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We report the first development of a novel, planar, microfluidic, graphitic carbon separations column utilizing an array of graphitic micropillars of diamond cross-section as the chromatographic stationary phase. 795 nm femtosecond laser ablation was employed to subtractively machine fluidic architectures and a micropillared array in a planar, graphitic substrate as a monolithic structure. A sample injector was integrated on-chip, together with fluid-flow distribution architectures to minimize band-broadening and ensure sample equi-distribution across the micro-pillared column width. The separations chip was interfaced directly to the ESI probe of a Thermofisher Surveyor mass spectrometer, enabling the detection of test-mixture analytes following their differential retention on the micro-pillared graphitic column, thus demonstrating the exciting potential of this novel separations format. Importantly, unlike porous, graphitic microspheres, the temperature and pressure resilience of the microfluidic device potentially enables use in subcritical H(2)O chromatography.

Native, uncoloured, proteins can be focused in a column containing a fluorescent packing material, using hydrodynamic flow and a counteracting non-linear electric field, and imaged along the length of the channel by fluorescence quenching.

SAR studies were conducted around lead compound 1 using high-throughput parallel solution and solid phase synthesis. Our lead optimization efforts led to the identification of several CCR2b antagonists with potent activity in both binding and functional assays [Compound 71 CCR2b Binding IC 50 3.2 nM; MCP-1-Induced Chemotaxis IC 50 0.83 nM; Ca 2+ Flux IC 50 7.5 nM].

N , N ′-Disubstituted homopiperazine derivatives have been discovered as CC-chemokine receptor 2b (CCR2b) inhibitors with submicromolar activity in the CCR2b binding assay. A 4-substituted benzyl group on one homopiperazine nitrogen was an important moiety for binding affinity to the CCR2b receptor. The SAR for CCR2b binding affinity correlated inversely with the σ factor of the functional group on this benzyl moiety. Introduction of hydroxy groups to appropriate positions in the 3,3-diphenylpropyl group on the other homopiperazine nitrogen increased CCR2b binding activity. The synthesis of an informer library to search for alternative substructures is also described.

Separation and focusing of proteins is described in a miniaturised dynamic field gradient focusing device with a 2.5 cm x 0.1 cm channel filled with a porous polymer monolith. The separation channel is in contact with a parallel electric field channel with five individually addressable electrodes through a porous glass membrane so that a variable field can be generated that drives charged proteins electroosmotically against a constant hydrodynamic flow. Separated pre-stained proteins were detected by means of a digital camera and background subtraction.

Monodisperse sphere-on-sphere (SOS) silica particles are produced in a one-pot reaction, removing the need for time-consuming preparation and classification steps. Analysis of peptides and proteins using HPLC displays faster separation at lower operating pressure than commercially available fused core materials.

Changes in electro-osmotic flow (EOF) induced in an open fused silica capillary by the introduction of a single packing retainer (referred to as a ‘frit’ throughout the article) are investigated. Frits located in a 34.5 cm long capillary close to the detection window, as used commonly in packed column capillary electro-chromatography, were made by sintering silica based packing materials having varying functionalities (bare silica, ODS-silica, a strong cation exchange packing (SCX) and a strong anion exchanger (SAX) at 430 °C for 15 s. The contributions of such frits to the total EOF measured in the capillary are discussed. Despite the high temperature of heating of the packing, sufficient residual functional groups remained on the frit such that distinctively different EOF behaviour was observed for each type of packing. Frits made from materials providing negative surface charges, such as silica, ODS-silica and SCX, increased the magnitude of the cathodic EOF compared to the open capillary. On the other hand, a substantial anodic EOF of −1.88 × 10−8 m2 V−1 s−1 was produced by introduction of a single frit made from the SAX material. An explanation of this behaviour is given, based on the hypothesis that the EOF generated by the frit determines the overall flow in the whole open capillary. The frit is considered to work as a pump and overrides the EOF generated at the capillary wall.

Liquid phase parallel synthesis has been developed to synthesize a novel series of iminodiacetic acid derivatives targeting the integrin receptors. This library was synthesized using a four-step reaction sequence. In each step of the sequence, the PEG-bound products were precipitated selectively and the excess reagents and the by-products were removed by simple filtration. The most notable result was that the library members were obtained in high purities (>95% pure).

An interface which permits both gradient elution capillary electrochromatography (CEC) and the coupling of high performance liquid chromatography (HPLC) to CEC is described. Mobile phase from an HPLC system with continuously variable composition is supplied on demand to the inlet of a CEC column, without the posibility of pressure driven flow. The interface also allows fractions separated by either reverse phase or normal phase HPLC to be transferred to a CEC column operated in reverse phase mode for further separation.

