Hydrodynamic chromatography separations in micro- and nanopillar arrays produced using deep-UV lithography

We report on a series of hydrodynamic chromatography separations conducted in micropillar array columns with an interpillar distance spacing of, respectively, 1.00, 0.70, and 0.47 μm. The columns have been produced using state-of-the-art deep-UV lithography and deep reactive ion etching techniques. Despite the fact that the efficiency was smaller than theoretically possible (due to fabrication limitations and significant injection and detection band broadening), it was nevertheless possible to separate mixtures of fluorescein isothiocyanate (used as the t(0) -marker) and 20- and 40-nm polystyrene beads. With the smallest interpillar distance, a resolution of R(s) = 0.5 between the 20- and 40-nm particles could be obtained in 90s over a column length of 4 cm. The selectivity obtained in the pillar array columns was found to be very similar to that observed in packed-bed columns. By detecting the fluorescent signals in a 90-μm-deep detection groove at the end of the column, the signal-to-noise ratio could be enhanced up to 150 times.     

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.     

Stepwise dansyl grafting on the kaolinite interlayer surface

A block copolymer (PS-b-poly(l-Glu)) composed of polystyrene and poly(l-glutamic acid) was used as a stabilizer for dispersion polymerization of styrene. When dispersion polymerization of styrene was conducted at 70 °C in 80% dimethylformamide-water with 0.5 wt% PS-b-poly(l-Glu), spherical polystyrene particles with D_n = 0.72 μm and narrow size distribution were obtained. Whereas AIBN concentration did not have any effects on particle size, molecular weight of the polystyrene particles was strongly dependent on the initiator concentration. As concentration of the PS-b-poly(l-Glu) increased from 0.2 to 1.0 wt%, particle size decreased from D_n = 0.91 to 0.69 μm with keeping surface area occupied by one poly(l-glutamic acid) chain about S = 50 nm². On the other hand, an increase in initial concentration of styrene from 2 to 20 wt% caused an increase in particle size from D_n = 0.48 to 1.36 μm and a decrease in surface area per poly(l-glutamic acid) block from S = 91 to 45 nm². Colloidal stability of the polystyrene particles in aqueous solution was responsive to pH due to the surface-grafted poly(l-glutamic acid). For dispersion polymerization of styrene, the PS-b-poly(l-Glu) functions as both a stabilizer and a surface modifier.     

Effect of an open tube in series with a packed capillary column on liquid chromatographic performance: The influence of particle diameter,temperature, and system pressure

A postcolumn reactor or a simple open tube connecting a capillary column to, for example, a mass spectrometer affects the performance of a capillary liquid chromatography system in two ways: stealing pressure from the column and adding band-spreading. This effect is especially intolerable in fast separations. Our calculations show that in the presence of a 25 μm radius postcolumn reactor, column (50 μm radius) efficiency (number of theoretical plates) is severely reduced by more than 75% with a t₀ of 10 s and a particle diameter from 1 to 5 μm for unretained solutes at room temperature. Therefore, it is necessary to minimize the reactor's effect and to improve the column efficiency by optimizing postcolumn conditions. We derived an equation that defines the observed number of theoretical plates (N_obs) taking into account the two effects stated above, which is a function of the maximum pressure P_m, the particle diameter d_p, the reactor radius a_r, the column radius a_c, the desired dead time t₀, the column temperature T and zone capacity factor k″. Poppe plots were obtained by calculations using this equation. The results show that for a t₀ shorter than 18 s, a P_m of 4000 psi, and a d_p of 1.7 μm, a 5 μm radius reactor has to be used. Such a small reactor is difficult to fabricate. Fortunately, high temperature helps to minimize the reactor effect so that reactors with manageable radius (larger than 12.5 μm) can be used in many practical conditions. Furthermore, solute retention diminishes the influence of a postcolumn reactor. Thus, a 12.5 μm reactor supersedes a 5 μm reactor for retained solutes even at a t₀ of 5 s (k″ > 3.8, or k′ > 2.0).     

