Practical assessment of frictional heating effects and thermostat design on the performance of conventional (3 μm and 5 μm) columns in reversed-phase high-performance liquid chromatography

A practical investigation of frictional heating effects in conventional C18 columns was undertaken, to investigate whether problems found for sub-2 μm columns were also present for those of particle size 3 μm and 5 μm and different internal diameter. The influence of a water bath, a still air heater, and a forced air heater on performance was investigated. Heating effects were substantial, with a decrease in k of almost 15% for toluene over the flow rate range ∼0.4–2.3 mL/min with a 15 cm × 0.46 cm ID column packed with 3 μm particles. Heating effects on retention increased with increasing solute k, with increase in the column ID, with decrease in the column particle size, and with decrease in the set column oven temperature. While the water bath minimised axial temperature gradients and thus its effect on k, radial temperature gradients were potentially serious with this system, especially at high mobile phase velocity, even with columns containing 5 μm particles. In contrast to the effects of axial temperature gradients in 4.6 mm columns, very little difference in Van Deemter plots was noted between the three different thermostats with 2 mm ID columns, even when 3 μm particles were used. However, the efficiency of 2 mm columns for peaks of low or moderate k (k < 4) can be compromised by the extra dead volume introduced by the heating systems, even with conventional HPLC systems with otherwise minimised extra column volume.     

Comparison of pyrimethanil-imprinted beads and bulk polymer as stationary phase by non-linear chromatography

A pyrimethanil-imprinted polymer (P1) was prepared by iniferter-mediated photografting a mixture of methacrylic acid and ethylene dimethacrylate onto homemade near-monodispersed chloromethylated polydivinylbenzene beads. The chromatographic behaviour of a column packed with these imprinted beads was compared with another column packed with irregular particles obtained by grinding a bulk pyrimethanil-imprinted polymer (P2). The comparison was made using the kinetic model of non-linear chromatography, studying the elution of the template and of two related substances, cyprodinil and mepanipyrim. Extension of the region of linearity, capacity factors for the template and the related substances, column selectivity, binding site heterogeneity, apparent affinity constant (K) and lumped kinetic association (k_a) and dissociation rate constant (k_d) were studied during a large interval of solute concentration, ranging between 1 and 2000 μg/ml. From the experimental results obtained, in the linearity region of solute concentration column selectivity and binding site heterogeneity remained essentially the same for the two columns, while column capacity (at 20 μg/ml, P1 = 23.1, P2 = 11.5), K (at 20 μg/ml, P1 = 8.3 × 10⁶ M⁻¹, P2 = 2.5 × 10⁶ M⁻¹) and k_a (at 20 μg/ml, P1 = 3.5 μM⁻¹ s⁻¹, P2 = 0.47 μM⁻¹ s⁻¹) significantly increased and k_d (at 20 μg/ml, P1 = 0.42 s⁻¹, P2 = 0.67 s⁻¹) decreased for the column packed with the imprinted beads. These results are consistent with an influence of the polymerisation method on the morphology of the resulting polymer and not on the molecular recognition properties due to the molecular imprinting process.     

Reactions of organo-rhodium complexes with multidentate N,N and N,S-heterocycles and exchange studies by NMR

