Femtosecond broadband stimulated Raman: a new approach for high-performance vibrational spectroscopy

Femtosecond stimulated Raman spectroscopy (FSRS) is a new technique that produces high-quality vibrational spectra free from background fluorescence. FSRS combines a narrow-bandwidth picosecond Raman pump pulse with an approximately 80 fs continuum probe pulse to produce stimulated Raman spectra from the pump-induced gain in the probe spectrum. The high intensity of the Raman pump combined with the broad bandwidth of the probe produces high signal-to-noise vibrational spectra with very short data acquisition times. FSRS spectra of standard solutions and solvents such as aqueous Na2SO4, aqueous KNO3, methanol, isopropanol, and cyclohexane are collected in seconds. Furthermore, stimulated Raman spectra can be obtained using just a single pump-probe pulse pair that illuminates the sample for only approximately 1 ps. Fluorescence rejection is demonstrated by collecting FSRS spectra of dyes (rhodamine 6G, chlorophyll a, and DTTCI) with varying degrees of fluorescence background and resonance enhancement. The high signal-to-noise, short data acquisition time, fluorescence rejection, and high spectral and temporal resolution of femtosecond stimulated Raman spectroscopy make it a valuable new vibrational spectroscopic technique.     

A comparison of the quantitative methods for the analysis of the platinum-containing anticancer drug [cis-[amminedichloro(2-methylpyridine)]platinum(II)] (ZD0473) by HPLC coupled to either a triple quadrupole mass spectrometer or an inductively coupled plasma mass spectrometer

The use of high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICPMS) as means for the quantitative determination of ZD0473, a platinum anticancer drug, and its related biologically active "aqua" compounds in biofluid samples is described. The performance of the resulting HPLC-ICPMS method was compared with that of a conventional HPLC-triple quadrupole mass spectrometer-based (HPLC-MS/MS) system for properties such as limit of detection, linearity, and reproducibility using spiked samples. The methods were then applied to the determination of plasma ultrafitrate concentrations of ZD0473 in dog plasma samples obtained following intravenous and oral administration at 0.5 and 6 mg/kg, respectively. These experiments showed that both methods were capable of providing accurate and precise results but that the HPLC-ICPMS method had advantages of extended linear range and superior sensitivity, providing a limit of quantification of 0.1 ng/mL for ZD0473, as compared to 5 ng/mL using the current HPLC-MS/MS method. In addition, by using a single combined HPLC-ICPMS/MS/MS system, it was possible to determine the relative MS/MS response of the aqua compounds for the first time.     

Simultaneous determination of the size and surface charge of individual nanoparticles using a carbon nanotube-based Coulter counter

A resistive-pulse Coulter counter based on a membrane containing a single multiwall carbon nanotube (MWNT) channel was used to simultaneously determine the size and surface charge of carboxy-terminated polystyrene nanoparticles. The membrane was prepared from an epoxy section containing a MWNT channel mounted on a poly(dimethylsiloxane) (PDMS) support structure. The PDMS support reduced the background noise level by a factor of > 20 compared to the Si/Si3N4 support structure used in our previous study. The lower noise level makes it possible to accurately measure the height and width of resistive-pulse signals resulting from transport of individual particles through the MWNT channel. Particle sizes, calculated from current pulse heights, were comparable to those determined by transmission electron microscope (TEM). The width of the current pulses is a measure of the nanoparticle transport time, and it permits calculation of the electrokinetic surface charge. Different types of polystyrene nanoparticles having nearly the same size, but different electrokinetic surface charge, could be resolved on the basis of the difference in their transport time.     

