Search

The desorption rate constants for a cationic dye from strong adsorption sites are compared for the same chromatographic interface but for two different substrates, fused silica and chromatographic silica gel. The dye is 1,1'-didodecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI). The interface consists of acetonitrile and a hydrocarbon monolayer ([C.sub.8]) covalently bound to the silica substrate. To measure slow desorption from fused silica, fluorescence imaging combined with correlation spectroscopy is used. To measure slow desorption from silica gel, fluorescence movies of silica gel particles are used. In both cases, the results show that there are two types of slow desorption processes on time scales exceeding 1 s. The desorption time from one type of site is within an experimental error of 7 s for both silica substrates. The adsorption kinetics for this type of site are slow, and the equilibrium population of DiI on these sites is comparable to that for DiI weakly adsorbed to the hydrocarbon monolayer. For the second type of site, for fused silica, the population of DiI is even higher than that of weakly adsorbed DiI, and the desorption time constant is ~2 min, although this is likely shortened by photobleaching. For silica gel, the relative population of DiI on this ultrastrong site is more than an order of magnitude lower, and the desorption time constant is 4.0 [+ or -] 0.1 min. Both silica substrates thus show two types of sites whose time constants agree within experimental error, suggesting that the strong adsorption sites on fused silica are chemically the same as those on chromatographic silica gel.

The effect of dosing vehicle excipients such as PEG400, propylene glycol, Tween 80, and hydroxypropyl-[beta]-cyclodextrin on the accuracy of LC/MS measurements used in pharmacokinetic studies is examined. Using PEG400 as a probe compound, the concentration-time profile of the excipient in plasma from rats dosed both orally and intravenously is determined. These excipient plasma concentrations can result in a 2-5-fold increase in calculated plasma clearance values when the excipient interferes with the quantitation of the dosed compound. This can result in false rejection of a compound in a drug discovery screen. Several plasma purification methods and enhanced chromatographic selectivity are examined as ways to minimize or avoid excipient effects, particularly for very polar compounds. The combination of efficient sample purification and selective chromatography provides an effective way to diminish the significant interference effects of PEG400 and Tween 80. When appropriate, using negative ion mode MS or changing a dosing vehicle excipient, such as substituting propylene glycol for PEG400, provides an alternative approach for eliminating signal interference. The mechanism of excipient-related signal interference is discussed in relation to both competition of gas-phase proton-transfer reactions and high viscosity of dosing excipients.

Commercial spectrophotometers typically use absorption-based wavelength calibration reference materials to provide wavelength accuracy for their applications. Low-mass fractions of holmium oxide ([Ho.sub.2][O.sub.3]) in dilute acidic aqueous solution and in glass matrixes have been favored for use as wavelength calibration materials on the basis of spectral coverage and absorption band shape. Both aqueous and glass [Ho.sub.2][O.sub.3] reference materials are available commercially and through various National Metrology Institutes (NMIs). Three NMIs of the North American Cooperation in Metrology (NORAMET) have evaluated the performance of [Ho.sup.3+](aq)-based Certified Reference Materials (CRMs) under 'routine' operating conditions using commercial instrumentation. The study was not intended to intercompare national wavelength scales but to demonstrate comparability of wavelength measurements among the participants and between two versions of the CRMs. It was also designed to acquire data from a variety of spectrophotometers for use in a NIST study of wavelength assignment algorithms and to provide a basis for a possible reassessment of NIST-certified [Ho.sup.3+](aq) band locations. The resulting data show a substantial level of agreement among laboratories, instruments, CRM preparations, and peak-location algorithms. At the same time, it is demonstrated that the wavelength comparability of the five participating instruments can actually be improved by calibrating all of the instruments to the consensus [Ho.sup.3+](aq) band locations. This finding supports the value of absorption-based wavelength standards for calibrating absorption spectrophotometers. Coupled with the demonstrated robustness of the band position values with respect to preparation and measurement conditions, it also supports the concept of extending the present approach to additional NMIs in order to certify properly prepared dilute acidic [Ho.sub.2][O.sub.3] solution as an intrinsic wavelength standard.

Single-molecule imaging is used for the first time to study the cationic dye, 1,1'-dioctadecyl-3,3,3'3'-tetramethyl-indocarbocyanine perchlorate (DiI), at the chromatographic interface consisting of acetonitrile/water and a hydrocarbon monolayer ([C.sub.18]) covalently bound to silica. Autocorrelations of burst data agree with our previous single-molecule counting results, showing that most dye molecules are diffusing and that there is a rare specific adsorption site associated with a 0.07-s desorption time. These autocorrelations go further in detecting an even rarer specific adsorption event associated with a 2.6-s desorption time. The latter desorption time would contribute much more significantly to peak tailing in chromatography. In water, the populations of DiI at these two specific adsorption sites are shown to be 11% and 4%, respectively, for the weaker and stronger sites, relative to the diffusing population of DiI. In 60% acetonitrile/ water, the relative populations of the specific adsorption sites are 11% and 17%, showing that acetonitrile enhances the population of the stronger specific adsorption site. Fluorescence movies of single and multiple molecules link the stronger specific adsorption sites to specific locations on the surface. The imaging makes rare observations frequent by pinpointing where the events occur spatially. This ability to observe rare events by imaging reveals the presence of a third type of specific adsorption site, for which DiI has a desorption time in excess of 20 s.