Dihalobridged binuclear complexes [Rh(diolefin)(μ-X)]₂ {diolefin = 1,5-cyclooctadiene (cod), X = Cl or Br; diolefin = norbornadiene (nbd), X = Cl}, undergo halide bridge cleavage reactions with multidentate N,N-heterocycles 1,3,5-tris(benzimidazolyl)benzene (L¹H₃), 1,3,5-tris(N-methylbenzimidazolyl)benzene (L²H₃) and N,S-heterocycle 1,3,5-tris(benzothiazolyl)benzene (L³H₃) to yield trinuclear heterocycle bridged complexes [{RhX(cod)}₃(μ-LH₃)] and [{RhCl(nbd)}₃(μ-LH₃)] (LH₃ = L¹H₃, L²H₃, L³H₃). ¹H NMR exchange measurements have shown resonances for olefinic protons 1″, 2″, 5″ and 6″ of cod at different chemical shifts, perhaps due to restricted Rh–N bond rotation. The olefinic and aliphatic protons would undergo exchange with each other and also with intermediate species. The exchange mechanism may be visualized to involve Rh–N bond breaking, rotation of the cod ligand of the T-shaped (three-coordinate) intermediate species followed by recomplexation. An alternate mechanism may be Rh–cod bond breaking at olefin positions 5″ and 6″, isomerisation of the T-complex such that 5″/6″ moves trans to X coupled with rotation of the heterocycle about the Rh–N bond (made easier by the reduced coordination number of the intermediate), followed by recoordination of 1″/2″ trans to N, followed by recomplexation. NMR signals from the intermediate species in one dimensional ¹H, ¹³C and 2D NMR spectra have supported the exchange of protons.     

Synthesis of B-trisubstituted borazines via the rhodium-catalyzed hydroboration of alkenes with N,N′,N″-trimethyl or N,N′,N″-triethylborazine

Hydroboration of terminal and internal alkenes with N,N′,N″-trimethyl- and N,N′,N″-triethylborazine was carried out at 50 °C in the presence of a rhodium(I) catalyst. Addition of dppb or DPEphos (1 equiv.) to RhH(CO)(PPh₃)₃ gave the best catalyst for hydroboration of ethylene at 50 °C, resulting in a quantitative yield of B,B′,B″-triethyl-N,N′,N″-trimethylborazine. On the other hand, a complex prepared from (t-Bu)₃P (4 equiv.) and [Rh(coe)₂Cl]₂ gave the best yield for hydroboration of terminal or internal alkenes.     

Separation of intact proteins on porous layer open tubular (PLOT) columns

Porous layer open tubular (PLOT) polystyrene divinylbenzene columns have been used for separating intact proteins with gradient elution. The 10 μm I.D. × 3 m columns were easily coupled to standard liquid chromatography–mass spectrometry (LC–MS) instrumentation with commercially available fittings. Standard proteins separated on PLOT columns appeared as narrow and symmetrical peaks with good resolution. Average peak width increased linearly with gradient time (t_G) from 0.14 to 0.33 min (t_G 20 and 120 min, respectively) using a 3 m column. With shorter columns, peak widths were larger and increased more steeply with gradient time. Theoretical peak capacity (n_c) increased with column length (tested up to 3 m). The n_c increased with t_G until a plateau was reached. The highest peak capacity achieved (n_c = 185) was obtained with a 3 m column, where a plateau was reached with t_G 90 min. The within- and between column retention time repeatabilities were below 0.6% and below 2.5% (relative standard deviation, RSD), respectively. The carry-over following injection of 0.5 ng per protein was less than 1.1%. The retention time dependence on column temperature was investigated in the range 20–50 °C. Proteins in a skimmed milk sample were separated using the method.     

Calorimetric investigation of the effect of hydroxyanthraquinones in Rheum officinale Baill on Staphylococcus aureus growth

The inhibitory effects of five hydroxyanthraquinones (HAQs) from root and rhizoma of Rheum officinale Baill, a traditional Chinese medicinal (TCM) herb, on Staphylococcus aureus growth were investigated by calorimetry. The power-time curves of S.aureus with and without HAQ were acquired and the extent and duration of inhibitory effects on the metabolism evaluated by growth rate constants (k₁, k₂), half inhibitory ratio (IC₅₀), maximum heat output (P_max) and peak time (t_p). The value of k₁ and k₂ of S. aureus in the presence of the five HAQs decreased with the increasing concentrations of HAQs. Moreover, P_max was reduced and the value of t_p increased with increasing concentrations of the five drugs. The inhibitory activity varied for different drugs. IC₅₀ of the five HAQs was 4 μg ml⁻¹ for emodin, 3.5 μg ml⁻¹ for rhein, 10 μg ml⁻¹ for aloe-emodin, 1000 μg ml⁻¹ for chrysophanol, 1600 μg ml⁻¹ for physcion. The sequence of antimicrobial activity of the five HAQs: rhein > emodin > aloe-emodin > chrysophanol > physicion.     

