Capillary zone electrophoresis method for determination of bitter (alpha- and beta-) acids in hop (Humulus lupulus L.) cone extracts.

PURPOSE: Humulus lupulus (H. lupulus), more commonly known as hop, is a member of the Cannabaceae family with male and female flowers on separate plants. It is native in Europe including Lithuania, Asia and North America. Hop has been recognized as a medicinal plant for centuries, nevertheless different medicinal activities of hop are currently investigated and discovered. An important class of hop compounds is the hop acids, which are classified as alpha-acids and beta-acids. Different varieties of hops vary in amount and composition of hop acids. METHODS: Simple capillary zone electrophoresis method has been optimized and applied for the analysis of hop acids in hop cone extracts. RESULTS: With this method the analysis takes ca. 10 min. Repeatability for migration times and peak areas expressed as relative standard deviation were up to 0.21% and 5.96%, respectively. CONCLUSIONS: Comparative results of capillary zone electrophoretic analysis of extracts of different hop varieties and conductometric titration, as a standard method for determination of alpha-acids, are presented. Both methods provide consistent results, however capillary zone electrophoresis is capable of separating co- form of humulones from other forms.

Capillary electrophoretic analysis of flavonoids in single-styled hawthorn (Crataegus monogyna Jacq.) ethanolic extracts.

Flavonoids are an important group of natural compounds, which can prevent coronary heart disease and have antioxidant properties. Hawthorn is a well known and widely used medicinal plant due to its cardiotonic activity. Previous studies refer mostly to the HPLC analysis of the flavonoids: vitexin, quercetin, hyperoside, oligomeric procyanidins, which appear to be primarily responsible for the cardiac action of the plant. Aqueous ethanolic extracts of single-styled hawthorn (Crataegus monogyna Jacq., f.: Rosaceae Juss.) leaves and sprouts were analyzed by means of capillary zone electrophoresis (CZE). Influence of vegetation period on the extract qualitative composition and flavonoids quantities was evaluated. Sample preparation by extraction using different concentration of aqueous ethanol (40-96%, v/v) and the influence of extractant composition on the recovery of flavonoids are discussed in detail. The results obtained using CZE are compared to the results of spectrophotometric and HPLC analysis of the extracts. The effect of storage conditions of extracts (solar irradiation, temperature and duration) on degradation of flavonoids was investigated.

Non-particulate (continuous bed or monolithic) acrylate-based capillary columns for reversed-phase liquid chromatography and electrochromatography.

Three approaches are described to synthesize acrylic non-particulate beds (also called continuous beds or monoliths) in aqueous polymerization media for reversed-phase capillary liquid chromatography/electrochromatography. In the first, hexyl acrylate comonomer was dissolved together with water soluble polar comonomers using a non-ionic detergent. In the second, a new alkyl ammonium salt comonomer, (3-allylamino-2-hydroxypropyl)dodecyldimethylammonium chloride was used, which is water soluble and has detergent properties itself. The alkyl group of this comonomer provides hydrophobicity while the ionic groups generate electroosmosis in the non-particulate bed. In the third approach, the alkyl comonomer was used as a detergent to dissolve another hydrophobic comonomer in an aqueous polymerization medium. All three approaches were evaluated with respect to hydrophobicity, efficiency and electroosmotic properties of the beds. Hydrophobicity expressed as methylene group selectivity for the three types of the beds in 50% methanol mobile phase was 1.86, 1.16 and 1.78, electroosmotic mobility -5.14 x 10(-5), 6.89 x 10(-5) and 6.37 x 10(-5) cm2 V(-1) s(-1) and efficiency for the retained compound (methylparabene) 67,000, 93,000 and 110,000 plates m(-1) correspondingly. The columns were tested using pressure driven capillary chromatography and capillary electrochromatography. The influence of polymerization temperature on hydrodynamic permeability, separation impedance and inverse size exclusion porosimetry characteristics were used to evaluate the separation columns. The increase of the polymerization temperature resulted higher permeability of the bed, separation impedance and lower polymeric skeleton porosity. Further characterisation was provided by examining the separation efficiency observed for a series of benzoic acid esters and alkyl parabens.

Dynamic electric field assisted multi-dimensional liquid chromatography of biological samples.

