Alternative method for microenzymatic inhibition activities monitoring from Baccharis trimera extracts by CZE-UV.

INTRODUCTION: Capillary zone electrophoresis with direct UV detection (CZE-UV) was used to investigate the hypothesis about the extract of Baccharis trimera enzymatic activities as an analytical approach to monitoring the phenomenon. OBJECTIVE: The aim of this work was to investigate enzymatic bioactivities of the hydroalcoholic and infusion extracts of B. trimera through screening evaluation of the inhibition of the enzymes acetylcholinesterase (AChE) and α-glycosidase (α-GLY). METHOD: An alternative approach using CZE-UV to hydroalcoholic and infusion extracts of B. trimera monitoring was applied to evaluate the inhibition ability of the enzymes AChE and α-GLY. The result of the reaction of acetylthiocholine (AThCh) with AChE was thiocholine (TCh) and acetic acid, and from the amount of TCh generated, the AChE inhibition was calculated. For the inhibition study of the two enzymes, the reactions of the extracts were optimised to be performed in situ, inside the capillary column, and the introduction of the solutions was performed through ordered sequential plug injections. RESULTS: Samples extracted with 70% ethanol presented 7.80% inhibition for AChE and 0.51% for α-GLY, while samples extracted with 96% ethanol resulted in 6.89% inhibition for AChE and no inhibition activity for α-GLY. CONCLUSION: In the present work, the potentialities of CZE-UV for the study of hydroalcoholic and infusion extracts of B. trimera were demonstrated. The experimental results were useful for the calculation of the percentage of the inhibition activities of the AChE and α-GLY enzymes.

The novel Outer Plug Standardization (OPS) calibration strategy applied in a new method for the high-throughput determination of Cl-, NO3- and SO4-2 in rainwater samples.

This paper presents the Outer Plug Standardization - OPS, a novel technique as an alternative for the classic internal standardization using multiple injection in capillary zone electrophoresis is proposed herein. This technique was applied in a new method for the determination of chloride, nitrate and sulfate in rainwater samples. After the injection accuracy was tested and proved to be a minor error source (average 1.26% RSD), the OPS was applied and it improved the intra-day and inter-day precision of the analytical method by 32.5% and 24.7%, respectively. Using a capillary with effective length of 23.5 cm the electrophoretic separation of the three inorganic anions could be achieved in <1 min, with detection limits of 0.05, 0.09 and 0.11 mg L-1 for chloride, nitrate and sulfate, respectively. Also, the statistical t-test was applied to the results obtained for 82 rainwater samples that were collected and analyzed applying both the method developed in this study and the official APHA 4140 method where no statistical difference was noted within a 95% confidence level.

Pre-colonial culture and technology through elemental and molecular analysis of ceramics with decorative paintings found at Tapera beach, Florianópolis, Brazil.

The chemical analysis of archaeological ceramic artifacts can help archaeologists to characterize the technology used in the manufacturing process, and to elucidate aspects related to the way of life of past peoples. In this context, six ceramic fragments found at Tapera Beach in Florianópolis were analyzed. FTIR spectroscopy was used to identify carbonates and oxalic acid originating from the biodegradation process. In the pictorial regions of the ceramics, iron oxides, manganese and chromium were present, along with calcium carbonate, which were used as pigments. In addition, the absence of characteristic bands for kaolinite suggests low firing temperatures (up to 900 °C). EDS confirmed the elements of the molecules identified by FTIR and showed the presence of Al and Si in the primary structure and Fe in the secondary structure of the ceramics. In addition, there were traces of rare earth elements in samples A3 and A5, which may be related to the geochemistry at the site from which the raw material was excavated. The GC-MS results showed the presence of lipids and the profile of the fatty acids detected suggested that the source of the oils adsorbed in the ceramic structure is vegetable.

Lavandula angustifolia essential oil inhalation reduces mechanical hyperalgesia in a model of inflammatory and neuropathic pain: The involvement of opioid and cannabinoid receptors.

