peptide chain, temperature, and mobile phase flow-rate. All these aspects were critically evaluated. Nearly complete separation of the individual conformers of GF(NMe)FY-NH2 was obtained applying fast chromatography on short column packed with 20–30 μm reversed-phase sorbent. [Copyright &y& Elsevier]

Abstract: Multivariate curve resolution-alternating least squares (MCR-ALS) is shown to be a powerful tool to resolve coelution problems in liquid chromatograpy–mass spectrometry (LC–MS) in scan mode. This investigation was performed using two types of LC columns, one traditional LC column of 25cm length with a slow gradient and a shorter LC column of 7.5cm with a rapid gradient which allowed much faster analysis and save of reagents and solvents. Mixtures of multiple biocide compounds were simultaneously analyzed in standard mixtures and in environmental samples (sediment and wastewater samples) with little sample pretreatment. Using the more traditional LC 25cm column, all biocide compounds were properly resolved by MCR-ALS and quantitatively analyzed with estimated errors always below 20%. When fast chromatography (LC column of 7.5cm) was used, MCR-ALS resolution of the more strongly coeluted compounds was also achieved but limitations were found in their simultaneous quantitative determination, specially for environmental samples. [Copyright &y& Elsevier]