Evaluation and comparison of very high pressure liquid chromatography systems for the separation and validation of pharmaceutical compounds

Chromatography using sub-2 microm particles is becoming increasingly popular due to the potential for increased speed, resolution, sensitivity, and peak capacity. To meet the demand, various vendors have re-engineered traditional LC systems to operate at pressures of up to 15000 psi to accommodate the elevated backpressures associated with using sub-2 microm particles. This report investigates and compares the performance of three very high pressure LC (VHPLC) systems: Waters Acquity, Agilent 1200 SL, and Thermo Accela. Specifications for the pump, autosampler, column compartment, detector, and software for each instrument are presented. To assess the chromatographic performance of the three instruments, method development and validation were performed for three pharmaceutical compounds and the results are compared and discussed. The material presented herein serves to highlight the different features of the VHPLC instruments, and assess their suitability for the analysis of pharmaceutical compounds.     

Automated analysis of individual particles using a commercial capillary electrophoresis system

Capillary electrophoretic analysis of individual submicrometer size particles has been previously done using custom-built instruments. Despite that these instruments provide an excellent signal-to-noise ratio for individual particle detection, they are not capable of performing automated analyses of particles. Here we report the use of a commercial Beckman P/ACE MDQ capillary electrophoresis (CE) instrument with on-column laser-induced fluorescence (LIF) detection for the automated analysis of individual particles. The CE instrument was modified with an external I/O board that allowed for faster data acquisition rates (e.g. 100 Hz) than those available with the standard instrument settings (e.g. 4 Hz). A series of eight hydrodynamic injections expected to contain 32 +/- 6 particles, each followed by an electrophoretic separation at -300 V cm(-1) with data acquired at 100 Hz, showed 28 +/- 5 peaks corresponding to 31.9 particles as predicted by the statistical overlap theory. In contrast, a similar series of hydrodynamic injections followed by data acquisition at 4 Hz revealed only 8 +/- 3 peaks suggesting that the modified system is needed for individual particle analysis. Comparison of electropherograms obtained at both data acquisition rates also indicate: (i) similar migration time ranges; (ii) lower variation in the fluorescence intensity of individual peaks for 100 Hz; and (iii) a better signal-to-noise ratio for 4 Hz raw data. S/N improved for 100 Hz when data were smoothed with a binomial filter but did not reach the S/N values previously reported for post-column LIF detection. The proof-of-principle of automated analysis of individual particles using a commercially available CE system described here opens exciting possibilities for those interested in the study and analyses of organelles, liposomes, and nanoparticles.     

Microparticle sampling by electrowetting-actuated droplet sweeping

This paper describes a new microparticle sampler where particles can be efficiently swept from a solid surface and sampled into a liquid medium using moving droplets actuated by the electrowetting principle. We successfully demonstrate that super hydrophilic (2 microm and 7.9 microm diameter glass beads of about 14 degrees contact angle), intermediate hydrophilic (7.5 microm diameter polystyrene beads of about 70 degrees contact angle), and super hydrophobic (7.9 microm diameter Teflon-coated glass beads and 3 microm size PTFE particles of over 110 degrees contact angles) particles on a solid surface are picked up by electrowetting-actuated moving droplets. For the glass beads as well as the polystyrene beads, the sampling efficiencies are over 93%, in particular over 98% for the 7.9 microm glass beads. For the PTFE particles, however, the sampling efficiency is measured at around 70%, relatively lower than that of the glass and polystyrene beads. This is due mainly to the non-uniformity in particle size and the particle hydrophobicity. In this case, the collected particles staying (adsorbing) on the air-to-water interface hinder the droplet from advancing. This particle sampler requires an extremely small amount of liquid volume (about 500 nanoliters) and will thus be highly compatible and easily integrated with lab-on-a-chip systems for follow-up biological/chemical analyses.     

Enrichment adsorption of a labile substance to the surface of particular mineral particles in river water as investigated by SEM-EDX and dilute-acid extraction/ICP-MS.

