Analysis of peptides and proteins using sub-2 μm fully porous and sub 3-μm shell particles

The objective of this study was to evaluate the potential of sub-2 μm totally porous particles and sub-3 μm shell particles for peptide and protein analysis. Specific analytical strategies must be developed for these biomolecules as their importance in the pharmaceutical industry increases and as their structural complexity involves some issues when classical LC conditions are employed. Attention was paid on comparing these different columns in various LC conditions (different temperatures, gradient times, and mobile phase flow rates). The comparison of the different supports was assessed considering columns characteristics (quality of packing, silanol activity, pore size, totally porous or shell particles). In this article, peptides were first analyzed with both column technologies. Similar results to those achieved with low molecular weight compounds were obtained (peak capacity >100 for t_grad around 3 min and columns dimensions of 2.1 mm id × 50 mm), but specific conditions were required (elevated temperature and the use of a volatile ion-pairing reagent, namely TFA). For peptide analysis following tryptic digestion, the goal was to improve peak capacity and resolution because of the large number of generated peptides. For this purpose, longer columns packed with porous sub-2 μm or shell sub-3 μm particles (i.e., 150 mm) and gradient times (i.e., up to 30 min) were tested. On the other hand, proteins in their intact forms have higher molecular weights (MW > 5000 Da) and a tertiary structure, thus requiring different conditions in terms of stationary phase hydrophobicity (C₄vs. C₁₈) and pore size (300 vs. 120 Å). In addition, there were issues with adsorption onto the LC system and/or the column itself. This study showed that proteins with MWs lower than 40,000 Da required chromatographic conditions close to those employed for peptide analysis. For larger proteins, a C₄ 300 Å stationary phase gave the best results, confirming theoretical predictions.     

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.     

Ion beam-induced magnetic patterning at the sub-0.1 μm level

We present a method that allows magnetic patterning of a continuous magnetic film without significant modification of the surface roughness or of the film's optical indices. It involves ion irradiation of Co/Pt multilayers, using either a standard ion implantation technology combined with high resolution masking, or a focussed ion beam. We fabricated arrays of lines or dots whose magnetic properties differ on a sub-100 nm scale. We describe the ion collision physics on which the techniques are based, as well as some of the observed consequences on the micromagnetic properties of the arrays and on the ultimate resolution. Possible applications to high-density information storage are briefly discussed.     

Histamine determination in human urine using sub-2 μm C18 column with fluorescence and mass spectrometric detection

A fast, sensitive, and selective method for the determination of histamine in human urine samples by ultrahigh pressure liquid chromatography (LC) with fluorescence and mass spectrometry (MS) detection is investigated. A fluorescent reagent, 4-(1-pyrene) butyric acid N-hydroxysuccinimide ester was conjugated to the primary and secondary amino moieties of histamine. The structure of dipyrene-labeled histamine in human urine was determined by quadrupole time-of-flight MS with electospray ionization interface. The determination of the dipyrene derivative of histamine in urine samples was achieved within 3.9 min on an ultrahigh pressure LC Eclipse Zorbax XDB-C(18) column with 1.8 μm particle diameter. In this work, histamine separation was achieved significantly faster (3.9 min) with improved detection limit (signal-to-noise = 3) of 0.04 nM than 19.5 min with a detection limit of 0.183 nM as reported in a previous method.     

Kinetic investigation of narrow-bore columns packed with prototype sub-2 μm superficially porous particles with various shell thickness

The recent successful breakthrough of sub-3 μm shell particles in HPLC has triggered considerable research efforts toward the design of new brands of core-shell particles. We investigated the mass transfer mechanism of a few analytes in narrow-bore columns packed with prototype 1.7 μm shell particles, made of 1.0, 1.2, and 1.4 μm solid nonporous cores surrounded by porous shells 350, 250, and 150 nm thick, respectively. Three probe solutes, uracil, naphthalene, and insulin, were chosen to assess the kinetic performance of these columns. Inverse size exclusion chromatography, peak parking experiments, and the numerical integration of the experimental peak profiles were carried out in order to measure the external, internal, and total column porosities, the true bulk diffusion coefficients of these analytes, the height equivalent to a theoretical plate, the longitudinal diffusion term, and the trans-particle mass transfer resistance term. The residual eddy diffusion term was measured by difference. The results show the existence of important trans-column velocity biases (7%) possibly due to the presence of particle multiplets in the slurry mixture used during the packing process. Our results illustrates some of the difficulties encountered by scientists preparing and packing shell particles into narrow-bore columns.     

