Characterization and multi-mode liquid chromatographic application of 4-propylaminomethyl benzoic acid bonded silica--a zwitterionic stationary phase.

4-Propylaminomethyl benzoic acid bonded silica (4-PAMBA-silica) was synthesized by reacting aminopropyl modified silica with 4-carboxybenzaldehyde and reducing the resulting Schiff base with sodium cyanoborohydride in situ. The structure of this bonded phase was confirmed by (13)C cross polarization magic angle spinning ((13)C CP MAS) NMR. Elemental analysis indicated a coupling efficiency of about 79%. Chromatographic characterization of a 4-PAMBA-silica stationary phase revealed that at a mobile phase pH of 3.0, basic compounds were unresolved and co-eluted near the void volume, while aromatic sulfonates were retained and were well-resolved. By contrast, at a mobile phase pH of 7.0, the aromatic sulfonates were unresolved and eluted at the void volume, while basic compounds were retained and were well-resolved. To further understand the chromatographic retention mechanism the retention factors for a series of cationic and anionic compounds were measured at pH 7.0 and 3.0 as a function of the charge and concentration of competing ions in the mobile phase. A plot of the logarithm of the retention factor versus the logarithm of the eluent ion concentration was linear with a negative slope that is equal to the ratio of effective charges of the solutes and the eluent ions. This indicates that an ion exchange mechanism contributes to the separation of both cations and anions at pH 7.0 and pH 3.0, respectively. The increase in retention of alkanoic acids with their number of carbons at a mobile phase pH of 7.0 and exclusion of alkanoic acids at a mobile phase pH of 3.0 suggests that an ion exclusion mode and hydrophobic interaction mode are also operational with 4-PAMBA-silica. The amino acids, L-arginine, L-phenylalanine, and L-tyrosine were retained and well-resolved with a mobile phase containing a high concentration of organic solvent. This behavior was further studied by measuring the retention factors of polar and charged compounds as a function of the organic solvent content in the aqueous mobile phase. An increase in retention with decreasing water content in the mobile phase was observed, consistent with a hydrophilic interaction liquid chromatographic (HILIC) mode of separation.

The influence of hydration on the conformation of bovine serum albumin studied by solid-state 13C-NMR spectroscopy.

13C proton-decoupled cross-polarization magic-angle spinning nmr spectra of bovine serum albumin are reported as a function of hydration. Increases in hydration level enhance the resolution of the peak centered at about 40 ppm but has little or no effect on the other spectral peaks. Hydration has little effect on either the rotating frame proton spin-lattice relaxation time or the cross-relaxation time for any of the peaks, suggesting that the efficiency of dipolar coupling is largely preserved on hydration of the protein. Resolution enhancement of the peak at 40 ppm is not understood, but possible sources of the behavior include a decrease in the line width of contributing resonances from lysine epsilon carbons due to increased motional averaging on hydration, reordering of disulfide bridges, and titration shifts induced by hydration. Hydration of bovine serum albumin appears to have little effect on the distribution of conformations sampled by the protein so that the broad distribution of conformations observed in the dry state is also observed in the fully hydrated state. This is in contrast to lysozyme where significant ordering of the conformation is seen on hydration.

The influence of hydration on the conformation of lysozyme studied by solid-state 13C-NMR spectroscopy.

13C proton decoupled cross-polarization magic-angle spinning nmr spectra of lysozyme are reported as a function of hydration. Increases in hydration level enhance the resolution of the spectra, particularly in the aliphatic region, but has no significant effect on either the rotating frame proton spin-lattice relaxation time or the cross-relaxation time. The enhancement in spectral resolution with hydration is attributed to a decrease in the distribution of isotropic chemical shifts, which reflects a decrease in the distribution of conformational states sampled by the protein. Changes in the distribution of isotropic chemical shifts occur after the addition of water to the charged groups as coverage of the polar side chains and peptide groups takes place. The onset of this behavior occurs at a hydration level of about 0.1-0.2 g water/g protein and is largely complete at about 0.3 g water/g protein, the same hydration range where changes in the heat capacity are observed. That hydrogen exchange of buried protons can occur at hydration levels significantly lower than those at which changes in the distribution of conformational states are first observed suggests that some motions that mediate exchange are already present in the dry protein. The preservation of efficient dipolar coupling indicates that the conformational rearrangements that do occur on hydration are small and do not involve any significant overall expansion of free volume or weakening of interactions that would increase the reorientational freedom of protein groups.

Applications of fluorinated compounds as phases and additives in chromatography and their uses in pharmaceutical analysis.

A review is presented of the use of fluorinated phases and fluorine-containing surfactants in chromatography. Additionally, new information is provided on the application of two nonionic perfluorosurfactants as reversed-phase mobile phase additives to enhance chromatographic performance of aromatic amines and some amines of pharmaceutical significance.

Nuclear magnetic resonance spectrometry of chemically and physically altered porous silica surfaces under gas chromatographic conditions.

Conventional nuclear magnetic resonance spectrometry in solution, in combination with (13)C labelling, has been utilized to investigate differences in freedom of motion of similar chemically altered and physically modified porous silica sorbents under dry conditions, and the results have been correlated with previously reported results for chemically altered solvated surfaces. Spin-lattice relaxation time of the labelled terminal methyl groups decreases in the order chemically modified solvated ligands, chemically bonded unsolvated ligands, and physically sorbed unsolvated molecules.