Lavandula angustifolia (LaEO) essential oil has been widely used by aromatherapy in the treatment of various clinical conditions, with evidence of its analgesic and anti-inflammatory potential. Our results demonstrate that sixty-five substances were identified in LaEO. Among the compounds found, the major ones were linalool (30.61%) and linalyl acetate (20.36%). We found that LaEO inhalation reduces mechanical hyperalgesia in conditions of chronic inflammatory and neuropathic pain. Furthermore, this effect seems to be mediated by peripheral and central opioid and cannabinoid 2 receptors. The findings of the present study suggests that the LaEO inhalation is effective on the chronic pain treatment.

Inhibitory and Cooperative Effects Regulated by pH in Host-Guest Complexation between Cationic Pillar[5]arene and Reactive 2-Carboxyphthalanilic Acid.

The study of host-guest complexation between reactive 2-carboxyphthalanilic acid (CPA) and two cationic pillararenes has been carried out. Host-guest complexation with significant kinetic effects was observed only with the smaller cavity size pillararene (P5A). Kinetics in the pH range 1.50-6.40, ESI-MS, 1H NMR titration, and ROESY experiments were performed to characterize the complexes. High binding stoichiometry (H:G2) was observed for all CPA protonation states. The system is pH-dependent, and inversion of cooperativity (negative to positive) occurs by increasing the dianionic CPA2- concentration (allosteric behavior). Toward physiological pH, association constant K1:1 does not change (104 M-1), and K1:2 increased from 102 to 104 M-1, as well as the inhibitory effect increased up to 222-fold. NMR results elucidated the structure of the complex and allowed us to create a map of H-H interactions that describes well the diversity and number of interactions in the complex.

Dual-opposite end multiple injection method applied to sequential determination of Na+, K+, Ca+2, Mg+2 ions and free and total glycerol in biodiesel by capillary zone electrophoresis.

A novel method for the sequential determination of sodium, potassium, calcium, and magnesium and free and total glycerol in biodiesel by capillary zone electrophoresis is proposed herein. The inorganic cations were separated along an effective length of 43.5 cm. The samples to quantify the free and total glycerol were injected into the opposite capillary end, close to the detection window, with an effective length of 8.5 cm. It was possible to achieve the separation of six analytes within 3 min. The quantification limits for the cations and glycerol ranged from 0.071 to 0.5 mg kg-1 to and 0.0017% to 0.017% w/w, respectively. Despite the complexity of the injection steps, the values for the instrumental, intraday and interday precision were better than 2.13, 4.49 and 5.68% (RSD), respectively, for the cations and the free and total glycerol. The method has good accuracy and specificity, which was statistically confirmed through an interlaboratory assay, where the method was compared with official methods.

In vivo antitumor activity of by-products of Passiflora edulis f. flavicarpa Deg. Rich in medium and long chain fatty acids evaluated through oxidative stress markers, cell cycle arrest and apoptosis induction.

Antiinflammatory and antitumor activity has been reported in Passiflora edulis (yellow passion fruit) nevertheless the intrinsic mechanisms of action are not fully elucidated. The present study aimeds to perform a comparison between the antitumor activity involving the crude extract (HCE) and the supercritical fluid extract with ethanol as co-solvent (SFEtOH) from P. edulis f. flavicarpa Deg. The in vitro cytotoxicity was evaluated in MCF-7 cells, while the in vivo antitumor activity was assessed in male Balb/c mice inoculated with Ehrlich carcinoma cells. SFEtOH exhibited higher antitumor activity compared to HCE. Wherein, SFEtOH showed an EC50 of 264.6 µg/mL against MCF-7 cells as well as an increased inhibition of tumor growth of 48.5% (p < 0.001) in male Balb/c mice, thereby promoting an increased mice lifespan to approximately 42%. Moreover, SFEtOH caused lipid (p < 0.001) and protein (p < 0.001) oxidation by increasing glutathione redox cycle activity while decreased the thioredoxin reductase activity (p < 0.001). SFEtOH also induced oxidative DNA damage in Ehrlich ascites carcinoma (EAC) cells leading to G2/M cycle arrest and has increased apoptotic cells up to 48.2%. These data suggest that the probable mechanisms of antitumor effect are associated to the lipid, protein and DNA damage, leading to cell cycle arrest and triggering apoptosis via mitochondrial pathway, should be probable due to the presence of medium and long chain fatty acids such as lauric acid.