Probing the kinetic performance limits for ion chromatography. I. Isocratic conditions for small ions

The first use of the kinetic plot method to characterise the performance of ion-exchange columns for separations of small inorganic anions is reported. The influence of analyte type (mono- and divalent), particle size (5 and 9 μm), temperature (30 and 60 °C) and maximum pressure drop upon theoretical extrapolations was investigated using data collected from anion-exchange polymeric particulate columns. The quality of extrapolations was found to depend upon the choice of analyte, but could be verified by coupling a series of columns to demonstrate some practical solutions for ion chromatography separations requiring relatively high efficiency. Separations of small anions yielding 25–40,000 theoretical plates using five serially connected columns (9 μm particles) were obtained and yielded deviations of <15% from the kinetic plot predictions. While this approach for achieving high efficiencies results in a very long analysis time (t₀ = 21 min), separations yielding approximately 10,000 theoretical plates using two serially connected columns (t₀ < 5 min) were shown to be more practically useful for isocratic separations when compared to use of a single column operated at optimum linear velocity (t₀ > 10 min).     

Downscaling a multicommuted flow injection analysis system for the photometric determination of iodate in table salt

In this work a downscaled multicommuted flow injection analysis setup for photometric determination is described. The setup consists of a flow system module and a LED based photometer, with a total internal volume of about 170 μL. The system was tested by developing an analytical procedure for the photometric determination of iodate in table salt using N,N-diethyl-henylenediamine (DPD) as the chromogenic reagent. Accuracy was accessed by applying the paired t-test between results obtained using the proposed procedure and a reference method, and no significant difference at the 95% confidence level was observed. Other profitable features, such as a low reagent consumption of 7.3 μg DPD per determination; a linear response ranging from 0.1 up to 3.0 m IO₃⁻, a relative standard deviation of 0.9% (n = 11) for samples containing 0.5 m IO₃⁻, a detection limit of 17 μg L⁻¹ IO₃⁻, a sampling throughput of 117 determination per hour, and a waste generation 600 μL per determination, were also achieved.     

Steric and electronic effects in stabilizing allyl-palladium complexes of “P-N-P” ligands, X2PN(Me)PX2 (X = OC6H5 or OC6H3Me2-2,6)

The chemistry of η³-allyl palladium complexes of the diphosphazane ligands, X₂PN(Me)PX₂ [X = OC₆H₅ (1) or OC₆H₃Me₂-2,6 (2)] has been investigated.The reactions of the phenoxy derivative, (PhO)₂PN(Me)P(OPh)₂ with [Pd(η³-1,3-R′,R″-C₃H₃)(μ-Cl)]₂ (R′ = R″ = H or Me; R′ = H, R″ = Me) give exclusively the palladium dimer, [Pd₂{μ-(PhO)₂PN(Me)P(OPh)₂}₂Cl₂] (3); however, the analogous reaction with [Pd(η³-1,3-R′,R″-C₃H₃)(μ-Cl)]₂ (R′ = R″ = Ph) gives the palladium dimer and the allyl palladium complex [Pd(η³-1,3-R′,R″-C₃H₃)(1)](PF₆) (R′ = R″ = Ph) (4). On the other hand, the 2,6-dimethylphenoxy substituted derivative 2 reacts with (allyl) palladium chloro dimers to give stable allyl palladium complexes, [Pd(η³-1,3-R′,R″-C₃H₃)(2)](PF₆) [R′ = R″ = H (5), Me (7) or Ph (8); R′ = H, R″ = Me (6)].Detailed NMR studies reveal that the complexes 6 and 7 exist as a mixture of isomers in solution; the relatively less favourable isomer, anti-[Pd(η³-1-Me-C₃H₄)(2)](PF₆) (6b) and syn/anti-[Pd(η³-1,3-Me₂-C₃H₃)(2)](PF₆) (7b) are present to the extent of 25% and 40%, respectively. This result can be explained on the basis of the steric congestion around the donor phosphorus atoms in 2. The structures of four complexes (4, 5, 7a and 8) have been determined by X-ray crystallography; only one isomer is observed in the solid state in each case.     

