Capacity factors and peak widths in the gradient elution reversed phase separation of high molar mass polystyrenes

Summary A simplex method for determining the constantsS andk _o from the equation lnk’=lnk _o−S ϕ (was developed and applied to the reversed phase separation of high molar mass polystyrenes using gradients of any curvature. Experimental retention times described using the equation had a standard deviation of 1.1%. The inclusion of a quadratic term in the equation was found to be unwarranted. BothS and lnk _o varied linearly with ln molar mass. The logarithm of peak width of an eluting polystyrene peak was found to be a linear function of the mobile phase composition at elution. The slope was equal toS.     

HPLC quantification of flavonoids and biflavonoids inCupressaceae leaves

Summary The aim of this investigation was to obtain qualitative and quantitative profiles of the flavonoid and biflavonoid composition of six cypress species—Cupressus funebris L.,Cupressus sempervirens L.,Cupressus glabra L.,Cupressus arizonica L.,Cupressus goveniana L., andCupressus lusitanica L. HPLC-diode-array detection (DAD), HPLC-MS, and HPTLC were used to identify the individual compounds. A chromatographic method was optimized for identification and quantification of the main flavonoid glycosides and biflavonoids. The flavonoids identified and calibrated were: rutin, quercetin glucoside, quercetin rhamnoside, and kaempferol 3-O-rhamnoside. The biflavonoids identified and calibrated were: cupressuflavone, amentoflavone, robustaflavone, hinokiflavone, methylrobustaflavone, methylamentoflavone, and dimethylcupressuflavone.     

Fast sample preparation involving MASE and coupled column normal phase liquid chromatography for the rapid trace analysis of dioxins in air-dust samples from fire catastrophe emissions

A new approach has been developed and tested for the urgent analysis of dioxins in samples of air-dust filters originating from catastrophe emissions. The procedure consists of a fast extraction of the sample with microwave solvent extraction (MASE) and acetone as solvent followed by a fast cleanup of the extract with normal phase coupled column liquid chromatography (LC/LC).

The multi-dimensional LC/LC system employs a 50 mm×4.6 mm i.d. column packed with 3 μm silica and a 150 mm×4.6 mm i.d. column packed with 5 μm PYE as the first and second analytical column, respectively. Iso-hexane is used on both columns to perform cleanup and dichloromethane to perform efficient back-flush elution of the compounds from the second column. The obtained polarity-based separation in the first dimension and molecular-structure based separation in the second dimension provides a fast and powerful cleanup.

Validation was done by analysing samples of homemade RIVM air-dust with aged residues (n=8, spiking level about 15 pg mg⁻¹ per compound) of dioxins/furans and samples of reference Urban Dust SRM 1649a (n=4) with both the new approach and the existing conventional procedure and were instrumentally analyzed with capillary gas chromatography and high resolution mass spectrometric detection (GC/HRMS).

In comparison to the existing conventional procedure, the new approach reduces sample processing from several days to several hours per sample.

As regards the aged-residue air-dust samples, the new method shows a good accuracy, precision and high selectivity providing a performance in good agreement with the existing procedure. In SRM air-dust, the concentration of a few compounds obtained by the new method was below (10–50%) the certified value.     

Siliceous materials with the surface polymer layer composed of dextran-polyimine mixture as column packings for liquid chromatography

Summary Forming a polymer layer on the surface of siliceous materials is one of the methods for protecting the silica skeleton from dissolution in alkaline mobile phases as well as eliminating the negative influence of silanol groups on separated molecules e.g. proteins. Polysaccharides, especially their derivatives bearing amine groups, can play the role of the surface layer. This paper discusses the possibilities of preparing such a layer by cross-linking a dextran-polyimine mixture (rather than the traditionally used DEAE-dextran) deposited on the surface of the solid material. The results presented prove the utility of synthesized materials as supports for affinity ligands in high performance affinity chromatography or as supports for complexed metal ions in ligand-exchange chromatography. The properties of the sorbents with a polymer layer can be changed both by the composition of the cross-linked mixture and by chemical modification.     

Comparison of adsorbents for on-line solid-phase extraction of polycyclic aromatic hydrocarbons before liquid chromatography with UV detection

Summary On-line solid-phase extraction (SPE) coupled with reversed-phase liquid chromatography and UV detection at 254 nm has been used for the determination of trace-level polycyclic aromatic hydrocarbons (PAH) in soil extracts. Five commercially available adsorbents (C₈, C₁₈, PLRP-S, PRP-1, and Bond-Elut Env) were evaluated. Results showed that recovery of the PAH decreased with increasing molecular weight, because of their poorer solubility. Recovery of high-molecular-weight PAH was significantly improved by addition of 10% (v/v) acetonitrile to the sample before loading of the SPE adsorbent. PAH recovery ranged from 64.0 to 108% when a 50 mL sample spiked with 1 μg L⁻¹ was applied to these adsorbents. Determination of PAH was possible with detection limits below 0.05 μg L⁻¹, which corresponds to 0.2 μg kg⁻¹ soil. The method was successfully used to determine PAH in soil extracts.     