The selective enrichment behavior of a labile substance, such as hydroxides, to the surface of particular mineral particles in river water was clarified by scanning electron microscopy/energy dispersive X-ray microanalysis (SEM-EDX). Individual particles other than diatom collected on a 0.45 microm filter from the Fuji and Sagami rivers, central Japan, were analyzed by SEM-EDX and classified into seventeen groups according to the chemical composition and shape. Phosphorus, sulfur, chlorine, manganese and copper detected in each particle collected on the 0.45 microm filter could be successfully used as effective indicators of labile substance secondarily formed and adsorbed afresh in river water, because the detection frequencies of such elements are quite low, or negligible, in fresh mineral particles derived from igneous rocks. The labile substance adsorbed on mineral particles collected on the 0.45 microm filter was also evaluated by dilute-acid leaching, followed by inductively coupled plasma mass spectrometry (ICP-MS). Almost all parts of the manganese detected in individual particles were those adsorbed afresh as hydroxides together with iron and aluminum. Also, anionic elements, such as phosphorus, sulfur and chlorine, formed complexes with the hydroxides and/or were incorporated in them. Mg and/or Ca-rich aluminosilicate groups were the most effective adsorbers of such labile species. However, Si-rich and Na-, K- and Na-Ca rich aluminosilicates did not significantly adsorb the labile substance. Consequently, the remarkable selectivity was clarified in the adsorption process of labile substance to individual mineral particles in river water.     

On visualization of sub-micron particles with dark-field light microscopy

Dark-field light microscopy is widely employed to visualize colloidal particles much smaller than the light wavelength used. In the captured images, the colloidal particles appear, against a dark background, as bright "specks" much larger than the geometrical size of the particles. To verify whether the "specks" are for individual particles or clusters of particles, experiments are performed which used low bulk concentrations of five suspensions of monodispersed particles (approximately 0.3 microm in diameter) and a dark-field video microscopic system with an optical resolution of approximately 0.5 microm to count the particles after they all have deposited onto the inner surfaces of a parallel-plate glass channel. The average size and the size distribution of the particles are also determined at the end of each experiment. The results confirmed that the visualized "specks" are for individual particles. The measured and prepared particle bulk concentrations in the five experiments closely matched, to within +/-5%, and the measured average size of the particles and their size distribution at the end of the five experiments were in agreement with the known values.     

Comparative study of the performance of columns packed with several new fine silica particles. Would the external roughness of the particles affect column properties?

We measured and compared the characteristics and performance of columns packed with particles of five different C(18)-bonded silica, 3 and 5 microm Luna, 3 microm Atlantis, 3.5 microm Zorbax, and 2.7 microm Halo. The average particle size of each material was derived from the SEM pictures of 200 individual particles. These pictures contrast the irregular morphology of the external surface of the Zorbax and Halo particles and the smooth surface of the Luna and Atlantis particles. In a wide range of mobile phase velocities (from 0.010 to 3 mL/min) and at ambient temperature, we measured the first and second central moments of the peaks of naphthalene, insulin, and bovine serum albumin (BSA). These moments were corrected for the contributions of the extra-column volumes to calculate the reduced HETPs. The C-terms of naphthalene and insulin are largest for the Halo and Zorbax materials and the A-term smallest for the Halo-packed column. The Halo column performs the best for the low molecular weight compound naphthalene (minimum reduced HETP, 1.4) but is not as good as the Atlantis or Luna columns for the large molecular weight compound insulin. The Zorbax column is the least efficient column because of its large C-term. The lowest sample diffusivity through these particles, alone, does not account for the results. It is most likely that the roughness of the external surface of the Halo and Zorbax particles limit the performance of these columns at high flow rates generating an unusually high film mass transfer resistance.     

Particle size analyses of porous silica and hybrid silica chromatographic support particles. Comparison of flow/hyperlayer field-flow fractionation with scanning electron microscopy, electrical sensing zone, and static light scattering

Porous silica and hybrid silica chromatographic support particles having particle diameters ranging approximately from 1 microm to 15 microm have been characterized by flow/hyperlayer field-flow fractionation (FFF). The particle size accuracy has been improved significantly in this work by a second-order polynomial calibration. Very good agreement between the FFF data and scanning electron microscopic (SEM) results has been achieved. The effects of particle porosity, pore sizes, and particle sizes on the particle size accuracy in electrical sensing zone (ESZ) analyses have been discussed. It has been demonstrated by computer simulation and experimental measurements that false peaks can be generated in certain particle size regions when the static light scattering (SLS) technique is applied to tightly distributed spherical chromatographic support particles.     