Determination of ternary mixtures of penicillin G, benzathine and procaine by liquid chromatography and factorial design study

In the present work, a rapid and sensitive method for simultaneous determination of penicillin G (PG), benzathine (BE) and procaine (PR) in drug and serum media is introduced. The polar hydro-organic (55/45) mobile phases containing an aqueous solution adjusted to pH = 3.7 and an organic solvent (MeOH) including triethylamine (TEA) and trifluroacetic acid (TFA) are used. The flow rate of 1 ml min⁻¹, a C₈ column (150 mm × 46 mm) with 5 μm i.d. and wavelength at 215 nm are selected for optimal separation condition. The limit of detection (LOD), linear concentration range and relative standard deviation (R.S.D.) of this method for the PG are 1.1 μg ml⁻¹, 10-2400 μg ml⁻¹ and 1.7% and for the BE are 1.2 μg ml⁻¹, 12-2100 μg ml⁻¹ and 1.8% and for the PR are 1.5 μg ml⁻¹, 20-2000 μg ml⁻¹ and 2%, respectively. The factorial design is used for the determination of main and interaction effects of pH, flow rate and concentration of MeOH, TEA and TFA in the separation at two levels. Also, the analysis of variance (ANOVA) table is obtained. The results show that TFA and TEA have higher effect than concentration of MeOH, pH and flow rate factors.     

Hydride addition at μ-vinyliminium ligand obtained from disubstituted alkynes

New μ-vinylalkylidene complexes cis-[Fe₂{μ-η¹:η³-C_γ(R′)C_β(R″)C_αHN(Me)(R)}(μ-CO)(CO)(Cp)₂] (R = Me, R′ = R″ = Me, 3a; R = Me, R′ = R″ = Et, 3b; R = Me, R′ = R″ = Ph, 3c; R = CH₂Ph, R′ = R″ = Me, 3d; R = CH₂Ph, R′ = R″ = COOMe, 3e; R = CH₂ Ph, R′ = SiMe₃, R″ = Me, 3f) have been obtained b yreacting the corresponding vinyliminium complexes [Fe₂{μ-η¹:η³-C_γ(R′)C_β(R″)C_αN(Me)(R)}(μ-CO)(CO)(Cp)₂][SO₃CF₃] (2a-f) with NaBH₄. The formation of 3a-f occurs via selective hydride addition at the iminium carbon (C_α) of the precursors 2a-f. By contrast, the vinyliminium cis-[Fe₂{μ-η¹:η³-C_γ (R′) = C_β(R″)C_α = N(Me)(Xyl)}(μ-CO)(CO)(Cp)₂][SO₃CF₃] (R′ = R″ = COOMe, 4a; R′ = R″ = Me, 4b; R′ = Prⁿ, R″ = Me, 4c; Prⁿ = CH₂CH₂CH₃, Xyl = 2,6-Me₂C₆H₃) undergo H⁻ addition at the adjacent C_β, affording the bis-alkylidene complexes cis-[Fe₂{μ-η¹:η²-C(R′)C(H)(R″)CN(Me)(Xyl)}(μ-CO)(CO)(Cp)₂], (5a-c). The cis and trans isomers of [Fe₂{μ-η¹:η³-C_γ(Et)C_β(Et)C_αN(Me)(Xyl)}(μ-CO)(CO)(Cp)₂][SO₃CF₃] (4d) react differently with NaBH₄: the former reacts at C_α yielding cis-[Fe₂{μ-η¹:η³-C_γ(Et)C_β(Et)C_αHN(Me)(Xyl)}(μ-CO)(CO)(Cp)₂], 6a, whereas the hydride attack occurs at C_β of the latter, leading to the formation of the bis alkylidene trans-[Fe₂{μ-η¹:η²-C(Et)C(H)(Et)CN(Me)(Xyl)}(μ-CO)(CO)(Cp)₂] (5d). The structure of 5d has been determined by an X-ray diffraction study. Other μ-vinylalkylidene complexes cis-[Fe₂{μ-η¹:η³-C_γ(R′)C_β(R″)C_αHN(Me)(Xyl)}(μ-CO)(CO)(Cp)₂], (R′ = R″ = Ph, 6b; R′ = R″ = Me, 6c) have been prepared, and the structure of 6c has been determined by X-ray diffraction. Compound 6b results from treatment of cis-[Fe₂{μ-η¹:η³-C_γ(Ph)C_β(Ph)C_αN(Me)(Xyl)}(μ-CO)(CO)(Cp)₂][SO₃CF₃] (4e) with NaBH₄, whereas 6c has been obtained by reacting 4b with LiHBEt₃. Both cis-4d and trans-4d react with LiHBEt₃ affording cis-6a.     