Synthesis and 1.1 μm near-infrared electrophosphorescence properties of a phenoxy-substituents copper phthalocyanine

A novel copper phthalocyanine bearing phenoxy-substituents was prepared and characterized by MS and Elemental analysis. Its UV/Vis absorption and photoluminescence (PL) spectra were investigated. Organic light-emitting devices (OLEDs) were demonstrated by employing this copper phthalocyanine doped into 4, 4′-N,N′-dicarbazole-biphenyl (CBP). Room-temperature electrophosphorescence was observed at about 1.1 μm due to transitions from the first excited triplet state to the singlet ground state (T ₁–S₀) of this CuPc. The intensity of NIR emission at lower doping concentrations (about 10 wt %) was extremely high compared with devices doped with fluorescent dyes. The results indicated that direct charge trapping appears to be the dominant mechanism. The article is published in the original.     

High temperature-high efficiency liquid chromatography using sub-2 μm coupled columns for the analysis of selected non-steroidal anti-inflammatory drugs and veterinary antibiotics in environmental samples

A high efficiency HPLC method was developed by coupling three sub-2 μm columns in series and operating them at high temperature for the separation of selected non-steroidal anti-inflammatory drugs and veterinary antibiotics in environmental samples. The separation was performed at 80 °C to reduce the solvent viscosity, thus reducing the column backpressure. The chromatographic performance of high temperature-extended column length HPLC method was used to determine the most widely used non-steroidal anti-inflammatory drugs and veterinary antibiotics such as sulphonamides in wastewater samples. The method could simultaneously determine 24 pharmaceuticals in short analysis time with high efficiency. The method involved pre-concentration and clean-up by solid phase extraction (SPE) using Oasis HLB extraction cartridges. It was validated based on linearity, precision, detection and quantification limits, selectivity and accuracy. Good recoveries were obtained for all analytes ranging from 72.7% to 98.2% with standard deviations not higher than 6%, except for acetaminophen and acetyl salicylic acid, for which low recovery was obtained. The detection limits of the studied pharmaceuticals ranged from 2 to 16 μg L⁻¹, while limits of quantification were in the range from 7 to 54 μg L⁻¹ with UV detection.     

Theoretical Investigation of Transparent (3–5 μm) and High Reflective (1.3 μm) Filter by Sol-Gel Process

When designing a cut-off interference filter, extra layers are needed to add on each side of periodic symmetrical multilayer to match the incident medium and the substrate. In this paper, we use sol-gel film with adjustable refractive index as the match layer. The influence of the refractive index and thickness of the match layer on the film system is discussed in detail. As an example, a special filter which is transparent (>80%) at the wavelength 3–5 m and high reflective (>90%) at the wavelength 1.3 m is designed. Finally a series of optimized film parameters are presented. The whole design process is a good reference to the other multilayer special filter made by sol-gel process.     

Metabolic profiling of amino acids in cellular samples via zwitterionic sub-2 μm particle size HILIC-MS/MS and a uniformly 13C labeled internal standard