Effect of matrix elasticity on affinity binding and release of bioparticles. Elution of bound cells by temperature-induced shrinkage of the smart macroporous hydrogel

The first step of bacterial or viral invasion is affinity and presumably multisite binding of bioparticles to an elastic matrix like a living tissue. We have demonstrated that model bioparticles such as inclusion bodies (spheres of about 1 microm in size) Escherichia coli cells (rods 1 x 3 microm), yeast cells (8 microm spheres), and synthetic microgel particles (0.4 microm spheres) are binding via different affinity interactions (IgG antibody-protein A, sugar-lectin, and metal ion-chelate) to a macroporous hydrogel (MH) matrix bearing appropriate ligands. The elastic deformation of the MH results in the detachment of affinity bound bioparticles. The particle detachment on elastic deformation is believed to be due to multipoint attachment of the particles to affinity matrix and the disturbance of the distance between affinity ligands when the matrix is deformed. No release of affinity bound protein occurred on elastic deformation. The efficiency of the particle release by the elastic deformation depends on the density of the ligands at the particle surface as well as on the elasticity of the matrix for relatively large particles. The release of the particles occurred irrespectively of whether the deformation was caused by external forces (mechanical deformation) or internal forces (the shrinkage of thermosensitive macroporous poly-N-isopropylacrylamide hydrogel on increase in temperature).     

Evaluation of ultra performance liquid chromatography. Part I. Possibilities and limitations

A practical evaluation of the possibilities and limitations of ultra performance liquid chromatography (UPLC) is presented. Acquity BEH columns packed with 1.7 microm particles are evaluated by means of van Deemter and Knox plots. The columns are characterised by high optimal velocities (3.7 mm/s) and low plate heights (4.4 microm). Minimum plate heights of 2d(p) were, however, not reached and reasons are presented and discussed. Furthermore, the use of 1.7 microm particles at 1000 bar is compared, from a theoretical viewpoint, to conventional LC (3.5 and 5 microm particles at 400 bar) in terms of analysis speed and practical maximum efficiency. Experimental data are used to construct kinetic- or "Poppe-plots", which facilitate investigation of the effect of pressure and particle size on speed and efficiency. It is found that UPLC conditions hold advantages in terms of speed of analysis, for required theoretical plate counts up to approximately 80,000.     

Chemistry at level of individual aerosol particle using multivariate curve resolution of confocal Raman image

Airborne particles with aerodynamic diameter in the 10-1 microm range have been collected in an industrial/urban zone by impaction and have been investigated by automated confocal Raman microspectrometry. The computer-microcontrolled XY scanning and Z focusing of Raman images provided many pixel Raman spectra which are characteristics of complex mixture at level of individual particle. The large heterogeneity was not resolved by the spatial resolution of the instrument which is limited by the optical diffraction. The severe spectral overlaps generated by heterogeneity were resolved by multivariate curve resolution (MCR) methods. The purity based method (SIMPLISMAX) was used to resolve both luminescence spectra and pure Raman spectra without prior information. The MCR-alternating least square (ALS) was used as a refined method of both spectra and spectral concentrations. The reconstructing Raman images of the respective spectral contribution supply a versatile potential to characterize the chemistry of atmospheric aerosols at the level of the individual particles.     

Preparation and characterization of structured hydrogel microparticles based on cross-linked hyperbranched polyglycerol