Research has been conducted on the (+)-pancratistatin. The results of the synthetic conversion of (+)-narciclasine to (+)-pancratistatin have indicated that pancratistatin modification leads to the decrease of the cancer cell growth inhibitory activity.

Fluorescence microscopy was used to investigate the origin of strong adsorption sites on silica. Fluorescence from a cationic dye, 1, 1[prime]-dioctadecyl-3,3,3[prime],3[prime]-tetramethylindocarbocyanine perchlorate, was imaged at the chemical interface between fused silica modified with chlorodimethyloctadecylsilane and water or acetonitrile. The fluorescence images reveal that the dye adsorbs strongly at lines and points on the surface. Comparison of an atomic force micrograph and a fluorescence micrograph of the same region show the same features, indicating that strong adsorption occurs at nanometer topographical indentations. Fluorescence recovery after photobleaching shows that recovery occurs through repopulation of the same adsorption sites. The rate of adsorption to the sites was found to be much slower for water than for acetonitrile as the mobile phase. End capping greatly reduces the number of strong adsorption sites, which indicates that exposed silica is the origin of irreversible adsorption. Similar but brighter patterns of strong adsorption appear when water is replaced with acetonitrile, and the patterns remain obvious even at surface concentrations approaching those used in HPLC. The adsorption behavior is the same for pH 7 and pH 2, showing that these topographical indentations behave as the putative 'active silanols'. Since the size distribution of the nanometer polishing marks overlap the pore size distribution of chromatographic silica gel, these results suggest that surface topography is a previously unrecognized factor in the tailing of organic bases.

Detection of gas-phase ammonia is particularly challenging because ambient ammonia concentrations may be less than 1 ppb (molecules of N[H.sub.3] per [10.sup.9] molecules of air), ammonia sticks to many materials commonly used to sample air, and particles containing ammonium may interfere with gas-phase measurements. We have built a new and sensitive photothermal interferometer to detect gas-phase ammonia in situ, under typical atmospheric conditions. Ammonia molecules in sampled air absorb infrared radiation from a C[O.sub.2] laser at 9.22 [[micro]meter], with consequent collisional heating, expansion, and refractive index change. This change in refractive index is detected as a phase shift in one arm of a homodyne interferometer. Measurements of vibrational and electrical noise in the interferometer correlate to an instrumental lower limit of detection of 6.6 ppt ammonia in 1 s. The C[O.sub.2] laser output is modulated at 1.2 kHz, and the ac signal from the interferometer is measured with a lock-in amplifier. The detector is zeroed by sampling through a [H.sub.3]P[O.sub.4]-coated denuder tube and is calibrated by dynamic dilution of two permeation tube outputs and by standard addition. Signal gain is insensitive to C[O.sub.2] or [H.sub.2]O in the sample, and the signal is linear over 5 orders of magnitude. The instrument 2[Sigma] precision is 31 ppt when the signal is integrated for 100 s and 250 ppt with a 1-s integration time. The windowless sample cell and inlet is fabricated entirely of glass to minimize sample loss and hysteresis. The instrument response time is demonstrated to be about 1 s.

Study of methyl-accepting polypeptides in yeast Saccharomyces cerevisiae cells reveals the presence of methyl-esterified RNAs and methyl-esterified polypeptides. The methyl-esterified 38-,33/31- and 26 kDa species are not methylated in the absence of STE14 gene function, indicating that they are substrates for STE14 isoprenylcysteine methyltransferase. The 35 kDa and 49 kDa yeast polypeptides are methylated even in species lacking STE14 methyl transferase, revealing the presence of a novel carboxyl methyltransferase in the yeast S. cerevisiae cells. Protein synthesis inhibitor does not affect the methylation of STE-14 independent substrates.

The synthesis of benzo- and norbornane-fused 1,3, 2-dioxaphosphepanes provides the materials for their stereochemical and conformational studies. Spectral data suggest an equilibrium between two chair forms where the phenyl, phenoxy and dimethylamino substituents align along the axial plane. There are signs, however, of some flexible twist forms. X-ray studies show that the norbornane-fused phenyl and phenoxy forms as well as the benzo-fused phenyl derivative are chair-shaped seven-membered species. The other benzo-fused dioxaphosphepane adopts a twist conformation.