Visualization and quantification of the onset and the extent of viscous fingering in micro-pillar array columns

New experimental data of the viscous fingering (VF) process have been generated by studying the VF process in perfectly ordered pillar array columns instead of in the traditionally employed packed bed columns. A detailed quantitative analysis of the contribution of VF to the observed band broadening could be made by following the injected species bands using a fluorescence microscope equipped with a CCD-camera. For a viscosity contrast of 0.16 cP, a plate height increase of about 1 μm can be observed, while for a contrast of respectively 0.5 cP and 1 cP, additional plate height contributions of the order of 5–20 μm were observed. Citing these values is however futile without noting that they also depend extremely strongly on the injection volume of injected sample. It was found that, for a given viscosity contrast of 0.314 cP, the maximal plate height increase varied between 0.5 μm and 18 μm if the injection volume was varied between 3.0 nl and 32.7 nl. These values furthermore also strongly vary with the distance along the column axis.     

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.     

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).     

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.     

An in-tube SPME device for the selective determination of chlorophyll a in aquatic systems

We report a new device for the estimation of the content of chlorophyll a pigment in water samples as an indicator of water quality. The extraction of the pigment from water was also optimized. 10 mL of water was filtered through a nylon filter (45 μm pore size and 13 mm of diameter), after the chlorophylls were dissolved by immersing the filter in 1 mL of a low non-hazardous solvent as ethanol. An in-valve in-tube SPME device coupled to capillary liquid chromatography with diode array detection was designed. A capillary column of 70 cm in length (0.32 mm i.d. coated with 5% diphenyl-95% polydimethylsiloxane, 3 μm coating thickness) was used as the loop of the injection valve for preconcentration and a Zorbax SB C₁₈ (SiO₂-based) 150 mm × 0.5 mm i.d., 5 μm column (Agilent) was used as analytical column. The achieved detection limit was 0.05 μg L⁻¹ and the working range of concentrations was 0.1-1 μg L⁻¹. % RSD values between 2 and 11 were obtained. Chlorophyll a in several water matrices was determined with good results in presence of other pigments such as chlorophyll b, pheophytin a and pheophytin b.     

Microfluidic device capable of sensing ultrafast chemiluminescence

Based on the principle of liquid core waveguide, a novel microfluidic device with micro-scale detection window capable of sensing flashlight emitted from rapid 1,1′-oxalyldi-4-methylimidazole (OD4MI) chemiluminescence (CL) reaction was fabricated. Light emitted from OD4MI CL reaction occurring in the micro-dimensional pentagonal detection window (length of each line segment: 900.0 μm, depth: 50.0 μm) of the microfluidic device with two inlets and one outlet was so bright that it was possible to take an image every 1/30 s at the optimal focusing distance (60 cm) using a commercial digital camera. Peaks obtained using a flow injection analysis (FIA) system with the micro-scale detection window and OD4MI CL detection show excellent resolution and reproducibility without any band-broadening observed in analytical devices having additional reaction channel(s) to measure light generated from slow CL reaction. Maximum height (H_max) and area (A) of peak, reproducibility and sensitivity observed in the FIA system with the microfluidic device and OD4MI CL detection depends on (1) the mole ratio between bis(2,4,6-trichlorophenyl) oxalate and 4-methyl imidazole yielding OD4MI, (2) the flow rate to mix OD4MI, H₂O₂ and 1-AP in the detection window of the microfluidic device, and (3) H₂O₂ concentration. We obtained linear calibration curves with wide dynamic ranges using H_max and A. The detection limit of 1-AP determined with H_max and A was as low as 0.05 fmole/injection (signal/background = 3.0).     