The aim of this work was to obtain well-defined HyPG-MA (methacrylated hyperbranched polyglycerol) microparticles with uniform sizes. Therefore, three different preparation methods were evaluated. First, we assessed a micromolding technique using rigid SU-8 (a photoresist based on epoxies) grids. Independent of the surface treatment of the SU-8 grid or the type of polymer used, approximately 50% of the microgels remained attached to the SU-8 grid or broke into smaller particles during the release process in which drying of the gels was followed by a sonication process. Although 90% methacrylate conversion could be obtained, this method has some additional drawbacks as the obtained dried microgels did not rehydrate completely after the drying step. Second, a soft micromolding technique was evaluated using elastomeric PDMS (poly(dimethyl siloxane)) grids. The use of these flexible grids resulted in a high yield (80-90% yield; >90% methacrylate conversion) of microgels with a well-defined size and shape (squares 100 microm x 100 microm x 50 microm or hexagons with ? 30 microm and a thickness of 20 microm) without the occurrence of water evaporation. However, a number of particles showed a less-defined shape as not all grids could be filled well. The microgels showed restricted swelling, implying that these gels are dimensionally stable. Third, an alternative method referred to as photolithography was evaluated. This method was suitable to tailor accurately the size and shape of HyPG-MA microgels and additionally gained 100% yield. Well-defined HyPG-MA microgels in the size range of 200-1400 microm (thickness of 6, 20, or 50 microm), with a methacrylate conversion of >90%, could easily be prepared by adding an inhibitor (e.g., 1% (w/v) of vitamin C) to the polymer solution to inhibit dark polymerization. Microgels in the size range of 30-100 microm (>90% conversion) could only be obtained when applying the photomask in direct contact with the polymer solution and using a higher (i.e., 2% (w/v)) concentration of vitamin C. Additionally, the microgels showed limited swelling, indicating that rather dimensionally stable particles were obtained. In conclusion, this paper shows that photolithography and soft micromolding, as compared to rigid micromolding, are the most appropriate techniques to fabricate structured HyPG-MA microgels with a tailorable and well-defined size and shape. These microgels have great potential in tissue engineering and drug delivery applications.     

Softening of fused-silica capillaries during particle packing

When a semipreparative capillary electrochromatography (CEC) capillary is packed with silica particles and exposed to solvent, its mechanical strength is markedly reduced. In our studies, a fused-silica capillary (internal diameter > 200 microm and wall thickness < 150 microm) was packed under pressure (approximately 200 psi) with spherical silica particles (1.5-5 microm) suspended in water or various common organic solvents. After one hour of exposure, the capillary can be readily deformed, and it keeps its deformed shape upon release of the force causing deformation. It is suggested that capillary softening is promoted through the propagation of internal microcracks that have been caused by action of the particles during packing in the presence of solvent. Application of a protective coating to the inside of the capillary is found to reduce or eliminate capillary softening.     

High throughput screening of active pharmaceutical ingredients by UPLC

Ultra performance LC (UPLC) was evaluated as an efficient screening approach to facilitate method development for drug candidates. Three stationary phases were screened: C-18, phenyl, and Shield RP 18 with column dimensions of 150 mm x 2.1 mm, 1.7 microm, which should theoretically generate 35,000 plates or 175% of the typical column plate count of a conventional 250 mm x 4.6 mm, 5 microm particle column. Thirteen different active pharmaceutical ingredients (APIs) were screened using this column set with a standardized mobile-phase gradient. The UPLC method selectivity results were compared to those obtained for these compounds via methods developed through laborious trial and error screening experiments using numerous conventional HPLC mobile and stationary phases. Peak capacity was compared for columns packed with 5 microm particles and columns packed with 1.7 microm particles. The impurities screened by UPLC were confirmed by LC/MS. The results demonstrate that simple, high efficiency UPLC gradients are a feasible and productive alternative to more conventional multiparametric chromatographic screening approaches for many compounds in the early stages of drug development.     

Ground, sieved, and C18 modified monolithic silica particles for packing material of microcolumn high-performance liquid chromatography

We here report a new type of stationary phase for microcolumns. C18 modified silica monolith particles were prepared by grinding and sieving the silica monolith followed by C18 modification and end-capping, and were used as packing material. Ground silica monolith particles were not spherical but irregular with some residual monolithic network structure. The separation efficiency of the stationary phase made of sieved monolith particles (5-10 microm) was better than that of the stationary phase made of unsieved particles. The microcolumn packed with the sieved C18 ground monolith particles (5-10 microm) showed quite good separation efficiency (height equivalent to theoretical plate, HETP, as low as 15 microm) and it was even superior to the microcolumn packed with a commercial spherical 5 microm C18 stationary phase. The column pressure drop of C18 monolith particles was about two-third of that of the commercial spherical C18 phase. The preparation method of C18 stationary phase with ground and sieved silica monolith particles presumably suggests advantages of simplicity and convenience in modification and washing procedures compared to bulk silica monolith. It also showed both improved separation efficiency and low back pressure.     