A novel analytical method using hydrophilic interaction liquid chromatography combined with electrospray tandem mass spectrometry for metabolic profiling of free, underivatized amino acids is presented. The separation uses a zwitterionic modified silica-based stationary phase with 1.8-μm particle size functionalized with ammonium sulfonic acid groups. Quantification is based on external standard calibration using a Pichia pastoris cell extract grown on uniformly ¹³C labeled glucose as an internal standard. The absolute limits of detection in the cellular matrix were in the subpicomolar range. Measurement accuracy was assessed by analyzing NIST Standard Reference Material 2389a, which provides certified values for 17 amino acids. The recovery of the amino acids ranged between 65 % (proline) and 120 % (lysine), with excellent repeatability precision below 2.5 % (n?=?5). Only, cystine showed poor recovery (29 %) and repeatability precision (13 %). Generally, the long-term precision obtained by hydrophilic interaction liquid chromatography–tandem mass spectrometry was excellent, being on average less than 9 % over 20 h of measurement time. Moreover, the novel separation method had average repeatability and reproducibility of the chromatographic peak width over time periods of 20 h and 6 months of 8 and 15 %, respectively, demonstrating its high robustness in routine analysis of cellular samples. Large concentration differences depending on the amino acid were found in the cell extracts, typically ranging from 0.002 nmol per milligram of cell dry weight (cystine) to 56 nmol per milligram of cell dry weight (arginine and glutamic acid).     

Progress of 1.5～1.6 μm Laser Crystals

Eye-safe 1.5～1.6 μm lasers have important applications in optical fiber communication, medicine, laser-range-finding, lidar, etc. Er^3＋ and Yb^3＋ co-doped crystal pumped by diode laser around 976 nm is an attractive method for obtaining 1.5～1.6 μm laser in compact device with high output beam quality. In this paper, the recent research and progress of several important Er^3＋ and Yb^3＋ co-doped laser crystals at 1.5～1.6 μm in authors’ lab are reported.     

Critical comparison of performances of superficially porous particles and sub-2 μm particles under optimized ultra-high pressure conditions

The performance of 2.7 μm superficially porous particles at 600 bar and sub-2 μm fully porous particles at 1000 bar were compared by the Poppe plot method. Theoretical Poppe plots were first constructed for each stationary phase to compare their kinetic performance at different analysis times. The theory was then verified by experiments under the optimized conditions identified from the Poppe plot calculation. We found that the 2.7 μm superficially porous particles at 600 bar can provide similar performance compared to the sub-2 μm fully porous particles at ultra-high pressure (1000 bar) when analysis times are very short (e.g. sub-minute). As analysis time increases, the superficially porous particles start to outperform the sub-2 μm particles and can give much higher efficiencies (e.g. > 2 times higher plate count) at very long analysis times (>3 h). The comparison was extended to gradient elution of a mixture of pharmaceutical interest by constructing gradient peak capacity Poppe plots and similar behavior was observed.     

Hydrophilic interaction chromatography of seized drugs and related compounds with sub 2 μm particle columns

The use of hydrophilic interaction chromatography (HILIC) with sub 2 μm particle columns for the analysis of drugs and related compounds of forensic interest is described. This technique uses a high organic/low aqueous buffered mobile phase with a polar stationary phase, and is excellent for the separation of many of the charged solutes that are found in forensic drug exhibits. In this study, HILIC is investigated for 11 solutes of forensic interest, including weak bases, weak acids, and a neutral solute. In addition, for columns containing either ethylene bridged hybrid particles with or without an amide bonded phase, the effects of acetonitrile concentration, buffer type, buffer concentration, linear velocity, and sample concentration were studied. Based on these studies, HILIC with sub 2 μm particle columns can offer highly efficient, selective, and rapid isocratic separations of drugs and related compounds of forensic interest, with excellent peak shapes and low back pressures. This is in contrast to reverse phase chromatography (RPLC), where gradient elution is usually required, which can result in extensive overlap between acidic, neutral, and basic solutes. In addition, since HILIC exhibits a much greater loading capacity than RPLC, it could be a preferred technique for drug profiling. Furthermore, because high organic content mobile phases are highly amenable to mass spectrometric detection, the use of HILIC with tandem mass spectrometric detection for the analysis of seized drugs is described.     