Eugenia brasiliensis leaves extract attenuates visceral and somatic inflammatory pain in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Eugenia brasiliensis Lam. (Myrtaceae) is a Brazilian tree distributed throughout Atlantic rain forest, since Bahia until Santa Catarina state, and is popularly known as "grumixaba, grumixameira, cumbixaba, ibaporoiti, and cereja-brasileira". The bark and leaves of Eugenia brasiliensis are used in folk medicine as adstringent, diuretic, energizing, anti-rheumatic and anti-inflammatory. This study aimed at investigating the chemical composition, antinociceptive and anti-inflammatory effect of the hydroalcoholic extract of Eugenia brasiliensis (HEEb). MATERIAL AND METHODS: Chemical composition of the HEEb was determined by High Performance Liquid Chromatography/ESI-Mass Spectrometry (HPLC-ESI-MS/MS). The antinociceptive and anti-inflammatory effects of HEEb (30-300 mg/kg) was verified in mice after oral administration by intra-gastric gavage (i.g.) 60 min prior to experimentation. It was investigated whether HEEb decreases visceral pain and leukocyte migration induced by an intraperitoneal (i.p.) injection of acetic acid (0.6%). We also evaluated whether HEEb decreases nociceptive behavior induced by formalin (including paw edema and temperature), prostaglandin E2 (PGE2), histamine, and compound 48/80. Finally, we evaluated the effect of HEEb in the chronic inflammatory (mechanical and thermal hypersensitivity) pain induced by complete Freund's adjuvant (CFA), as well as quantifying the concentration of the pro-inflammatory cytokines TNF-α and IL-6 in the paw by ELISA method. RESULTS: Seven polyphenols were identified in HEEb by HPLC-ESI-MS/MS analysis. HEEb treatment alleviated nocifensive behavior and leukocyte migration caused by acetic acid. Moreover, HEEb also reduced the inflammatory pain and paw temperature induced by formalin, as well as it decreased nociceptive behavior induced by histamine and compound 48/80. Finally, acute and repeated treatment of animals with HEEb (100 mg/kg, i.g.) markedly reduced the mechanical and thermal (heat) hypersensitivity, besides decrease paw edema and temperature induced by CFA, and this effect was evident until the day 7. Moreover, repeated treatment with HEEb (100 mg/kg, i.g.) significantly reduced the levels of IL-6 and TNF-α in the paw when compared to the CFA group. CONCLUSIONS: This is the first report showing that HEEb presents antinociceptive and anti-inflammatory effects in the visceral and somatic inflammatory pain in mice, possibly involving the inhibition of histamine receptors and pro-inflammatory cytokines activated pathways. Our results are of interest because they support the use of Eugenia brasiliensis as a potential source of phytomedicine for inflammatory diseases and pain.

Chemical composition, antioxidant and antibacterial activities of essential oils from leaves and flowers of Eugenia klotzschiana Berg (Myrtaceae)

ABSTRACT Many essential oils (EOs) of different plant species possess interesting antimicrobial effects on buccal bacteria and antioxidant properties. Eugenia klotzschiana Berg (pêra-do-cerrado, in Portuguese) is a species of Myrtaceae with restricted distribution in the Cerrado. The essential oils were extracted through the hydrodistillation technique using a modified Clevenger apparatus (2 hours) and chemically characterized by GC-MS. The major compounds were α-copaene (10.6 %) found in oil from leaves in natura, β-bisabolene (17.4 %) in the essential oil from dry leaves and α-(E)-bergamotene (29.9 %) in oil from flowers. The antioxidant activity of essential oils showed similarities in both methods under analysis (DPPH and ABTS˙+) and the results suggested moderate to high antioxidant activity. The antibacterial activity was evaluated by determining minimum inhibitory concentrations (MICs), using the microdilution method. MIC values below 400 µg/mL were obtained against Streptococcus salivarius (200 µg/mL), S. mutans (50 µg/mL), S. mitis (200 µg/mL) and Prevotella nigrescens (50 µg/mL). This is the first report of the chemical composition and antibacterial and antioxidant activities of the essential oils of E. klotzschiana. These results suggest that E. klotzschiana, a Brazilian plant, provide initial evidence of a new and alternative source of substances with medicinal interest.