Encapsulation and dispersion of carbon black by an in situ controlling radical polymerization of AA/BA/St with DPE as a control agent

In this paper, a new strategy to encapsulate and disperse carbon black by an in situ controlling free radical polymerization of 1,1-Diphenylenthyene (DPE) method was developed. Firstly, a living amphipathic precursor polymer of P (AA-BA) containing DPE unit was synthesized. This precursor could be grafted or absorbed on the surface of small carbon black particles to prevent further aggregation of carbon black. And the DPE unit in the living amphipathic precursor could initiate following monomer to form polymer shell via in situ polymerization. Carbon black/polymer core-shell composite particles with 69.6 wt.% polymer shell were prepared. The encapsulated carbon black had a small particle size and high performance on dispersibility and stability. Encapsulation mechanism of this method was confirmed by analyses of TEM, UV–vis, ¹H NMR, ¹³C NMR, TGA, and other instruments.     

Molecular state and distribution of fullerenes entrapped in sol-gel samples

A novel synthetic method that can encapsulate fullerene molecules (pure C60, pure C70, or their mixture) over a wide range of concentrations ranging from micromolar to millimolar in hybrid glass by a sol-gel method without any time-consuming, complicated, and unwanted extra steps (e.g., addition of a surfactant or derivatization of the fullerenes) has been successfully developed. The molecular state and distribution of encapsulated fullerene molecules in these sol-gel samples were unequivocally characterized using newly developed multispectral imaging techniques. The high sensitivity (single-pixel resolution) and ability of these instruments to record multispectral images at different spatial resolutions (approximately 10 microm with the macroscopic instrument and approximately 0.8 microm with the microscopic instrument) make them uniquely suited for this task. Specifically, the imaging instruments can be used to simultaneously measure multispectral images of sol-gel-encapsulated C60 and C70 molecules at many different positions within a sol-gel sample in an area either as large as 3 mm x 4 mm (with the macroscopic imaging instrument) or as small as 0.8 microm x 0.8 microm (with the microscopic instrument). The absorption spectrum of the fullerene molecule at each position can then be calculated either by averaging the intensity of a 15 x 15 square of pixels (which corresponds to an area of 3 mm x 4 mm) or from the intensity of a single pixel (i.e., an area of about 0.8 microm x 0.8 microm), respectively. The molecular state and distribution of fullerene molecules within sol-gel samples can then be determined from the calculated spectra. It was found that spectra of encapsulated C60 and C70 measured at five different positions within a sol-gel sample were similar not only to one another but also to spectra measured at six different times during the sol-gel reaction process (from t = 0 to 10 days). Furthermore, these spectra are similar to the corresponding spectra of monomeric C60 or C70 molecules in solution. Similarly, spectra of sol-gel samples containing a mixture of C60 and C70 were found to be the same at five different positions, as well as similar to spectra calculated from an average of the spectra of C60 and C70 either encapsulated in a sol-gel or in solution. It is evident from these results that C60 and C70 molecules do not undergo aggregation upon encapsulation into a sol-gel but rather remain in their monomeric state. Furthermore, entrapped C60 and C70 molecules in their monomeric state were distributed homogeneously throughout the entire sol-gel samples. Such a conclusion can be readily, quickly, and easily obtained, not with traditional spectroscopic techniques based on the use of a single-channel detector (absorption, fluorescence, infrared, Raman) but rather with the newly developed multispectral imaging technique. More importantly, the novel synthetic method reported here makes it possible, for the first time, to homogenously entrap monomeric fullerene molecules (C60, C70, or their mixture) in a sol-gel at various concentrations ranging from as low as 2.2 mM C60 (or 190 microM C70) to as high as 4.2 mM C60 (or 360 microM C70).     