High-performance liquid chromatography determination of triptolide in vitro permeation studies

A simple, rapid and sensitive high-performance liquid chromatography (HPLC) method has been developed for the determination of triptolide. Triptolide was separated from skin endogenous and blank matrices on a 5 μm LiChrospher RP-C₁₈ column by a mobile phase of methanol-water (65:35, v/v). The permeation samples were injected directly without pretreatment. The limit of quantitation (LOQ) and detection (LOD) for triptolide in permeation samples were far below (0.01 and 0.005 μg/mL, respectively). The method was linear over the range of 0.1-104.2 μg/mL with r² = 0.9999. This HPLC assay is promising for measuring in vitro percutaneous penetration of triptolide through mice skins and also can be performed in the triptolide-loaded microemulsions formulation screening.     

Surfactant induced softening in gelatin hydrogels

Effect of binding of three surfactants, alpha olefin sulfonate (AOS, anionic), Triton-X100 (TX-100, non-ionic) and cetyl trimethyl ammonium bromide (CTAB, cationic) to the hydrogels of gelatin was studied at room temperature (25 °C) by dynamic light scattering and oscillatory rheology with surfactant concentrations (20-100 mM) much larger than the critical micellar concentrations (cmc) of these surfactants. The measured intensity auto-correlation function of light scattered from gels revealed the presence of finite heterodyne contribution ≈0.11 ± 0.01 that increased to ≈0.25 ± 0.02 after transition to the soft gel state indicating a softening process for surfactant concentrations exceeding 50 mM. The dynamic structure factor S(q, t) of micelle bound gelatin gels revealed two clearly identifiable relaxation modes namely; the fast mode, S(q, t) ∼ exp · (−D_fq²t) for t ? 1 ms and a stretched exponential mode, S(q, t) ∼ exp · −(t/τ_c)^β for 1 ms ? t ? 1 s. This behaviour was universal with β ≈ 0.85 ± 0.04 independent of the surfactant type. The low frequency (1.5 rad/s) storage modulus G′, loss modulus G″ and tan δ behaviour revealed a gradual softening of the gel independent of the surfactant type. The exponent (β) fast mode diffusivity (D_f) and stretched exponential mode relaxation time were found to be less sensitive to this softening transition.     