Simultaneous determination of aspartame, cyclamate, saccharin and acesulfame-K in powder tabletop sweeteners by FT-Raman spectroscopy associated with the multivariate calibration: PLS, iPLS and siPLS models were compared.

For the first time, a procedure for simultaneous determination of the main artificial sweeteners, aspartame (ASP), cyclamate (CYC), saccharin (SAC), and acesulfame-K (ACSK) by a spectroscopic method associated with the multivariate calibration is proposed. These analytes were quantified in tabletop sweeteners samples using FT-Raman spectroscopy. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used as reference method. Partial least squares (PLS), interval PLS (iPLS), and synergism PLS (siPLS) methods were evaluated in a comparative study where the selected interval models presented better results. Multivariate regression models, such as PLS, iPLS and siPLS were built and the lower root mean square errors for prediction (RMSEP) found were 0.027-0.031% w/w, 0.316-0.363% w/w, 0.082-0.184% w/w, and 0.040-0.049% w/w to ASP, CYC, SAC, and ACSK, respectively. The coefficient of determination for prediction (R2p) varied between 0.978 and 0.979, 0.969-0.977, 0.952-0.994, and 0.959-0.965 for ASP, CYC, SAC and ACSK, respectively. The analysis of model's residues was made by bias and permutation tests to evaluate systematic and trend errors. The selected intervals by iPLS and siPLS were evaluated and the bands related to the vibrational modes of the analytes were assigned with the aid of density functional theory calculations (DFT).

Chemical composition, antioxidant and antibacterial activities of essential oils from leaves and flowers of Eugenia klotzschiana Berg (Myrtaceae).

Many essential oils (EOs) of different plant species possess interesting antimicrobial effects on buccal bacteria and antioxidant properties. Eugenia klotzschiana Berg (pêra-do-cerrado, in Portuguese) is a species of Myrtaceae with restricted distribution in the Cerrado. The essential oils were extracted through the hydrodistillation technique using a modified Clevenger apparatus (2 hours) and chemically characterized by GC-MS. The major compounds were α-copaene (10.6 %) found in oil from leaves in natura, ß-bisabolene (17.4 %) in the essential oil from dry leaves and α-(E)-bergamotene (29.9 %) in oil from flowers. The antioxidant activity of essential oils showed similarities in both methods under analysis (DPPH and ABTS˙+) and the results suggested moderate to high antioxidant activity. The antibacterial activity was evaluated by determining minimum inhibitory concentrations (MICs), using the microdilution method. MIC values below 400 µg/mL were obtained against Streptococcus salivarius (200 µg/mL), S. mutans (50 µg/mL), S. mitis (200 µg/mL) and Prevotella nigrescens (50 µg/mL). This is the first report of the chemical composition and antibacterial and antioxidant activities of the essential oils of E. klotzschiana. These results suggest that E. klotzschiana, a Brazilian plant, provide initial evidence of a new and alternative source of substances with medicinal interest.

Determination of phenolic profile by HPLC-ESI-MS/MS and anti-inflammatory activity of crude hydroalcoholic extract and ethyl acetate fraction from leaves of Eugenia brasiliensis

ABSTRACT Eugenia brasiliensis Lam., Myrtaceae, is used in folk medicine for anti-inflammatory diseases such as arthritis and rheumatism. This study investigated the anti-inflammatory activity and phenolic profile of the crude hydroalcoholic extract and ethyl acetate fraction from E. brasiliensis leaves. Crude hydroalcoholic extract and the ethyl acetate fraction were analyzed by HPLC-ESI-MS/MS in comparison to standard phenolic compounds. The anti-inflammatory activity of the crude hydroalcoholic extract (1, 10 and 25 mg kg-1) and the ethyl acetate fraction (10, 25 and 50 mg kg-1) was evaluated in a swiss mouse model of acute pleurisy induced by carrageenan, being the total cell count, exudation and analysis of nitrite/nitrate the inflammation parameters. HPLC-ESI-MS/MS analysis revealed apigenin, catechin, galangin, isoquercetin, myricetin, quercetin and rutin. Crude hydroalcoholic extract and ethyl acetate fraction were effective in inhibiting cell migration in all tested doses. Crude hydroalcoholic extract was effective in inhibiting exudation only at the 10 mg kg-1 dose; ethyl acetate fraction was effective in all tested doses. Results for nitrite/nitrate levels reveals that only the ethyl acetate fraction was effective at the tested doses. This is the first report of the presence of isoquercetin, galangin and apigenin in this species. Results from the phytochemical analysis enhance the chemical knowledge of this species. In the future, together with more studies, validation of its popular use in inflammatory diseases is possible.