An intensive investigation into the electroosmotic flow in capillary electrochromatography has been performed on C 18 packed columns. Linear velocities obtained when varying voltage and electrolyte characteristics were comparable to those found in capillary electrophoresis. Only at low ionic strength was a deviation from the expected relationship observed; this was attributed to double-layer overlap in the particle pores and turbulent flow. Van Deemter studies on C 18 phases confirmed that linear velocity was unrelated to particle size, but did not support current theory that smaller particles will result in improved efficiency. The role of particle porosity remains ambiguous; lower flow-rates were generated on a C 18 material with 30 nm pores compared to an 8 nm pore material, yet silica did not display the same trend. Our results suggest that high efficiencies can be obtained on 3 μm particles although the columns may be poorly packed. Reproducible separations are most likely to be achieved on well-packed columns of larger particle size. Studies on silica highlighted problems in column manufacture; duplicate columns gave comparable flows but not separation parameters. New procedures for the use of very small particles (1.5 μm) are desirable.

The problems of memory effects when using ion-exchange columns have been addressed. Studies on capillaries packed with a strong cation-exchange (SCX) material have shown voltage conditioning to be sufficient to produce an equilibrated column and reliable results. Storage in acetonitrile–water (80:20, v/v) for six weeks had no detrimental effect on the column performance. Columns used for capillary electrochromatography typically consist of an open and packed section, each contributing to the electroosmotic flow (EOF). Studies performed on SCX columns of varying proportions of packed section (25–100% of the total length) showed that at pH 7.5 the field strengths are similar in both sections and the linear velocity changes little with the packed section length. In contrast, at pH extremes the field strengths were greater in the packed section and the velocity decreased with length. A comparison of the conductance ratios for C18 and SCX columns indicated little difference between them at high pH. However, at low pH results were not routinely obtained for the C18 column, whereas the short SCX column gave rise to far higher EOF than expected.

Capillary electrochromatography (CEC) has seen a resurgence of interest during the 1990s, despite having origins in the 1970s. The technique combines the desirable features of both high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE): the separation process is based on differential interactions between the stationary and mobile phases, whilst the electroosmotic flow transports the mobile phase through the capillary. Thus, it has demonstrated advantages over both HPLC and CE, which are yet to be fully exploited over a wide field of application; already the popularity of CEC is on the increase, as reflected in the number of scientific publications and seminars held. The aim of this tutorial review is to increase awareness and understanding of both theoretical and practical aspects of CEC. Whilst it does not provide an in-depth account of CEC, it does attempt to cover the more important, relevant work available in the open literature: only major advancements associated with CEC applications are highlighted. Material presented in the review was typically obtained by literature searches involving Analytical Abstracts, Chemical Abstracts and ‘BIDS’ (for academic use only).

Tapered and narrow restrictor capillaries have been investigated for use in capillary electrochromatography (CEC) and have been shown to offer viable, more reproducible, alternatives to silica frits and may aid in suppression of bubble formation, a recognised problem in CEC. In addition, these capillaries have been coupled to mass spectrometry, especially for low-flow and nano-flow electrospray ionisation, where results have shown better maintenance of chromatographic efficiency and higher sensitivity, compared with more conventional interfaces. The use of tapered CEC column inlets is suggested as being useful in sampling from micro-environments, such as cell contents.

A commercial supercritical fluid chromatograph has been adapted for preparative SFC. A simple fractionating and collection system which can use the liquid supercritical fluid modifier as a trapping is described. The fractionation of polycarboxylic acid mixtures is used to illustrate the technique. Acids, isolated and obtained in this way, were of a purity close to 100%. The effect on recovery rates of using additonal liquid collection solvent in the collection vessels and of surrounding them with a dry ice jacket was investigated.

A simple, versatile, and general approach to the solution phase, parallel synthesis of chemical libraries conducted on a generalized or universal template, which allows the preparation of multi-mil...

A model is devised using molecular mechanics to simulate chromatographic separations of enantiomers. Theoretical results derived from this model are compared with experimental findings obtained using supercritical fluid chromatography. The model is then developed to incorporate the effects of binding the stationary phase to a matrix. Computed results show that addition of the matrix into the model has significant effects on the ability of the stationary phase to separate racemic mixtures.