Reanalysis of Chiral Discrimination of Phenoxypropionic Acid Herbicides on a Teicoplanin Phase Using a Bi-Langmuir Approach

Summary The chiral discrimination of phenoxypropionic acid herbicides by reversed-phase chromatography on a teicoplanin phase has been re-examined using the perturbation method to calculate the solute distribution isotherms. The effects of both temperature and the methanol (organic modifier) content of the mobile phase on the chiral discrimination mechanism were well described by the bi-Langmuir model. The method confirmed a change in the mechanism of enantiomer retention at a critical temperature, T^*, and showed that the mechanism was independent of (i) herbicide molecular structure, i.e. the position of the chloro group on the phenol ring, and (ii) the absolute configuration of the carbon atom. Enantioselectivity was enhanced by increasing the mobile phase methanol content. Use of this approach also revealed that secondary sites on the teicoplanin surface were involved in the processes determining both retention and selectivity. It was clearly demonstrated that these secondary sites of low affinity were not affected by the temperature change and were not involved in the chiral recognition mechanism.     

Optimization of the Synthesis of 2,6–Dinitro-4-trifluoromethylphenyl Ether Substituted Cyclodextrin Bonded Chiral Stationary Phases

The comparisons of five different chiral stationary phases (CSPs) based on 2,6-dinitro-4-trifluoromethylphenyl (DNP-TFM) ether substituted β-cyclodextrin are presented. The five CSPs differ from each other in the linkage/spacer chemistry, or on the position of the substituents on β-cyclodextrin, or in the sequence of the synthetic procedure. The results show that there are two optimum combinations: (1) DNP-TFM randomly substituted on the β-cyclodextrin as the chiral selector along with a carbamate linkage chain bonding it to the silica support; and (2) β-cyclodextrin derivatized by DNP-TFM substituents only on the C-2 and C-3 positions of the cyclodextrin with an ether linkage chain anchoring it to the silica gel. These two combinations show complementary separations for some enantiomers. The spacer chain effect is much more pronounced for the CSP based on the β-cyclodextrin derivatives with DNP-TFM substituents only on C-2 and C-3 positions than its randomly substituted counterpart. The sequence of derivatizing the cyclodextrin and attaching it to silica gel also affects its selectivity and efficiency. The β-cyclodextrin should be derivatized before it is linked to the silica gel.     

HPLC-DAD–MS Analysis of Anthocyanins in Rose Wine Made From cv. Öküzgözü Grapes,and Effect of Maceration Time on Anthocyanin Content

The anthocyanin composition of rose wine made from cv. Öküzgözü, a variety of grape native to Turkey, and the effect of maceration time (3, 6, 12, 18, or 24 h) on the anthocyanin content of the wine have been investigated. High-performance liquid chromatography with diode-array detection (HPLC-DAD) and mass spectrometry (MS) were used for anthocyanin analysis. Thirteen different anthocyanins, including five glucosides, five acetyl glucosides, and three coumaroyl glucosides were identified and quantified. It was found that an important characteristic of Öküzgözü rose wine was the presence of a large amount of malvidin-3-glucoside and its acylated esters. The total anthocyanin content of the wine increased with increasing maceration time. On the basis of sensory analysis, the most preferred wine was that produced with 3-h maceration, because of its better color and general flavor attributes.     

Analysis of Zoledronic Acid and Its Related Substances by Ion-Pair RP-LC

A simple high-performance liquid chromatographic method – ion-pair reversed- phase high performance liquid chromatography (RPIC) has been developed and employed for the analysis of zoledronic acid and its related substances in bulk material and commercial dosage forms. The mobile phase was a mixture of methanol (20%) and 5 mmol L^–1 phosphate buffer (80%) containing 6 mmol L^–1 tetrabutylammonium bromide, adjusted to pH 7.0 with sodium hydroxide. C₈ column was used as the stationary phase. The chromatographic conditions were optimized. The active ingredient – zoledronic acid was successfully separated from its related substances, including remained imidazol-1-yiacetic acid in the synthesis of zoledronic acid and other possible impurities of oxidation and decomposition. The excipients did not interfere with the determination of zoledronic acid in commercial dosage formulations. The method was rapid, simple, accurate and reproducible. It was not only successfully employed for the assay of zoledronic acid in bulk material and pharmaceutical dosage forms but also for the determination of its related substances.     

Cystine and cysteine analysis as the same phenylthiocarbamyl derivatives by HPLC in protein hydrolyzates

Summary A new approach is described, and a novel explanation presented, for the high performance liquid chromatographic analysis of cystine and cysteine as their phenylthiocarbamyl derivatives. PTC cystine and cysteine have been eluted with the same retention times and molar responses, most probably due to electrophilic attack of phenylisothiocyanate on cystine resulting in the scission of the disulfide bond yielding two moles of cysteine. Further, total PTC cystine and cysteine have been measured both in model solutions and in standard protein hydrolyzates (lysozyme, bovine albumin, ribonuclease) with the same linearity as the other ineteen amino acids. The reproducibility of the measurements, at the 250–750 pmole level, proved to be 4.1% (Relative Standard Deviation %) or less.