Shifts in peptide and protein charge state distributions with varying spray tip orifice diameter in nanoelectrospray Fourier transform ion cyclotron resonance mass spectrometry

The influence of the diameter of the spray tip employed for nano-electrospray mass spectrometry (nano-ES-MS) upon mass spectral charge state distributions was investigated using angiotensin I (M(r) = 1296), insulin (M(r) = 5774), and ubiquitin (M(r) = 8560) as test analytes. Under a variety of experimental conditions, the charge state distributions of the test peptides and protein consistently shifted toward higher values as the tip orifice diameter decreased. This finding indicates that the use of narrow diameter capillaries can promote the formation of higher charge state ions that are more reactive precursors in tandem mass spectrometry experiments. A detailed comparison of charge state distributions obtained for nanospray capillaries of varying diameters was undertaken while systematically varying experimental parameters such as sample flow rate, analyte concentration, solvent composition, and electrospray current. The general tendency to obtain higher charge states from narrow diameter capillaries was conserved throughout, but tips with smaller orifices were more sensitive to sample flow rate (the average charge state was lowered significantly as flow was raised), whereas tips with bigger orifices were more sensitive to analyte concentration and pH of the solution (as each was lowered, the average charge state increased).     

Comparison of ultraviolet femtosecond and nanosecond laser ablation inductively coupled plasma mass spectrometry analysis in glass, monazite, and zircon

We compared the analytical performance of ultraviolet femtosecond and nanosecond laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS). The benefit of ultrafast lasers was evaluated regarding thermal-induced chemical fractionation, that is otherwise well known to limit LA-ICPMS. Both lasers had a Gaussian beam energy profile and were tested using the same ablation system and ICPMS analyzer. Resulting crater morphologies and analytical signals showed more straightforward femtosecond laser ablation processes, with minimal thermal effects. Despite a less stable energy output, the ultrafast laser yielded elemental (Pb/U, Pb/Th) and Pb isotopic ratios that were more precise, repeatable, and accurate, even when compared to the best analytical conditions for the nanosecond laser. Measurements on NIST glasses, monazites, and zircon also showed that femtosecond LA-ICPMS calibration was less matrix-matched dependent and therefore more versatile.     

Cavity ring-down spectroscopy as a detector for liquid chromatography

We have demonstrated the use of cavity ring-down spectroscopy (CRDS) as a detector for high performance liquid chromatography (HPLC). For this use, we have designed and implemented a Brewster's angle flow cell such that cavity ring-down spectroscopy can be performed on microliter volumes of liquids. The system exhibits a linear dynamic range of 3 orders of magnitude (30 nM to 30 microM quinalizarin at 470 nm) for static measurements and 2 orders of magnitude (0.5 microM to 50 microM) for HPLC measurements. For the static measurements, the baseline noise is 2.8 x 10(-6) AU rms and 1.0 x 10(-5) AU peak-to-peak, and for the HPLC separations, it is 3.2 x 10(-6) AU rms and 1.3 x 10(-5) AU peak-to-peak. The baseline noise is determined after the data are smoothed by an 11-point boxcar average. The peak areas detected from HPLC separations are reproducible to within 2-3%. The HPLC mass detection limit for a molecule with epsilon = 9 x 10(3) M(-1) cm(-1) in a 300-microm path length cell (illuminated volume, 0.5 microL) is reported as 2.5 x 10(-8) g/mL. These results were obtained using a simple pulsed CRDS system and are comparable to, if not better than, a high-quality commercial UV-vis absorption detector for the same path length.     

Mass and composition matrix-assisted laser desorption ionization time of flight analysis enabling inference of the sequence of most peptides where the protein of origin is known

Mass spectrometers combining matrix-assisted laser-desorption ionization (MALDI) and time-of-flight analysis are among the most widely used in peptide analysis. They excel at accurate mass determinations on complex samples but, compared with tandem instruments, have very limited capacity to determine amino acid sequence through daughter ion analysis. Here we have investigated the sequence information that can be inferred from the masses of peptides in the special circumstance in which the peptides are known to be sub-sequences of known parent sequences. We show how sequence can be inferred from the measured m/z of a peptide (mass analysis) and examine the parameters that influence the level of confidence that can be placed in "inferred sequences". We further describe how specific amino acid modifications can be used with MALDI-TOF analysis to obtain partial composition information and demonstrate that combined mass and composition (MAC) analysis enables the sequences of most peptide ions to be inferred with very high confidence.     