Fast gradient screening of pharmaceuticals with 5 cm long, narrow bore reversed-phase columns packed with sub-3 μm core-shell and sub-2 μm totally porous particles

The performance of 5 cm long narrow-bore columns packed with 2.6-2.7 μm core-shell particles and a column packed with 1.7 μm totally porous particles was compared in very fast gradient separations of polar neutral active pharmaceutical compounds. Peak capacities as a function of flow-rate and gradient time were measured. Peak capacities around 160-170 could be achieved within 25 min with these 5 cm long columns. The highest peak capacity was obtained with the Kinetex column however it was found that as the flow-rate increases, the peak capacity of the new Poroshell-120 column is getting closer to that obtained with the Kinetex column. Considering the column permeability, peak capacity per unit time and per unit pressure was also calculated. In this comparison the advantage of sub-3 μm core-shell particles is more significant compared to sub-2 μm totally porous particles. Moreover it was found that the very similar sized (d_p = 2.7 μm) and structured (ρ = 0.63) new Poroshell-120 and the earlier introduced Ascentis Express particles showed different efficiency. Results obtained showed that the 5 cm long narrow bore columns packed with sub-3 μm core-shell particles offer the chance of very fast and efficient gradient separations, thus these columns can be applied for fast screening measurements of routine pharmaceutical analysis such as cleaning validation.     

Measurement of methyl mercury (I) and mercury (II) in fish tissues and sediments by HPLC-ICPMS and HPLC-HGAAS

A procedure for the extraction and determination of methyl mercury and mercury (II) in fish muscle tissues and sediment samples is presented. The procedure involves extraction with 5% (v/v) 2-mercaptoethanol, separation and determination of mercury species by HPLC-ICPMS using a Perkin-Elmer 3 μm C8 (33 mm × 3 mm) column and a mobile phase 3 containing 0.5% (v/v) 2-mercaptoethanol and 5% (v/v) CH₃OH (pH 5.5) at a flow rate 1.5 ml min⁻¹ and a temperature of 25 °C. Calibration curves for methyl mercury (I) and mercury (II) standards were linear in the range of 0-100 μg l⁻¹ (r² = 0.9990 and r² = 0.9995 respectively). The lowest measurable mercury was 0.4 μg l⁻¹ which corresponds to 0.01 μg g⁻¹ in fish tissues and sediments. Methyl mercury concentrations measured in biological certified reference materials, NRCC DORM - 2 Dogfish muscle (4.4 ± 0.8 μg g⁻¹), NRCC Dolt - 3 Dogfish liver (1.55 ± 0.09 μg g⁻¹), NIST RM 50 Albacore Tuna (0.89 ± 0.08 μg g⁻¹) and IRMM IMEP-20 Tuna fish (3.6 ± 0.6 μg g⁻¹) were in agreement with the certified value (4.47 ± 0.32 μg g⁻¹, 1.59 ± 0.12 μg g⁻¹, 0.87 ± 0.03 μg g⁻¹, 4.24 ± 0.27 μg g⁻¹ respectively). For the sediment reference material ERM CC 580, a methyl mercury concentration of 0.070 ± 0.002 μg g⁻¹ was measured which corresponds to an extraction efficiency of 92 ± 3% of certified values (0.076 ± 0.04 μg g⁻¹) but within the range of published values (0.040-0.084 μg g⁻¹; mean ± s.d.: 0.073 ± 0.05 μg g⁻¹, n = 40) for this material. The extraction procedure for the fish tissues was also compared against an enzymatic extraction using Protease type XIV that has been previously published and similar results were obtained. The use of HPLC-HGAAS with a Phenomenox 5 μm Luna C18 (250 mm × 4.6 mm) column and a mobile phase containing 0.06 mol l⁻¹ ammonium acetate (Merck Pty Limited, Australia) in 5% (v/v) methanol and 0.1% (w/v) l-cysteine at 25 °C was evaluated as a complementary alternative to HPLC-ICPMS for the measurement of mercury species in fish tissues. The lowest measurable mercury concentration was 2 μg l⁻¹ and this corresponds to 0.1 μg g⁻¹ in fish tissues. Analysis of enzymatic extracts analysed by HPLC-HGAAS and HPLC-ICPMS gave equivalent results.     