Spatially selected synthesis of LaF3 and Er-doped CaF2 crystals in oxyfluoride glasses by laser-induced crystallization

Oxyfluoride glasses with a small amount of NiO are prepared using a conventional melt quenching technique, and the spatially selected crystallization of LaF₃ and CaF₂ crystals is induced on the glass surface by irradiations of continuous wave lasers with a wavelength of λ=1064 or 1080 nm. Dots and lines including LaF₃ crystals are patterned by heat-assisted (300 °C) laser irradiations (λ=1064 nm) with a power of P=1 W and an irradiation time of 10 s for dots and a scanning speed of S=5 μm/s for lines. Lines consisting of CaF₂ crystals are also patterned in an ErF₃-doped oxyfluoride glass by laser irradiations (λ=1080 nm) with a power of P=1.7 W and a scanning speed of S=2 μm/s, and the incorporation of Er³⁺ ions into CaF₂ crystals is confirmed from micro-photoluminescence spectrum measurements. It is proposed that the lines patterned by laser irradiations in this study are consisted of the composite of LaF₃ or CaF₂ nanocrystals and SiO₂-based oxide glassy phase. It is demonstrated that a combination of Ni²⁺-dopings and laser irradiations is effective in spatially selected local crystallizations of fluorides in oxyfluoride glasses.     

Fibre coupled micro-light emitting diode array light source with integrated band-pass filter for fluorescence detection in miniaturised analytical systems

In this work, a new type of miniaturized fibre-coupled solid-state light source is demonstrated as an excitation source for fluorescence detection in capillary electrophoresis. It is based on a parabolically shaped micro-light emitting diode (μ-LED) array with a custom band-pass optical interference filter (IF) deposited at the back of the LED substrate. The GaN μ-LED array consisted of 270 individual μ-LED elements with a peak emission at 470 nm, each about 14 μm in diameter and operated as a single unit. Light was extracted through the transparent substrate material, and coupled to an optical fibre (OF, 400 μm in diameter, numerical aperture NA = 0.37), to form an integrated μ-LED-IF-OF light source component. This packaged μ-LED-IF-OF light source emitted approximately 225 μW of optical power at a bias current of 20 mA. The bandpass IF filter was designed to reduce undesirable LED light emissions in the wavelength range above 490 nm. Devices with and without IF were compared in terms of the optical power output, spectral characteristics as well as LOD values. While the IF consisted of only 7.5 pairs (15 layers) of SiO₂/HfO₂ layers, it resulted in an improvement of the baseline noise as well as the detection limit measured using fluorescein as test analyte, both by approximately one order of magnitude, with a LOD of 1 × 10⁻⁸ mol L⁻¹ obtained under optimised conditions. The μ-LED-IF-OF light source was then demonstrated for use in capillary electrophoresis with fluorimetric detection. The limits of detection obtained by this device were compared to those obtained with a commercial fibre coupled LED device.     

Synthesis, magnetism and electronic structure of YbNi2−xFexAl8 (x=0.91) isolated from Al flux

The combination of ytterbium, nickel, iron in liquid aluminum resulted in the formation of the new intermetallic compound YbNi_2−xFe_xAl₈ (x=0.91) which adopts the CaCo₂Al₈ structure type with a=14.458(3) Å, b=12.455(3) Å, c=3.9818(8) Å and space group Pbam. Its resistivity drops with decreasing temperature, saturating to a constant value at lower temperatures. Above 50 K, the inverse magnetic susceptibility data follows Curie-Weiss Law, with a calculated μ_eff=2.19 μ_B. Although the observed reduced moment in magnetic susceptibility measurement suggests that the Yb ions in this compound are of mixed-valent nature, ab initio electronic structure calculations within density functional theory using LDA+U approximation give an f¹³ configuration in the ground state.     

Compromise between conjugation length and charge-transfer in nonlinear optical η-monocyclopentadienyliron(II) complexes with substituted oligo-thiophene nitrile ligands: Synthesis, electrochemical studies and first hyperpolarizabilities