Detection of G proteins by affinity probe capillary electrophoresis using a fluorescently labeled GTP analogue

An affinity probe capillary electrophoresis (APCE) assay for guanine-nucleotide-binding proteins (G proteins) was developed using BODIPY FL GTPgammaS (BGTPgammaS), a fluorescently labeled GTP analogue, as the affinity probe. In the assay, BGTPgammaS was incubated with samples containing G proteins and the resulting mixtures of BGTPgammaS-G protein complexes and free BGTPgammaS were separated by capillary electrophoresis and detected with laser-induced fluorescence detection. Separations were completed in less than 30 s using 25 mM Tris, 192 mM glycine at pH 8.5 as the electrophoresis buffer and applying 555 V/cm over a 4-cm separation distance. BGTPgammaS-Galpha(o) peak heights increased linearly with Galpha(o) up to approximately 200 nM using a 50 nM BGTPgammaS probe. The detection limit for Galpha(o) was 2 nM, corresponding to a mass detection limit of 3 amol. The high speed of the APCE assays allowed reaction kinetics and the dissociation constant (Kd) to be determined. The on-rate and off-rate of BGTPgammaS to Galpha(o) were 0.0068 +/- 0.0004 and 0.000 23 +/- 0.000 01 s(-1), respectively. The half-life of the BGTPgammaS-Galpha(o) complex was 3060 +/- 240 s and Kd was 8.6 +/- 0.7 nM. The estimates of these parameters are in good agreement with those obtained using established techniques, indicating the suitability of this method for such measurements. Lowering the temperature of the separation improved the detection of the complex, allowing the assay to be performed on a commercial instrument with longer separation times. Additionally, the capability of the technique to detect several G proteins based on their binding to BGTPgammaS was demonstrated with assays for Galpha and Galpha(i1) and for Ras and Rab3A.     

Direct dating of archaeological pottery by compound-specific 14C analysis of preserved lipids

A methodology is described demonstrating the utility of the compound-specific 14C technique as a direct means of dating archaeological pottery. The method uses automated preparative capillary gas chromatography employing wide-bore capillary columns to isolate individual compounds from lipid extracts of archaeological potsherds in high purity (>95%) and amounts (>200 microg) sufficient for radiocarbon dating using accelerator mass spectrometry (AMS). A protocol was developed and tested on n-alkanes and n-carboxylic acids possessing a broad range of 14C ages. Analytical blanks and controls allowed background 14C measurements to be assessed and potential sources of errors to be detected, i.e., contamination with modern or dead 14C, isotopic fraction effects, etc. A "Russian doll" method was developed to transfer isolated target compounds onto tin powder/capsules prior to combustion and AMS analyses. The major advantage of the compound-specific technique is that 14C dates obtained for individual compounds can be directly linked to the commodities processed in the vessels during their use, e.g., animal fats. The compound-specific 14C dating protocol was validated on a suite of ancient pottery whose predicted ages spanned a 5000-year date range. Initial results indicate that meaningful correlations can be obtained between the predicted date of pottery and that of the preserved lipids. These findings constitute an important step forward to the direct dating of archaeological pottery.     

Addition of trace metals increases denitrification rate in closed marine systems

We investigated the effect of trace metals (Fe, Mn, Cu, Zn and Mo) on the denitrification unit at the Montreal Biodome. Two dosages of the five trace metals were tested on a denitrifying bacterial population which was extracted from the denitrification unit and cultured in 250 mL chemostats with artificial seawater. The low dosage showed a 20% increase in the denitrification rate whereas the high dosage had a more pronounced effect with a 250% increase. No increase in bacterial growth was observed, suggesting that the trace metals had an effect on the denitrification activity. When the trace metals were tested separately, only iron had a significant effect similar to the increase in the denitrification rate observed when the five trace metals were added. The combination of Fe and Mn caused a small but significant increase compared to the five trace metals. We then tested the effect of adding Fe, Mn and Cu to the denitrification unit at the Montreal Biodome. A high dosage of these trace metals showed a 250% increase in the denitrification rate, which went from 200 to 700 g NO(x)-N/d. Our results showed that the addition of trace metals is crucial for denitrification activities.