Development of a fast method for simultaneous determination of hippuric acid, mandelic acid, and creatinine in urine by capillary zone electrophoresis using polymer multilayer-coated capillary.

This paper reports the development of a fast separation method employing capillary zone electrophoresis for the simultaneous determination of hippuric acid, mandelic acid, and creatinine in samples of urine using a coated capillary. The background electrolyte was composed of 10 mmol L-1 tris(hydroxymethyl)aminomethane and 30 mmol L-1 2-hydroxy-isobutyric acid at pH 3.6. The internal standard was 3,5-dihydroxybenzoic acid. Separations were performed in a fused silica capillary (32 cm total length, 8.5 cm effective length, and 50 µm internal diameter) coated with crosslinked hydroxypropyltrimethyl ammonium chloride chitosan and κ-carrageenan. Direct UV detection was performed at a wavelength of 200 nm. Samples and standards were injected hydrodynamically (-50 mbar, 3 s) using the short-end injection procedure. The electrophoretic system was operated under constant voltage of 30 kV with positive polarity on the injection side. The separation time for hippuric acid, mandelic acid, and creatinine was less than 70 s. The evaluation of some analytical parameters of the method for the three analytes showed good linearity (R 2 > 0.99), limit of detections of 0.21 to 0.63 mg L-1, inter-day precision better than 3.0% (peak area), and recovery in the range of 98 to 106%. The method developed was applied in the analysis of the three analytes in urine samples. Graphical Abstract New method using capillary zone electrophoresis for analysis of creatinine, hippuric acid and mandelic acid in urine.

High-Throughput Analysis of Lidocaine in Pharmaceutical Formulation by Capillary Zone Electrophoresis Using Multiple Injections in a Single Run.

This paper reports the development of a subminute separation method by capillary zone electrophoresis in an uncoated capillary using multiple injection procedure for the determination of lidocaine in samples of pharmaceutical formulations. The separation was performed in less than a minute leading to doing four injections in a single run. The cathodic electroosmotic flow contributed to reducing the analyses time. The background electrolyte was composed of 20 mmol L(-1) 2-amino-2-(hydroxymethyl)-1,3-propanediol and 40 mmol L(-1) 2-(N-morpholino)ethanesulfonic acid at pH 6.1. The internal standard used was benzylamine. Separations were performed in a fused uncoated silica capillary (32 cm total length, 23.5 cm effective length, and 50 µm internal diameter) with direct UV detection at 200 nm. Samples and standards were injected hydrodynamically using 40 mbar/3 s interspersed with spacer electrolyte using 40 mbar/7 s. The electrophoretic system was operated under constant voltage of 30 kV with positive polarity on the injection side. The evaluation of some analytical parameters of the method showed good linearity (r (2) > 0.999), a limit of detection 0.92 mg L(-1), intermediate precision better than 3.2% (peak area), and recovery in the range of 92-102%.

Antinociceptive effect of hydroalcoholic extract and isoflavone isolated from Polygala molluginifolia in mice: evidence for the involvement of opioid receptors and TRPV1 and TRPA1 channels.