A systematic series of η⁵-monocyclopentadienyliron(II) complexes with substituted oligo-thiophene nitrile ligands of general formula [FeCp(P_P)(NC{SC₄H₂}_nNO₂)][PF₆] (P_P = dppe, (+)-diop; n = 1-3) has been synthesized and characterized. The electrochemical behaviour of the new compounds was explored by cyclic voltammetry. Quadratic hyperpolarizabilities (β) of the complexes with dppe coligands have been determined by hyper-Rayleigh scattering (HRS) measurements at two fundamental wavelengths of 1.064 and 1.550 μm, to uncover the two-photon resonance effect and to estimate static β values. The obtained overall results are found to be better than for the related η⁵-monocyclopentadienyliron(II) complexes with p-benzonitrile derivatives. Although an increase of the resonant β at 1.064 μm with increasing number of thiophene units in the conjugated ligand was found (up to 910 × 10⁻³⁰ esu), the static values β₀ remain practically unchanged, as shown by the 1.550 μm measurements. Combined with the electrochemical and spectroscopic data (IR, NMR, UV-vis), this remarkable evolution of β shows that the increase of conjugation length is balanced by a decrease in charge-transfer efficiency.     

Ferromagnetic ordering in ThSi2 type CeAu0.28Ge1.72

The compound CeAu_0.28Ge_1.72 crystallizes in the ThSi₂ structure type in the tetragonal space group I4₁/amd with lattice parameters a=b=4.2415(6) Å c=14.640(3) Å. CeAu_0.28Ge_1.72 is a polar intermetallic compound having a three-dimensional Ge/Au polyanion sub-network filled with Ce atoms. The magnetic susceptibility data show Curie-Weiss law behavior above 50 K. The compound orders ferromagnetically at ∼8 K with estimated magnetic moment of 2.48 μ_B/Ce. The ferromagnetic ordering is confirmed by the heat capacity data which show a rise at ∼8 K. The electronic specific heat coefficient (γ) value obtained from the paramagnetic temperature range 15-25 K is∼124(5) mJ/ mol K². The entropy change due to the ferromagnetic transition is ∼4.2 J/mol K which is appreciably reduced compared to the value of R ln(2) expected for a crystal-field-split doublet ground state and/or Kondo exchange interactions.     

Optimized chromatographic and bioluminescent methods for inorganic pyrophosphate based on its conversion to ATP by firefly luciferase

Two new methods for inorganic pyrophosphate (PPi) quantification are described. They are based on the enzymatic conversion of PPi into ATP by firefly luciferase (Luc, E.C. 1.13.12.7) in the presence of dehydroluciferyl-adenylate (L-AMP) followed by the determination of ATP by one of two different procedures, either UV-monitored (260 nm) ion-pair-HPLC (IP-HPLC) (method A) or luciferase-dependent bioluminescence in the presence of its substrate, firefly luciferin (d-LH₂) (method B). These methods were subjected to optimization using experimental design methodologies to obtain optimum values for the selected factors: method A—incubation time (t_inc = 15 min), inactivation time of the enzyme (t_inac = 2 min), pH of the reaction mixture (pH 7.50) and the concentrations of L-AMP ([L-AMP] = 40 μM) and luciferase ([Luc] = 0.1 μM); method B—concentrations of L-AMP ([L-AMP] = 2 μM), luciferase ([Luc] = 50 nM) and luciferin ([LH₂] = 30 μM). Method A has a linear response over the range of 0.1-20 μM of PPi, with a limit of detection (LOD) of 0.5 μM and a limit of quantitation (LOQ) of 1.8 μM. Precision, expressed as relative standard deviation (R.S.D.), is 7.4% at 1 μM PPi and 5.9% at 8 μM PPi. Method B has a linear response over the range of 0.75-6.0 μM of PPi, with LOD and LOQ of 0.624 and 2.23 μM, respectively, and a R.S.D. of 5.1% at 2.5 μM PPi and 4.9% at 5 μM PPi. Under optimized conditions sensitive and robust methods can be obtained for the analysis of PPi impurities in commercial nucleotides and tripolyphosphate (P₃).     