Complex biological samples require very high resolution separation strategies. The platform introduced here capitalises on the hyphenation of liquid chromatographic (LC) and electric potential gradient electrochromatographic multi-dimensional separation genres. First-dimension selectivity is provided by simultaneous size exclusion (SEC) and strong cation exchange (SCX) chromatography modes, while the second dimension comprises reversed phase (RP) characteristics in a dynamic (time-variant) electric field. The time-variant potential gradient with reversal of polarity is applied across the second dimension monolithic capillary throughout the duration of the solvent strength gradient elution. Hence, the platform offers comprehensive on-line sample clean-up (matrix depletion, analyte enrichment), fractionation (first dimention LC), and separation (second dimension LC) with the prospect of altering selectivity via polarity reversal dynamic electric field tuning.

Polyrotaxane approach for synthesis of continuous beds for capillary electrochromatography.

The polyrotaxane formation approach was evaluated for synthesis of continuous beds for capillary electrochromatography. This approach has the advantage of generating diverse electroosmotic and chromatographic properties without chemical reactions. The polyrotaxane derivatized continuous beds were formed adding the macrocyclic compounds to the solution of neutral acrylic monomers and crosslinker prior to the initiation of the polymerisation. Cationic and anionic derivatives of beta-cyclodextrin were used as macrocyclic compounds. Investigation of the electroosmotic properties indicated a template directed and enthalpy controlled self-assembly of the polyrotaxanes during the polymerisation of the continuous beds. This process was monomer-composition dependent and favored by the hydrophobicity of the polymeric skeleton. The morphology of the continuous beds was evaluated using high-resolution optical microscopy with CCD camera and atomic force microscopy. Reversed-phase capillary chromatography driven by electroosmosis, originating from the polyrotaxane structure, was performed using several test mixtures. Not primarily designed for the chiral chromatography the polyrotaxane derivatized continuous beds demonstrated enantioselective separation of D,L-metoprolol. The stability of the polyrotaxane derivatized continuous beds was tested. The beds demonstrated reproducible electroosmotic properties in the range from pH 4 to pH 9 (RSD=0.69%).

Synthesis and characterization of polyrotaxane-based polymeric continuous beds for capillary electrochromatography.

The continuous bed technique with its attractive features, such as fritless design, one-step in situ synthesis, low back pressure and no need for pressurising the electrode vessels to suppress bubble formation was applied to form polyrotaxane-based stationary phases for capillary electrochromatography (CEC). Rotaxanes are synthesized from two classes of substances, namely linear reactive monomers and inert cyclic compounds. Upon polymerisation, a gel forms with the cyclic molecules mechanically immobilized (see Fig. 1). We have employed this simple approach, using charged derivatives of cyclodextrins in order to introduce charged groups into continuous beds and thus render them appropriate for electrochromatography. The self-assembly of supramolecular structures to form rotaxanes during the synthesis of the continuous beds is treated. The electroosmotic and chromatographic properties of the various polyrotaxane-based stationary phases synthesized are discussed, as well as the synthesis of the continuous beds, including how to affect their porosity and its influence on the efficiency of the electrokinetic separation. The applicability of the rotaxane-based continuous bed is demonstrated by separation of model compounds by reversed- and normal-phase chromatography. A separation of enantiomers is also presented. This experiment is of particular interest because it indicates that the interaction with the cavity of beta-cyclodextrin (beta-CD) is not a fundamental requirement for enantioseparations.

Continuous beds with vancomycin as chiral stationary phase for capillary electrochromatography.

Enantiomeric separations in capillary electrochromatography (CEC) carried out using a continuous-bed chiral stationary phase (CSP) based on the macrocyclic antibiotic, vancomycin, is presented. The continuous beds were prepared from methacryloxypropyl modified fused silica capillaries (100 microm ID) by in situ copolymerization of N-(hydroxymethyl)acrylamide and piperazine diacrylamide with vinyl sulfonic acid comonomer used to introduce ionic functionality and thus a strong electroosmotic flow (EOF). The CSP was subsequently prepared by immobilizing the vancomycin stationary phase by reductive amination. Preliminary results have indicated that an extremely strong EOF is obtained in both the nonaqueous polar organic (15.2 x 10(-5) cm2 V(-1) s(-1) and the aqueous reversed-phase modes of operation (8.5 x 10(-5) cm2 V(-1) s(-1)). Enantioselectivity was obtained for four racemic compounds, the best of which was in the case of thalidomide which was separated in 10 minutes with high resolution (Rs = 2.5) and efficiency (120,000 plates meter(-1)) values.