PURPOSE: The plants of the genus Polygala (Polygalaceae) have been used for a long time in folk medicine to treat pain and inflammation. The species Polygala molluginifolia is native to southern Brazil and is popularly known as "cânfora". The presented study analyzes the antinociceptive effect of hydroalcoholic extract from Polygala molluginifolia (HEPm) and an isoflavone (ISO) isolated from the extract, in behavioral models of pain in mice, as well as the mechanism underlying this effect. MATERIALS AND METHODS: The phytochemical analysis of HEPm was performed through a capillary electrophoresis analysis and colorimetric test. The antinociceptive effects of HEPm and ISO (10-1000 mg/kg, i.g.) were evaluated by applying the formalin test; mechanical and thermal hyperalgesia to postoperative pain in mice. The possible involvement of opioid receptors, TRPV1 and TRPA1 channels in the antinociceptive effect of HEPm and ISO were also evaluated. Finally, the nonspecific effects of HEPm and ISO were evaluated by measuring locomotor activity (Open-field Test) and corporal temperature. RESULTS: The 5,3',4'-trihydroxy-6″,6″-dimethylpyrano[2″,3″:7,6] isoflavone (ISO) was identified in HEPm by capillary electrophoresis analysis and selected for the experimental tests. The oral administration of HEPm or of ISO significantly inhibited the neurogenic and inflammatory phases of formalin-induced pain, edema formation and local hyperemia, without causing any change to locomotor activity. Acute and repeated treatment of animals with HEPm reduced mechanical and thermal (heat and cold) hyperalgesia in the postoperative pain. In addition, administering HEPm or ISO markedly reduced nociceptive behavior induced by the peripheral and central injection of TRPV1 and TRPA1 channels activators. Finally, the antinociception provided by the administration of HEPm or ISO was reversed by the preadministration of naloxone. CONCLUSIONS: Taken together, these results provide the first experimental evidence of the significant antinociceptive effect of HEPm and ISO in animal models of acute pain without causing sedation or locomotor dysfunction. This effect appears to be mediated, at least in part, by the activation of opioid receptors and/or by the inhibition of TRPV1 and TRPA1 channels. Moreover, this study adds new scientific evidence and highlights the therapeutic potential of the medicinal plant Polygala molluginifolia in the development of phytomedicines with analgesic properties.

Inhalation of Cedrus atlantica essential oil alleviates pain behavior through activation of descending pain modulation pathways in a mouse model of postoperative pain.

ETHNOPHARMACOLOGICAL RELEVANCE: Cedrus atlantica essential oil (CaEO) presents analgesic and anti-inflammatory sedative properties. However, it remains unknown whether CaEO alleviates acute postoperative pain. MATERIALS AND METHODS: Here, we investigated the effect of CaEO on postoperative pain and its mechanisms related to the descending pain control in Swiss males mice induced by a plantar incision surgery (PIS) in the hindpaw. RESULTS: Inhalation of CaEO (5', 30' or 60') markedly reduced mechanical hypersensitivity. This effect was prevented by pre-treatment with naloxone or p-chlorophenylalanine methyl ester (PCPA, 100mg/kg, i.p.)-induced depletion of serotonin. In addition, p-alpha-methyl-para-tyrosin (AMPT, 100mg/kg, i.p.)-induced depletion of norepinephrine, intraperitoneal injection of the α2-adrenergic receptor antagonist yohimbine (0.15 mg/kg, i.p.) or haloperidol (1mg/kg, i.p.) an antagonist of dopaminergic (D1 and D2) receptors prevented the effect of CaEO on hypersensitivity. CONCLUSIONS: These findings suggest that CaEO alleviates postoperative pain by activating the descending pain modulation pathways on the opioidergic, serotonergic, noradrenergic (α2-adrenergic) and dopaminergic (dopamine D1 and D2 receptors) systems.

Superficially Porous Particle Based Hydroxypropyl-ß-cyclodextrin Stationary Phase for High-Efficiency Enantiomeric Separations.

A superficially porous particle (SPP)-based hydroxypropyl-ß-cyclodextrin (HPBCD) chiral stationary phase (CSP) was produced and its chromatographic performance was compared to both 5 µm and 3 µm fully porous particle (FPP)-based CSPs. The relative surface coverage of the HPBCD chiral selector on each particle was approximately equal, which resulted in equivalent enantiomeric selectivity (α) values on each phase when constant mobile phase conditions were used. Under such conditions, the SPP column resulted in greatly reduced analysis times and three times greater efficiencies compared to the FPP columns. When higher flow rates were used, efficiency gains per analysis times were five times greater for the SPP column compared to the FPP-based columns. When the mobile phases were altered to give similar analysis times on each column, resolution values were doubled for the SPP column. Finally, the novel SPP based HPBCD column proved to be stable for 500 injections under high flow rate (4.5 mL/min) and high pressure (400 bar) conditions used for an ultrafast (~45 sec) enantiomeric separation.

Comparison of superficially porous and fully porous silica supports used for a cyclofructan 6 hydrophilic interaction liquid chromatographic stationary phase.