Comparison of μm and mm sized disk electrodes for end-column electrochemical detection in capillary electrophoresis

End-column electrochemical detection based on either the use of a 25 μm microdisk electrode or a 0.5 mm macrodisk electrode has been compared with respect to performance and influence on non-aqueous capillary electrophoretic separations. Despite the much higher coulometric efficiency obtained with the larger disk electrode, the microdisk electrode configuration offers comparable limits of detection (LOD) for the neutral and positively charged ferrocene compounds employed in conjunction with a non-aqueous acetonitrile-based buffer. The LODs for ferrocene were found to be 4.0 × 10^–8 M and 6.7 × 10^–8 M for the microdisk and macrodisk detector, respectively. In addition, both detector arrangements showed different relative responses for neutral and positively charged analytes. The macroelectrode-based detector introduced additional zone broadening while this was not found to be the case with the microelectrode arrangement. Using the microelectrode detector, the band broadening in an electro-osmotically driven flow system was compared to that in a gravity flow-based system. It was demonstrated that the zone broadening under gravity flow conditions was approximately twice as large as under electro-osmotic flow conditions for a typical set of experimental parameters. Received: 1 June 1998 / Revised: 20 August 1998 / Accepted: 24 August 1998     

Comparison of μm and mm sized disk electrodes for end-column electrochemical detection in capillary electrophoresis

End-column electrochemical detection based on either the use of a 25 μm microdisk electrode or a 0.5 mm macrodisk electrode has been compared with respect to performance and influence on non-aqueous capillary electrophoretic separations. Despite the much higher coulometric efficiency obtained with the larger disk electrode, the microdisk electrode configuration offers comparable limits of detection (LOD) for the neutral and positively charged ferrocene compounds employed in conjunction with a non-aqueous acetonitrile-based buffer. The LODs for ferrocene were found to be 4.0 × 10^–8 M and 6.7 × 10^–8 M for the microdisk and macrodisk detector, respectively. In addition, both detector arrangements showed different relative responses for neutral and positively charged analytes. The macroelectrode-based detector introduced additional zone broadening while this was not found to be the case with the microelectrode arrangement. Using the microelectrode detector, the band broadening in an electro-osmotically driven flow system was compared to that in a gravity flow-based system. It was demonstrated that the zone broadening under gravity flow conditions was approximately twice as large as under electro-osmotic flow conditions for a typical set of experimental parameters.     

Determination of thyroxine and 3,5,3′-triiodothyronine by sub- and super-equivalence isotope dilution analysis

Trace amounts of thyroxine in model samples (160.0 ng.ml^–1 and 20.0 ng.ml^–1), and thyroxine and 3,5,3-triiodothyronine in blood plasma were determined by sub- superequivalence isotope dilution analysis and radio-immunoassay technique. Hormones were labelled by¹²⁵I. The separation of antibody-bound hormone from free hormone was performed by ultracentrifugation. The results show higher accuracy of the sub- super-equivalence isotope dilution method over that of radioimunoassay.     

Absolute line intensities of the ν9 band of propyne at 15.5 μm

A tunable diode laser spectrometer was used to perform measurements of absolute line intensities for 20 transitions in the ν₉-fundamental of propyne centered at 15.5 μm. From these data, the vibrational band-strength S^v_o was significantly determined to be S^v_o = 196.8 ± 6.0 cm⁻² atm⁻¹ at 297 K. These results will be used for further applications of astrophysical interest such as a refined contribution for modelling Titan's atmosphere.     

Structural origin and energy transfer processes of 1.8 μm emission in Tm3+ doped germanate glasses

A detailed characterization of structural analysis and energy transfer (ET) process in Tm(3+) doped germanate glasses has been presented. The thermal stability and Raman spectroscopic analysis of the host glass structure has been discussed. It is observed that replacing GeO(2) by Ga(2)O(3) resulted in decreasing of glass transition temperature and largest phonon energy. Increasing concentration of Ga(2)O(3) decreases the density of cross-linking of the germanate glass network, and weaker Ga-O bonds result in lower phonon energy. The influences of the concentrations of Ga(2)O(3) and the glass structure on the ET have also been analyzed. The extended overlap integral method is used to calculate the microparameters of the energy transfer and the critical distance. The results show that the transference Tm → Tm ((3)H(4), (3)H(6) → (3)H(6), (3)H(4)) energy migration is not phonon dependent in the quasiresonant processes, while the cross relaxation Tm → Tm ((3)H(4), (3)H(6) → (3)F(4), (3)F(4)) is a multiphonon mechanism dominated by one-phonon creation (～96%). It is demonstrated that the stretching frequencies and the ET processes become lower when increasing the concentration of Ga(2)O(3).     