Silica modified with zirconium oxide for on-line determination of inorganic arsenic using a hydride generation-atomic absorption system

In the present work, an on-line pre-concentration system, using SiO₂/ZrO₂ as solid phase adsorbent, for the determination of low concentrations of arsenic was developed.In this procedure, total inorganic arsenic present in the sample is reduced to As (III) with l-cysteine and the solution is passed into a glass column (?_i = 3 mm; h = 35 mm) containing 70 mg of SiO₂/ZrO₂ (100-230 mesh) to retain the As (III), with the solution flowing at 3.2 mL min⁻¹. After 2 min, the concentrated species are eluted with 3 mol L⁻¹ HCl (3.2 mL min⁻¹) loading a 150 μL loop while a 1.0% (m/v) sodium borohydride solution loads the other loop of a FI-HG AAS system. The elution step is carried out during 7 s and, at the same time, the SiO₂/ZrO₂ phase is regenerated for the next concentration cycle. The arsine generated is carried with N₂ (flow rate = 90 mL min⁻¹) to a quartz cell where the electrothermal atomization process occurs.Under these conditions, a 20-fold enrichment factor is obtained considering the increase of sensitivity in the analytical curve after the concentration step. The limits of detection and quantification were 0.05 and 0.35 μg L⁻¹, respectively, with an injection frequency of about 28 h⁻¹ and relative standard deviation lower than 8%. Four metallurgical reference materials were analyzed with the proposed method after their acid dissolution. The results obtained were in good agreement with certified values.     

Synthesis of nanocrystals in KNb(Ge,Si)O5 glasses and chemical etching of nanocrystallized glass fibers

The nanocrystallization behavior of 25K₂O−25Nb₂O₅-(50−x)GeO₂-xSiO₂ glasses with x=0,25,and50 (i.e., KNb(Ge,Si)O₅ glasses) and the chemical etching behavior of transparent nanocrystallized glass fibers have been examined. All glasses show nanocrystallization, and the degree of transparency of the glasses studied depends on the heat treatment temperature. Transparent nanocrystallized glasses can be obtained if the glasses are heat treated at the first crystallization peak temperature. Transparent nanocrystallized glass fibers with a diameter of about 100 μm in 25K₂O-25Nb₂O₅-50GeO₂ are fabricated, and fibers with sharpened tips (e.g., the taper length is about 450 μm and the tip angle is about 12°) are obtained using a meniscus chemical etching method, in which etching solutions of 10 wt%-HF/hexane and 10 M-NaOH/hexane are used. Although the tip (aperture size) has not a nanoscaled size, the present study suggests that KNb(Ge,Si)O₅ nanocrystallized glass fibers have a potential for new near-field optical fiber probes with high refractive indices of around n=1.8 and high dielectric constants of around ε=58 (1 kHz, room temperature).     

Analysis of salicylic acid based on the fluorescence enhancement of the As(III)-salicylic acid system

A new, simple and sensitive spectrofluorimetric method for the determination of salicylic acid (λ_ex = 315 nm, λ_em = 408 nm) using As(III) as a sensitizing reagent has been investigated by measuring the increase of fluorescence intensity of salicylic acid due to the complexation of As(III)-salicylic acid in presence of sodium dodecyl sulfate (SDS) 10⁻³ M. Under optimum conditions, a significant relationship was obtained between the fluorescence intensity and salicylic acid concentration. A linear calibration curve was obtained in the range 13.8-13812 μg l⁻¹ with product-moment correlation coefficient (R) 0.99985 and detection limit 4.2 μg l⁻¹. The R.S.D. is 2.35% (n = 5).The method was applied successfully to the determination of salicylic acid in human serum.     

Novel tetrahedral tetranickel cluster with alkylidyne ligand (NiCp)4(μ3-CR)

A novel tetrahedral (μ₃-alkylidyne)tetranickel cluster (NiCp)₄(μ₃-C(CH₂)₄CH₃) (4) was obtained in the reaction of nickelocene with potassium and 1-hexene. Compound 4 was characterised by means of MS and X-ray diffraction analysis. It crystallizes in the orthorhombic crystal system and Pna2₁ (No. 33) space group. Unit cell dimensions: a = 28.406(6) Å, b = 8.928(2) Å, c = 9.541(2) Å; Z = 4. The compound possesses 63 valence electrons, three more than the expected “magic number” for such type of clusters, and three of the four nickel atoms do not fulfil the 18VE rule. It is paramagnetic with the magnetic moment 3.54 μ_B, what corresponds to three unpaired electrons per molecule. This was confirmed by molecular orbital calculations using the density functional theory (DFT).