A new HILIC stationary phase comprised of native cyclofructan-6 (CF6) bonded to superficially porous silica particles (2.7µm) was developed. Its performance was evaluated and compared to fully porous silica particles with 5µm (commercially available as FRULIC-N) and 3µm diameters. Faster and more efficient chromatography was achieved with the superficially porous particles (SPPs). The columns were also evaluated in the normal phase mode. The peak efficiency, analysis time, resolution, and overall separation capabilities in both HILIC and normal phase modes were compared. The analysis times using the superficially porous based column in HILIC mode were shorter and the theoretical plates/min were higher over the entire range of flow rates studied. The column containing the superficially porous particles demonstrated higher optimum flow rates than the fully porous particle packed columns. At higher flow rates, the advantages of the superficially porous particles was more pronounced in normal phase separations than in HILIC, clearly demonstrating the influence that the mode of chromatography has on band broadening. However, the minimum reduced plate heights (hmin) were typically lower in HILIC than in the normal phase mode. Overall, the superficially porous particle based CF6 column showed clear advantages over the fully porous particle columns, in terms of high throughput and efficient separations of polar compounds in the HILIC mode.

Superficially porous particles vs. fully porous particles for bonded high performance liquid chromatographic chiral stationary phases: isopropyl cyclofructan 6.

This work reports a comparison of HPLC separations of enantiomers with chiral stationary phases (CSPs) prepared by chemically bonding cyclofructan-6, functionalized with isopropyl carbamate groups on fully and superficially porous particles (SPPs). The chromatographic performance of the superficially porous CSP based column was compared with columns packed with 5 µm and 3 µm fully porous particles (FPPs). At a flow rate of 3.0 mL/min the number of plates on column afforded by the SPP column was ∼7× greater than the number of plates on column (same length) obtained when using the 5 µm FPP based column. The flow rate providing the highest efficiency separation was ∼1.0 mL/min for the SPP column while it was ∼0.5 mL/min for both FPP columns. It was found that the selectivity and resolution of the separations were comparable between fully porous and superficially porous based columns (under constant mobile phase conditions), even though the SPP column contained lower absolute amounts of chiral selector. When tested under constant retention conditions, the SPP based CSP greatly improved resolution compared to the FPP based columns. At high flow rates the efficiency gained by using superficially porous CSP was accentuated. The advantages of columns based on SPPs become more obvious from the viewpoint of plate numbers and resolution per analysis time.

New multilayer coating using quaternary ammonium chitosan and κ-carrageenan in capillary electrophoresis: application in fast analysis of betaine and methionine.

The aim of this study was to develop a new multilayer coating with crosslinked quaternary ammonium chitosan (hydroxypropyltrimethyl ammonium chloride chitosan; HACC) and κ-carrageenan for use in capillary electrophoresis. A new semi-permanent multilayer coating was formed using the procedure developed and the method does not require the presence of polymers in the background electrolyte (BGE). The new capillary multilayer coating showed a cathodic electroosmotic flow (EOF) of around 30×10(-9) m(2) V(-1) s(-1) which is pH-independent in the range of pH 2 to 10. The enhanced EOF at low pH obtained contributed significantly to the development of a fast method of separation. The multilayer coating was then applied in the development of a fast separation method to determine betaine and methionine in pharmaceutical formulations by capillary zone electrophoresis (CZE). The BGE used to determine the betaine and methionine concentrations was composed of 10 mmol L(-1) tris(hydroxymethyl) aminomethane, 40 mmol L(-1) phosphoric acid and 10% (v/v) ethanol, at pH 2.1. A fused-silica capillary of 32 cm (50 µm ID×375 µm OD) was used in the experiments and samples and standards were analyzed employing the short-end injection procedure (8.5 cm effective length). The instrumental analysis time of the optimized method was 1.53 min (approx. 39 runs per hour). The validation of the proposed method for the determination of betaine and methionine showed good linearity (R(2)>0.999), adequate limit of detection (LOD <8 mg L(-1)) for the concentration in the samples and inter-day precision values lower than 3.5% (peak area and time migration). The results for the quantification of the amino acids in the samples determined by the CZE-UV method developed were statistically equal to those obtained with the comparative LC-MS/MS method according to the paired t-test with a confidence level of 95%.