Synthesis, structure, and bridge-terminal alkyl exchange kinetics of pyrazolate-bridged dialuminum complexes containing bridging n-alkyl groups

Treatment of triethylaluminum with 3,5-diphenylpyrazole in a 2:1 stoichiometry afforded the ethyl-bridged complex Et₂Al(μ-Ph₂pz)(μ-Et)AlEt₂ (79%) as a colorless crystalline solid. Treatment of tri-n-propylaluminum with 3,5-di-tert-butylpyrazole in a 2:1 stoichiometry afforded the n-propyl-bridged complex (nPr)₂Al(μ-tBu₂pz)(μ-nPr)Al(nPr)₂ (63%) and the dimeric complex [(nPr)₂Al(μ-tBu₂pz)]₂ (3%), respectively, as colorless crystalline solids. Treatment of tri-n-propylaluminum (1 equiv.) or triisobutylaluminum (1 or 2 equiv.) with 3,5-di-tert-butylpyrazole afforded exclusively the dimeric complexes [(nPr)₂Al(μ-tBu₂pz)]₂ (68%) or [(iBu)₂Al(μ-tBu₂pz)]₂ (96%), respectively, as colorless crystalline solids. The solid state structures of Et₂Al(μ-Ph₂pz)(μ-Et)AlEt₂ and (nPr)₂Al(μ-tBu₂pz)(μ-nPr)Al(nPr)₂ consist of 3,5-disubstituted pyrazolato ligands with a di-n-alkylalumino group bonded to each nitrogen atom. An ethyl or n-propyl group acts as a bridge between the two aluminum atoms. The kinetics of the bridge-terminal exchange was determined for the bridging n-alkyl complexes by ¹³C NMR spectroscopy, and afforded ΔH^‡ = 1.5 ± 0.1 kcal/mol, ΔS^‡ = −46.8 ± 39.0 cal/K mol, and for Et₂Al(μ-Ph₂pz)(μ-Et)AlEt₂ and ΔH^‡ = 1.7 ± 0.1 kcal/mol, ΔS^‡ = −46.6 ± 43.4 cal/K mol, and for (nPr)₂Al(μ-tBu₂pz)(μ-nPr)Al(nPr)₂. The negative values of ΔS^‡ imply ordered transition states relative to the ground states, and rotation along the N-AlR₃ vector without aluminum-nitrogen bond cleavage is proposed.     

Enthalpy change and mechanism of oxidation of o-phenylenediamine by hydrogen peroxide catalyzed by horseradish peroxidase

The hydrogen peroxide-oxidation of o-phenylenediamine (OPD) catalyzed by horseradish peroxidase (HRP) at 37 °C in 50 mM phosphate buffer (pH 7.0) was studied by calorimetry. The apparent molar reaction enthalpy with respect to OPD and hydrogen peroxide were −447 ± 8 kJ mol⁻¹ and −298 ± 9 kJ mol⁻¹, respectively. Oxidation of OPD by H₂O₂ catalyzed by HRP (1.25 nM) at pH 7.0 and 37 °C follows a ping-pong mechanism. The maximum rate V_max (0.91 ± 0.05 μM s⁻¹), Michaelis constant for OPD K_m,S (51 ± 3 μM), Michaelis constant for hydrogen peroxide Km,H₂O₂ (136 ± 8 μM), the catalytic constant k_cat (364 ± 18 s⁻¹) and the second-order rate constants k₊₁ = (2.7 ± 0.3) × 10⁶ M⁻¹ s⁻¹ and k₊₅ = (7.1 ± 0.8) × 10⁶ M⁻¹ s⁻¹ were obtained by the initial rate method.     

