A study of the methylene/perfluormethylene selectivity of porous polymer monolithic stationary phases exhibiting different fluorous/hydrophobic content.

Porous polymer monolithic columns are prepared from a variety of monomers and cross-linkers and can be customized to exhibit different selectivities for separate analyte classes. The composition of the monolith can be precisely controlled by selecting different monomers and or cross-linker ratios. In this work monoliths exhibiting both fluorous and hydrophobic character were prepared using butyl methacrylate and its fluorous analogue (monomer) and 1,3-butanediol diacrylate and its fluorous analogue (cross-linker) in different ratios. The selectivity of the monoliths was probed using capillary electrochromatography with several fluorous and alkyl benzene analytes. Hydrophobic stationary phases exhibited greater methylene selectivity ( [Formula: see text] ) while those with increasing fluorous character show enhanced pefluoromethylene selectivity ( [Formula: see text] ). The Gibbs free energy change associated with the sorption of the analytes on each stationary phase composition can be calculated from migration times (i.e. capacity factor) for the addition of an individual -CF2- or -CH2- moiety. Furthermore, the Gibbs free energy change associated with a single -CF2- or -CH2- moiety (analyte) interacting with an individual -CF2- or -CH2- (stationary phase) can also be estimated by plotting fluorous column composition against [Formula: see text] . Furthermore [Formula: see text] and [Formula: see text] can be plotted versus H2O percentage in mobile phase, and a new concept, hypothetical water percentage (HWP) is proposed to evaluate the hydrophobicity/fluorophilicity of a stationary phase.

A fluorous porous polymer monolith photo-patterned chromatographic column for the separation of a flourous/fluorescently labeled peptide within a microchip.

A fluorous porous polymer stationary phase is photo-patterned within a glass microfluidic chip to conduct CEC. During free radical-initiated polymerization, extraneous polymer forms and contributes to excessive microfluidic channel clogging. Nitrobenzene is explored as free radical quencher to limit clogging by minimizing extraneous polymer formation and a number of initiator to quencher ratios are explored with a 0.5:1 quencher (nitrobenzene): initiator (benzoin methyl ether) molar ratio shown to be optimal. The microchip patterned with a fluorous monolith was used to carry out the electrochromatographic analysis of a mixture containing fluorescent and fluorous labeling products. The fluorous monolithic column shows fluorous selectivity for compounds labeled with perfluoromethylene tags and a custom peptide is synthesized that possesses functional groups that can be both fluorescently and fluorously labeled. MALDI MS was used to identify the labeled fragments and microchip based electrochromatography was used to analyze the resulting labeling mixture. This is the first report to our knowledge that uses fluorous porous polymer monolith within a microchip to separate analytes using fluorous-fluorous interactions.

Development of fluorous porous polymer monolith (FPPM) for the capillary electrochromatographic separation of fluorous analytes based on fluorous-fluorous interaction.

This is the first report on the CEC separation of fluorous analytes on a fluorous porous polymer monolith (FPPM) stationary phase based on fluorous-fluorous interaction. Monolithic columns do not require retaining frits and can be conveniently photo-patterned within a capillary. Two groups of fluorous compounds, a N-f-Cbz-4-nitro-benzylamine (N) series and a N-f-Cbz-4-phenyl-benzylamine (P) series, each series having compounds differing only by the length of their perfluorinated tag, were employed to evaluate the ability of the fluorinated column to separate fluorous analytes using a variety of mobile phase compositions and separation conditions. Fluorous monoliths showed enhanced separation performance by providing better selectivity, higher resolution and shorter analysis time compared to a similar non-fluorous (reversed phase) monolithic column. Under optimal conditions, column efficiency as high as 234,000 plates per metre was achieved, and all four compounds of the N series were fully resolved in <5 minutes. Perfluoromethylene selectivity was used to quantitatively evaluate the interaction between the perfluorinated chain on the analytes and both the FPPM and non-FPPM columns. It was found that the non-FPPM column resolves fluorous analytes mainly based on reversed phase interaction while the FPPM column resolves them mainly based on fluorous-fluorous interaction. Results are compared to fluorous monolith columns used in a nano-liquid-chromatographic (nano-LC) separation with gradient elution. The FPPM column required less than one fifth the analysis time in CEC mode than was required in nanoLC mode, with superior separation efficiency and resolution. FPPM stationary phases provide an attractive option for the analysis of perfluorinated analytes, which is expected to be useful in areas such as proteomics for the separation of fluorously tagged proteins, and in environmental analysis where fluorinated species are of increasing concern.

Fluorous monolith specificity: the effects of polymer density and secondary interactions on column performance and amenability to biological samples.

Continuing from the foundation laid by our previous work in the field, we present here an examination of the effects of monolith density and overall composition on the efficacy of performance in the realm of fluorous separations. By variation of the proportions of monomer and cross-linking agent relative to a static porogenic solvent composition, it was found that a composition of 30% polymer-forming material provides the optimal results in terms of resolution and peak shape for fluorous chromatography of a mixture of similarly labeled benzylamines. The presence of so-called "secondary interactions" that can compete with fluorous specificity in columns of this type were also examined and discussed, with similar results to those observed for commercial fluorous columns being noted. We suggest that these effects may actually be positive if they can be properly harnessed, as the ability to provide a second dimension for fluorous separations based on polarity may allow more complex analyses of labeled proteomic samples to be effectively undertaken. Finally, we present some initial results on the effectiveness of our optimized fluorous monoliths in a series of tagging and separation experiments using a custom-synthesized peptide. With successful resolution of labeled biological samples from their nonfluorous counterparts achieved, we discuss the potential expansion and further applicability of fluorous monoliths of this type in proteomic avenues, as well as their amenability to the greater analytical community.