Synthesis and Structure of μ-Peroxo-bis(4-bromophenoxotri-p-tolylantimony)

-Peroxo-bis(4-bromophenoxotri-p-tolylantimony) was synthesized by reacting 4-bromophenol with tri-p-tolylstibine in the presence of hydrogen peroxide. According to X-ray diffraction data, the Sb atoms have trigonal bipyramidal coordination. In the centrosymmetric molecule, the two Sb atoms are joined via bridging peroxo group disordered over two sites. The Sb–O(1,2') bond lengths are equal to 1.999(6) and 2.03(1) Å, the Sb···O(2,1') distances are equal to 2.605(9) and 2.626(5) Å, respectively. The lengths of the terminal Sb–O(3) bonds are 2.093(3) Å.     

Synthesis and x-ray structures of some homonuclear μ-alkylidenetungsten complexes

The complexes W₂{μ-CHCHC(CH₃)₂](CO)₁₀} and W₂{μ-CHCHC(CH₃)(CH₂)₃CH₃][(CO)₁₀} have been synthesized, and an X-ray diffraction study has revealed the presence of five CO groups on each metal center. The analogy between W₂{μ-CHCHC(CH₃)₂][(CO)₉]} anda complex of W(CO)₄ and a tungstabutadiene (CO)₅WCHCHC(CCH₃)₂ prompted the synthesis of the first heteroatom-substitued μ-alkylidene complexes of tungsten, starting from conjugated Fischer-typ carbene complexes (CO)₅WC(OR)CHCHR. The X-ray structure of the complex W₂{μ-C(OEt)CHCH(CH₃)][(CO)₉]} has also been determined. In the case of the simplest conjugated compelx (CO)₅WC(OR)CHCH₂, an interesting rearrangement initiated by addition of W(CO)₅ to the terminal CC double bond giving a dinuclear complex W(CO)₅[η²-CH₂CHC(OMe)W(CO)₅] in which the two metal centers are not directly linked, has been observed.     

On the extra-column band-broadening contributions of modern, very high pressure liquid chromatographs using 2.1 mm I.D. columns packed with sub-2 μm particles

The efficiencies of two narrow bore columns (100 mm and 50 mm × 2.1 mm) packed with 1.7 μm totally porous BEH-C₁₈ particles were measured on two very high pressure liquid chromatographs (Acquity from Waters and 1290 Infinity HPLC System from Agilent) operating at maximum pressures of 1034 and 1200 bar, respectively. The probe compounds were a mixture of uracil, acetophenone, toluene, and naphthalene eluted in a 50/50 (v/v) solution of acetonitrile and water at 303 K with a flow rate of 0.40 mL/min. The apparent efficiencies of columns, which lumps the consequences of band broadening due to the column and the system contributions, may depend much on the extra-column volumes of the instruments used. Actually, it is known for a long time that the apparent column performance is strongly affected by the instrument characteristics, including the diameter of the connecting tubes, the injection technique (with or without needle seat capillary), and the detection cell volume. When the 1290 Infinity HPLC System is equipped with a needle seat, an inlet and an outlet connecting capillary tube with inner diameters around 115 μm, its extra-column variance for a 0.1 μL injection volume is 9.2 μL² while that of the Acquity instrument is 6.9 μL². Minor modifications suggested by their respective manufacturers allowed significant reductions of these variances, to 6.2 and 3.9 μL², respectively. Yet, in their optimized configurations and for weakly retained compounds (k ? 1), these modern, sophisticated instruments cannot provide more than 75% (1290 Infinity) and 85% (Acquity) of the maximum efficiency of a 2.1 mm × 50 mm BEH column. For more strongly retained compounds (k > 4), in contrast, they are both able to provide more than 95% of the maximum expected efficiency.