The kinematic viscosity of the slurry used for the packing of microbore columns for liquid chromatography has been correlated with the packing efficiency. The columns were packed into polyetheretherketone (PEEK) tubing with an internal diameter of 0.5 mm using unbonded and bonded spherical silica gels under constant pressure conditions. Higher efficiencies were obtained using slurries suspended in aqueous solutions of surfactants, such as polyoxyethylene sorbitane monolaurte (Tween-20) and sodium dodecyl sulphate, in comparison with organic solvents usually used for column packing. The concentration dependencies of the relative kinematic viscosities of six slurries were compared using capillary viscometry. The slurries with low relative viscosities possessed rheological properties which enabled high packing densities, typical of a stable dense random packing arrangement, to be obtained at moderate packing pressures. The unusually low relative viscosities are discussed in terms of a “porous hydrodynamic bearing” model. The techniques used should also be generally applicable to the slurry packing of conventional columns.

The synthesis and antitumor activities of the novel water soluble camptothecin derivatives 7-[(4-methylpiperazino)methyl]-10,11-(methylenedioxy)-(20S)-campto thecin trifluoroacetate (6) and 7-[(4-methylpiperazino)methyl]-10,11-(ethylenedioxy)-(20S)-camptot hecin trifluoroacetate (7) are described. The solubilities of compounds 6 and 7 were measured to be 4.5 and 5.8 mg/mL, respectively, in pH 5 acetate buffer in contrast to < 0.003 mg/mL for camptothecin in the same buffer. In the purified topoisomerase I cleavable complex enzyme assay, compounds 6 and 7 demonstrated potent inhibition of topoisomerase I with IC50's of 300 and 416 nM, respectively, in comparison to 679 nM for camptothecin and 1028 nM for topotecan. In human tumor cell cytotoxicity assays, compounds 6 and 7 demonstrated potent antitumor activity against ovarian (SKOV3), ovarian with upregulated MDRp-glycoprotein (SKVLB), melanoma (LOX), breast (T47D), and colon (HT29) with IC50's ranging from 0.5 to 102 nM. Compounds 6 and 7 induced tumor regressions in the HT29 human colon tumor xenograft model and demonstrated similar rank order of potency compared to in vitro assay results.

There is a continuing challenge in improving the separation speed while keeping both the resolution high and the back pressure low in high performance liquid chromatography (HPLC). The recent renaissance of core–shell (or fused-core) silica particles has shown great promise in this respect. However, the fused-core silica particles are typically synthesized by the time-consuming multiple-step layer-by-layer technique. An one-pot synthesis of spheres-on-sphere (SOS) silica microspheres is presented here. The preparation parameters including Si precursors, mixing methods (magnetic stirring, mechanical stirring and homogenization), heating (microwave heating and conventional heating), and reaction temperature are investigated in order to control the morphology and improve the size distribution of the SOS particles. The improvement and influence on SOS morphology, particle size and particle size distribution are discussed. Furthermore, the calcined and modified SOS particles are packed into stainless steel columns, which are then assessed for the separation of various test mixtures containing small molecules and proteins under normal phase, reversed phase, and HILIC conditions. The HPLC tests demonstrate fast and efficient separation with very low back pressure, suggesting that SOS particles are a type of new and highly promising packing materials for liquid chromatography.

The separation in 6 minutes of the sixteen US Environmental Protection Agency (EPA) polycyclic aromatic hydrocarbons (PAHs) on a single packed column by supercritical fluid chromatography (SFC) is described and compared to that obtained using liquid chromatography with the same column. Several environmental samples containing polycyclic aromatic hydrocarbons obtained from various sources including soil, water, petrochemicals and coal have been analysed by SFC under optimised conditions. Packed column SFC is shown to be an efficient and promising method for the rapid monitoring of environmental polycyclic aromatic hydrocarbons.

This paper presents the first study of the UV imaging of spots on thin-layer chromatographic plates whilst still wet with solvent. Imaging of spots of benzophenone during and after development was carried out using a charge-coupled device camera. Limits of detection were found to be 5 ng on a wetted plate and 3 ng for a dry plate and the relationship between peak area and sample loading was found to be linear in the low nanograrn range over an order of magnitude for both wet and dry modes with r(2) values > 0.99. It was found that UV measurements on wet glass-backed plates suffer from low sensitivity; however, the use of aluminium-backed plates gave increased sensitivity. The apparent absorption coefficient (epsilon(app)) of 10AU m(2) g(-1) at 254 nm is consistent with reflection of the light from the aluminium surface with a double pass through the sorbent layer, and suggests that use of aluminium-backed plates should enable monitoring of separations by UV absorbance during TLC development. (c) 2008 Elsevier B.V. All rights reserved.