Continuous particle separation in spiral microchannels using Dean flows and differential migration

Microparticle separation and concentration based on size has become indispensable in many biomedical and environmental applications. In this paper we describe a passive microfluidic device with spiral microchannel geometry for complete separation of particles. The design takes advantage of the inertial lift and viscous drag forces acting on particles of various sizes to achieve differential migration, and hence separation, of microparticles. The dominant inertial forces and the Dean rotation force due to the spiral microchannel geometry cause the larger particles to occupy a single equilibrium position near the inner microchannel wall. The smaller particles migrate to the outer half of the channel under the influence of Dean forces resulting in the formation of two distinct particle streams which are collected in two separate outputs. This is the first demonstration that takes advantage of the dual role of Dean forces for focusing larger particles in a single equilibrium position and transposing the smaller particles from the inner half to the outer half of the microchannel cross-section. The 5-loop spiral microchannel 100 microm wide and 50 microm high was used to successfully demonstrate a complete separation of 7.32 microm and 1.9 microm particles at Dean number De = 0.47. Analytical analysis supporting the experiments and models is also presented. The simple planar structure of the separator offers simple fabrication and makes it ideal for integration with on-chip microfluidic systems, such as micro total analysis systems (muTAS) or lab-on-a-chip (LOC) for continuous filtration and separation applications.     

Heterogeneous chemistry between PbSO4 and calcite microparticles using Raman microimaging

Currently, the smelting activities of lead and zinc are the loudest sources of local pollution by emission in the troposphere of dust of micrometer size containing PbSO(4). As the particles evolve in the troposphere, their chemical and physical properties - and hence their characteristics such as toxicity - change by accumulation of atmospheric heterogeneous reactions. Calcite (CaCO(3)) represents a large part of the mineral fraction in tropospheric aerosols with aerodynamic diameters less than 10 microm. The calcite particles are expected to react with PbSO(4) particles. In an effort to model the chemical behaviour of PbSO(4) individual particles in the troposphere, we present the in situ Raman imaging results during the course of the reactions in a water droplet of PbSO(4) particles with a calcite microcrystal surface. The computer-microcontrolled XY scanning and Z focusing of confocal Raman imaging combined with multivariate curve resolution (MCR) of Raman images have resolved the severe spectral overlaps of the Raman spectra which are not resolved by the spatial resolution of the instrument ( approximately 1 microm(3)). The results pointed out the identification and the mapping of Pb(3)(CO(3))(2)(OH)(2), PbCO(3) and CaSO(4).2H(2)O (gypsum) on the calcite surface.     

Method to predict and compare the influence of the particle size on the isocratic peak capacity of high-performance liquid chromatography columns

A kinetic plot based method has been used to experimentally predict the optimal particle size yielding the maximal isocratic peak capacity in a given analysis time. Applying the method to columns of three different manufacturers and characterizing them by separating a 4-component paraben mixture at 30 degrees C, it was consistently found that the classical 3 and 3.5 microm particles provide the highest peak capacity for typical isocratic separation run times between 30 and 60 min when operating the columns at a conventional pressure of 400 bar. Even at 1000 bar, the sub-2 microm particles only have a distinct advantage for runs lasting 30 min or less, while for runs lasting 45 min or longer the 3 and 3.5 microm again are to be preferred. This finding points at the advantage for high-resolution separations that could be obtained by producing 3 and 3.5 microm particle columns that can be operated at elevated pressures.     

Continuous hydrophoretic separation and sizing of microparticles using slanted obstacles in a microchannel

We report a microfluidic separation and sizing method of microparticles with hydrophoresis--the movement of suspended particles under the influence of a microstructure-induced pressure field. By exploiting slanted obstacles in a microchannel, we can generate a lateral pressure gradient so that microparticles can be deflected and arranged along the lateral flows induced by the gradient. Using such movements of particles, we completely separated polystyrene microbeads with 9 and 12 microm diameters. Also, we discriminated polystyrene microbeads with diameter differences of approximately 7.3%. Additionally, we measured the diameter of 10.4 microm beads with high coefficient of variation and compared the result with a conventional laser diffraction method. The slanted obstacle as a microfluidic control element in a microchannel is analogous to the electric, magnetic, optical, or acoustic counterparts in that their function is to generate a field gradient. Since our method is based on intrinsic pressure fields, we could eliminate the need for external potential fields to induce the movement of particles. Therefore, our hydrophoretic method will offer a new opportunity for power-free and biocompatible particle control within integrated microfluidic devices.