Ultrahigh-pressure liquid chromatography: fundamental aspects of compression and decompression heating.

Ultrahigh-pressure liquid chromatography is an emerging technique for carrying out rapid and highly efficient separations. Unfortunately, one of the simplifying assumptions made in conventional high-performance liquid chromatography, incompressibility of the mobile phase, is not valid when higher and higher pressures are used. Rather, both compression and decompression of the eluent must be considered in terms of both heating and changes in the solvent's structure. The first of these problems, eluent heating during the compression and decompression cycles, which occurs in the pump and column, respectively, are considered in terms of a combined first principle-empirical approach that is solved (i.e., an analytic solution obtained from the resulting integral equation) using 0.01 Bar pressure steps. The approach is used to estimate compression and decompression heating for methanol and water.

Infrared studies of the polymorphic states of the fenamates.

Infrared spectroscopy has been used to characterize the polymorphic purity as well as to study the thermal conversion of three of the more common fenamates between their different crystalline forms via measuring changes in the NH stretch region that occur between 3300 and 3350 wavenumbers. Shifts in band frequency for mefenamic acid result from differences in internal hydrogen bonding between the NH group and either the carbonyl or hydroxyl groups of the acid moiety. Due to out-of-plane rotations about the central N-C(ring2) bond additional polymorphic states have been suggested for flufenamic and tolfenamic acids. Rates of conversion are given for flufenamic, mefenamic, and tolfenamic acids at temperatures between 85 and 160 degrees C depending on the polymorphic transition for a particular analyte. Subsequently, these rates are used to calculate the activation energy for the observed polymorphic transition. Values of 71.6, 49.0, and 50.8 kcal/mol are obtained respectively for (1) the polymorph I to II transition of mefenamic acid, (2) the polymorph I to II transition of tolfenamic acid, and (3) the polymorph III to I transition of flufenamic acid.

Rapid ESI-MS method for examining the thermal decomposition of pharmaceuticals.

A Finnigan MAT TSQ 700 triple quadrupole mass spectrometer equipped with a Harvard 22 syringe pump has been used to investigate the thermal decomposition of three common pharmaceuticals, acetaminophen, indomethacin, and mefenamic acid. In addition, comparative measurements on these same drugs were carried out by HPLC after thermally degrading them at elevated temperatures in order to evaluate the usefulness of the ESI-MS as a rapid means of determining the relative stability and identifying thermal decomposition products. Similar results were obtained between the two techniques (i.e., the relative rates of decomposition were mefenamic acid > indomethacin > acetaminophen), with the advantage that the ESI-MS approach was much quicker to perform.

Studies of the thermal degradation of acetaminophen using a conventional HPLC approach and electrospray ionization-mass spectrometry.

The thermal degradation of acetaminophen is studied via conventional accelerated aging studies by initially thermally stressing the compound at temperatures between 160 degrees C and 190 degrees C and measuring the rate of decomposition by reversed-phase high-performance liquid chromatography. Rates of decomposition of the compound in the dry state and the activation energy for the process are determined and compared with previously published kinetic and thermodynamic data for the degradation of acetaminophen in solution. In addition, the thermal fragmentation of acetaminophen under electrospray ionization (ESI) conditions using an interface with a heated capillary inlet is studied and the apparent activation energy for this process also is characterized. A comparison of the data shows that acetaminophen is significantly more stable in the dry state than in solution. However, the gas-phase fragmentation of acetaminophen under ESI conditions occurs more readily than either dry- or solution-state degradation. Although the resulting electrospray fragmentation mimics the breakdown product that is formed when the compound undergoes either acid or base catalyzed hydrolysis in aqueous solutions, the mechanism that produces the fragment ion appears to involve a two-step process. Initially, the parent ion forms of the analyte are produced in the spray region of the interface followed by wall-catalyzed decomposition and re-ionization in the heated inlet capillary of the spectrometer.

Liquid chromatographic studies of the effect of phosphate on the binding properties of silica-immobilized bovine serum albumin.

High-performance liquid chromatography has been used to examine how phosphate ions affect the binding properties of bovine serum albumin (BSA) immobilized to porous silica. In doing this, the time dependence of the protein to reach conformational equilibrium is measured as a function of the concentration of phosphate in the eluent using the D- and L-isomers of tryptophan and kynurenine as solutes. The overall binding and chiral selectivity (alphaD,L) of the protein toward these solutes appear to be related to two types of effects: one being those that are site-selective and only influence the retention of the L-isomers and the other being those that are nonselective and influence the retention of both enantiomers. An interesting feature of the concentration-dependent data is a maximum in alphaD,L at intermediate phosphate concentrations (i.e., 10 to 50mM phosphate) indicative of both cooperative and antagonistic binding effects. Phosphate eluents within this concentration range provide selectivity advantages, and those at higher concentrations decrease the time required for the protein or column to reach equilibrium. A final set of studies has also been carried out using four alternate buffer systems (i.e., borate, carbonate, acetate, and arsenate eluents). Although the borate eluents affect the BSA's binding properties and alphaD,L similar to the phosphate eluents, the other buffers result in poor separations. Observations from this study are useful in helping to optimize separations carried out on immobilized BSA as well as addressing biological and mechanistic questions related to how anions influence the native binding properties of serum albumins.

Species dependency of the liquid chromatographic properties of silica-immobilized serum albumins.

A series of chemically bonded serum albumins from different animal sources (i.e., bovine, human, sheep, and pig) have been prepared via a three-step procedure. Subsequently, the differences in the amino acid residues forming the binding pockets for L-tryptophan and related analogues (subdomain IIIA) were investigated using liquid chromatography. Even though there is a high homology among the different species of serum albumins obtained from a number of animal sources, single changes in the relevant sequence were found to significantly alter the proteins' binding selectivity. Van't Hoff plots for both the L- and the D-enantiomers show expected properties. However, changes in the amino acid sequence are reflected in changes of the specific binding/chromatographic selectivity for differently substituted L-tryptophans.

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.