Electrophoretic field gradient focusing has been used to separate the two oxidation states of myoglobin (Mb), and to separate Mb from bromophenol blue (BPB). Polyacrylamide and Sephadex were shown to be suitable packing materials whilst silica led to band broadening with Mb. BPB and Mb could be simultaneously focused apart using either a fixed 21-electrode setup or a dynamic 6-electrode setup. Using a dynamic three-electrode setup either analyte could be focused but not both simultaneously. It was shown that a higher ionic strength buffer in the separation channel compared to the coolant channel enhanced focusing between electrodes due to a conductivity gradient. Different running buffers were investigated and it was found that using a pH 8.6 buffer containing N,N,N-tris(hydroxymethyl)aminomethane (Tris) and phosphate ions the oxidation states of Mb could be separated but the separation of Mb from BPB was not as good as would be hoped for. Using a pH 8.6 buffer containing Tris, N-2-hydroxyethylpiperazine-N'-3-propanesulphonate and chloride ions as running buffer, BPB and Mb could be well separated but the two oxidation states of Mb merged.

This paper presents the first study of imaging of spots on thin-layer chromatographic plates whilst still wet with solvent. Imaging and quantification of Sudan II after development with dichloromethane was carried out in both reflectance and transmission modes, using a charge coupled device (CCD) camera. The relationship between peak area and sample loading was established at low sample loading, and found to be linear over an order of magnitude for both wet and dry modes with r2-values > 0.99. All data processing was carried out using the Beer-Lambert equation. Curvature at high loadings in the plots of integrated absorbance as a function of sample loading was accounted for using an empirical expression designed for use with the Kubelka-Munk treatment and apparent absorbance of the stationary phase due to scattering. Results are consistent with an effective pathlength significantly longer than the thickness of the sorbent layer. The limit of detection on a dry plate (0.5 ng) was found to be lower than on a wetted plate (2 ng). Precision was found to be 1-4% RSD intra-plate and 8-14% RSD inter-plate. Results are compared with quantification of the same analyte on dried plates.

This paper describes the micro-fabrication and preliminary assessment of a miniature polydimethylsiloxane (PDMS) device for performing rapid, parallel liquid phase chromatographic separations driven by centrifugal force in microchannels. Device components include a main separating channel, into which a high performance liquid chromatography (HPLC) particulate stationary phase was packed under pressure by application of centrifugal force, in addition to solvent and sample reservoirs. Also described are methods for sealing such devices based upon partial polymerisation of PDMS. The mobile phase flow rate through a typical device was measured and several important chromatographic parameters determined from a test separation. An expression describing mobile phase flow through packed channels was also developed, based upon work on liquid flow in open micro-channels. Good agreement between predicted and measured flow rates were observed. Some predictions for potential uses of such devices and possibilities for further miniaturisation are discussed.

Structure-activity relationships (SAR) of a weakly active class of CCR2b inhibitors were utilized to initiate a lead evolution program employing the Drug Discovery Engine. Several alternative structural series have been discovered that display nanomolar activity in the CCR2b binding and CCR2b-mediated chemotaxis assays.

The present state of the theory of capillary electrochromatography (CEC) is reviewed. Emphasis is placed on electroosmosis and the electrical double layer, and the generally good understanding of the factors affecting the electroosmotic flow in CEC columns. The relation of CEC to other electrically driven separations are described, along with band broadening, and the influence of column temperature in CEC. The theoretical potential of CEC is assessed from the standpoint of current and future column technology, and likely future application areas are described.

Much capillary electrochromatography (CEC) work is carried out on bonded silica packings which offer many advantages: the number of such packings which are available; the fact that the chemistry of bonding and the separation process are fairly well understood; and the possibility of the transfer to CEC of existing HPLC methods. Packing methods for the preparation of CEC columns have been investigated. The problems inherent in the use of burned-in frits remains an obstacle, but can be at least partially overcome by minimising the length and by silanisation. The influence of a variety of mobile phase variables on aspects of CEC is in agreement with theory for: ionic strength, organic content (including isoeluotropy), and pH. Temperature can be used as a variable to change column selectivity in CEC. The influence of pH on electroosmotic flow (EOF) by changing the degree of ionisation of residual silanol groups is similar for a wide range of neutral bonded groups, but is much less marked for bonded sulphonic acid groups. The EOF may be reversed for bonded groups containing nitrogen.

A study was carried out in which 50 microm I.D. fused-silica capillaries were packed with 3 microm octadecylsilane bonded silica, from the same batch, by four methods; liquid slurry and carbon dioxide supercritical carrier, each with and without the use of an ultrasonic probe. A neutral test mixture was analysed by capillary column in reversed-phase mode, and the reproducibility of the electroosmotic flow and of migration time, column efficiency and retention factors, was determined. Initially results suggested that there was no significant difference between properties of columns packed by different methods, and a more thorough statistical evaluation confirmed this; differences observed in the column performance were attributed to random variations between replicate columns, and not between packing methods. However, the variation was least when applying the ultrasonication during liquid slurry.