We have developed porous polymer monoliths (PPMs) that are versatile and robust reversed-phase chromatography media. The PPMs are cast-to-shape, UV-cured polymers that form uniform packings within pretreated glass capillaries and fused-silica chips. No applied pressure is ever needed to flush the PPMs since they support electroosmotic flow as cast. Such characteristics make the PPMs useful for chip-based devices. Our results show efficiencies greater than or equal to 150,000 plates/m for both capillary and chip-based separations of polycyclic aromatic hydrocarbons. By changing the monomers, the hydrophobicity of the polymers, and the direction of the electroosmotic flow can be altered without degrading chromatographic performance. We describe here the development of these acrylate-based materials along with both physical and chromatographic characterization.

Quantitative measures of salt-bridge-type interactions in a highly exposed aqueous environment have been obtained by modifying the well-studied cyclophane platform 1 to include carboxylates in close proximity to bound, cationic guests, producing hosts 2 and 3. Many guests show significantly enhanced binding to 2 and 3, but cations of the RNMe3 + type show little or no enhancement. We propose that the latter observations result from the fact that RNMe3 + compounds have very diffuse positive charges. Guests that show enhanced binding have focused regions of large, positive electrostatic potential. The highly charged 3 is able to bind very polar, very well-solvated guests, including a series of arginine-based dipeptides. Neutral, water-soluble host 4 was prepared and found to show a decreased affinity for cationic guests. We propose a novel induced dipole mechanism to rationalize these results.

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The first rigorous evaluation of a UV-initiated porous polymer monolith (PPM) as a stationary phase for chip electrochromatography (ChEC) is described. All channels in an offset T-injector-design-chip (25-microm deep by 50-microm wide channels) were filled by capillary action with an acrylate-based PPM precursor solution and polymerized in situ using 365 nm light for several minutes. Photodefinability of the monolith cast in the channels during the polymerization process was also demonstrated by masking off the injection arms during photoinitiation. The chromatographic performance of this chip was compared with that of chips completely filled with monolith. The detection window was photodefined after polymerization using the detection laser (257 nm doubled argon ion laser) to depolymerize the detection window. A successful ChEC separation of 10 out of 13 polycyclic aromatic hydrocarbons (PAH) was performed with on-column, off-packing laser-induced fluorescence detection at 257 nm. Van Deemter plots for early-, middle-, and late-eluting compounds showed the minimum plate height to be 5 microm. The average number of theoretical plates per meter for the PAH was 200,000. Several factors contributed to irreproducible results. Oxygen was observed to dynamically quench the fluorescence of the sample over time. Improved sealing of the reservoirs solved this problem. A within-chip variability in the retention time of 2-10% RSD was observed. These results demonstrate the feasibility and reliability of the PPM as a solid reversed-phase for electroosmotic flow-driven chip-based chromatography in microscale total analysis systems.

Cyclophanes 1-6 catalyze the nucleophilic dealkylation of a simple sulfonium compound by potassium iodide. The cation-pi interaction is important in substrate binding, but the primarily electrostatic nature of this effect does not explain all observations concerning catalysis. As a series of substituents are placed on the cyclophane framework, a systematic variation in catalyst effectiveness is seen, such that more polarizable substituents produce more potent catalysts. This provides support for the notion that transition states are especially polarizable, and catalysis can be enhanced by maximizing London dispersion forces. The reactions studied here are very similar to the broad class of biological methylations mediated by S-adenosylmethionine, and the biological catalysts may use forces similar to those described here.

The results implicate a late, equilibrium folding intermediate in IB formation, in contrast to the apparent involvement of transient folding intermediates in other IB systems described to date. As equilibrium unfolding intermediates have also been implicated in light chain amyloidosis and deposition diseases, IB formation may prove a useful model for these human diseases.

Surface-enhanced Raman spectroscopy (SERS) has the potential to make a significant impact in biology research due to its ability to provide information orthogonal to that obtained by traditional techniques such as mass spectrometry (MS). While SERS has been well studied for its use in chemical applications, detailed investigations with biological molecules are less common. In addition, a clear understanding of how methodology and molecular characteristics impact the intensity, the number of peaks, and the signal-to-noise of SERS spectra is largely missing. By varying the concentration and order of addition of the SERS-enhancer salt (LiCl) with colloidal silver, we were able to evaluate the impact of these variables on peptide spectra using a quantitative measure of spectra quality based on the number of peaks and peak intensity. The LiCl concentration and order of addition that produced the best SERS spectra were applied to a panel of synthetic peptides with a range of charges and isoelectric points (pIs) where the pI was directly correlated with higher spectral quality. Those peptides with moderate to high pIs and spectra quality scores were differentiated from each other using the improved method and a hierarchical clustering algorithm. In addition, the same method and algorithm was applied to a set of highly similar phosphorylated peptides, and it was possible to successfully classify the majority of peptides on the basis of species-specific peak differences.

A strategy for improved surface-enhanced Raman spectroscopy (SERS) measurements that extends the variety of analytes accessible to SERS analysis is developed. The strategy involves inducing aggregation by mixing positively charged nanoparticles which form SERS-active clusters when mixed with negatively charged silver nanoparticles fabricated using the Lee-Meisel process. To make positively charged nanoparticles, silver nanoparticles using the traditional Lee-Meisel process are fabricated and coated with a thin layer of silica and the silica modified with silane chemistry. Analytes with a significant amount of negative charge exhibit strong Raman bands when the strategy using these fabricated, positively charged nanoparticles for inducing cluster formation is used, enabling their detection and analysis. We envision the use of positively charged nanoparticles in cluster formation for expanding the range of analytes that can be detected using SERS and hence the range of applications SERS can play a role in.