Lipophilicity data for some preservatives estimated by reversed-phase liquid chromatography and different computation methods

The chromatographic behavior of some preservatives was performed by reversed-phase high-performance liquid chromatography on C18 (LiChroCART, Purosphere RP-18e), C8 (Zorbax, Eclipse XDB-C8), CN100 (Säulentechnik, Lichrosphere) and NH₂ (Supelcosil LC-NH₂) columns. The lipophilicity estimated for the first time on the first three columns are comparable and very well correlated. The mobile phase was a mixture of methanol–water (0.1% formic acid) in different volume proportions from 40% to 60% (v/v) for RP-C18, RP-C8 and RP-CN100 column (exception for parabens on RP-C8 column—the methanol concentrations being from 55% to 65%) and from 30% to 50% (v/v) for RP-NH₂. Highly significant correlations were obtained between different experimental indices of lipophilicity (log k_w, S, φ₀, mean of k and log k, and scores of k and log k corresponding to the first principal component) and computed log P values, and C8 column seems to be more suited for estimating the lipophilicity of the investigated compounds. These direct correlations offer a very good opportunity to derive powerful predictive models via Collander-type equations. The reliability of scores values as lipophilic indices is shown by their high correlation with the log K_ow obtained using classical “shake-flask” technique, log k_w and also some of the computed log P values. In addition, the results obtained applying PCA to the retention data may be used in interpreting the molecular mechanism of interactions between eluents and stationary phases with different polarity and to explain the chromatographic behavior of compounds. Finally, the “congeneric lipophilicity chart” described by the scores corresponding to the first principal component has the effect of separating compounds from each other more effectively from congeneric ((dis)similarity) point of view. The parabens and tert-butylhydroquinone appeared to be the most lipophilic preservatives.     

Characterization of new types of stationary phases for fast and ultra-fast liquid chromatography by signal processing based on AutoCovariance Function: A case study of application to Passiflora incarnata L. extract separations

In this study, a comparative investigation was performed of HPLC Ascentis^® (2.7 μm particles) columns based on fused-core particle technology and Acquity^® (1.7 μm particles) columns requiring UPLC instruments, in comparison with Chromolith™ RP-18e columns. The study was carried out on mother and vegetal tinctures of Passiflora incarnata L. on one single or two coupled columns. The fundamental attributions of the chromatographic profiles are evaluated using a chemometric procedure, based on the AutoCovariance Function (ACVF). Different chromatographic systems are compared in terms of their separation parameters, i.e., number of total chemical components (m_tot), separation efficiency (σ), peak capacity (n_c), overlap degree of peaks and peak purity. The obtained results show the improvements achieved by HPLC columns with narrow size particles in terms of total analysis time and chromatographic efficiency: comparable performance are achieved by Ascentis^® (2.7 μm particle) column and Acquity^® (1.7 μm particle) column requiring UPLC instruments. The ACVF plot is proposed as a simplified tool describing the chromatographic fingerprint to be used for evaluating and comparing chemical composition of plant extracts by using the parameters D% – relative abundance of the deterministic component – and c_EACF – similarity index computed on ACVF.     

A rapid, acetonitrile-free, HPLC method for determination of melamine in infant formula

A simple, precise, accurate and validated, acetonitrile-free, reverse phase high performance liquid chromatography (HPLC) method is developed for the determination of melamine in dry and liquid infant formula. The separation is performed on a Kromasil C18 column (150 mm × 3.2 mm I.D., 5 μm particle size) at room temperature. The mobile phase (0.1% TFA/methanol 90:10) is pumped at a flow rate of 0.3 mL min⁻¹ with detection at 240 nm. Melamine elutes at 3.7 min. A linear response (r > 0.999) is observed for samples ranging from 1.0 to 80 μg mL⁻¹. The method provides recoveries of 97.2-101.2% in the concentration range of 5-40 μg mL⁻¹, intra- and inter-day variation in <1.0% R.S.D. The limit of detection (LOD) and limit of quantification (LOQ) values are 0.1 μg mL⁻¹ and 0.2 μg mL⁻¹, respectively.