News modulator vpi^ivij, trie researchers produce a heterodyne signal between a weak
CD-induced polarization grating and a stronger intensity grating, thus enhancing the CD signal. The pump laser is a pulsed Nd:YAG, so the method can be used for timeresolved CD measurements that are limited only by the pulse duration of the pump laser. The authors did not thoroughly study the method's detection limit, but the experimental results were background limited. They say that artifacts caused by polarization optics and sample cells must be Experimental setup. (P1 and P2, polarizers; IF, overcome. (J. Am. Chem. Soc. interference filter; SF, spatial filter; CF, color filter to 1997 119 8293-300) block excitation at 266 nm.)
In search of a combinatorial screening method Combinatorial chemistry techniques have led to an explosion of compounds that must be screened for biological activity. The analytical challenge lies in devising fast methods for screening these libraries. Solid-phase screening methods have been the mainstay of combinatorial chemistry, but solution phase assays could improve the specificity of screens. Paul Vouros and co-workers at Northeastern University and Cubist Pharmaceuticals combine MS with CE or LC to screen ligands in solution, but they add an extra step. The large protein complexes are isolated from the unbound ligands with gel-filtration spin columns. The protein-ligand complexes are then disso-
SCIENCE
Going mobile The aerosol time-of-flight mass spectrometers (ATOFMS) designed by Kimberly Prather's group at the University of California-Riverside frequently take field trips. When Analyttcal Chemistry spoke with Prather, the instruments were in central Los Angeles and Azusa (a small city located between Los Angeles and Riverside) participating in the Southern California Ozone Study (SCOS97-NARSTO, North American Research Strategy for Tropospheric Ozone), an effort to study real-time ozone formation. Prather's group uses MS to characterize the composition and source of atmospheric particles. 652 A
ciated, and the ligands are identified by LC/MS or CE/MS. They discovered that a substantial amount of the protein was lost in the spin columns; however, the low cost, speed, small sample volumes, and disposability of the columns still make them preferable to standard size exclusion chromatography columns. Human serum albumin was used as a test receptor for six ligands of varying affinity for the protein. The authors believe that the method is well suited to ligands with high binding affinity. The analysis could be performed with as little as 900 pmol of the receptor protein an amount that the authors could easily be reduced. LC sPPTT1s
better suited to the analysis than CE because it is more tolerant of matrix components and high salt concentrations (Rabid Commun Mass Sbectrom 1997 11 1178-84) The ATOFMS instruments, which use a dual reflectron design, are described in the Oct. 15 issue of Analytical Chemistry y(. 4083). The eositive end negative ions generated by the desorption and ionization of a single particle are analyzed simultaneously, which is particularly useful in identifying the source of atmospheric particles. "The dual reflectron with online detection gives you more than double the information," says Prather. "By knowing twice as much about the composition of the particle, it gives you that much more information about the origin of the particle and subsequent chemistry it may have undergone in the atmosphere." The biggest challenge that Prather's group faced in building the instrument
Analytical Chemistry News & Features, November 1, 1997
Binding constants by CZE Currently,fivemethods are available to determine binding constants by capillary zone electrophoresis (CZE): frontal analysis (FA), Hummel and Dreyer (HD), affinity CE (ACE), vacancy peak (VP), and vacancy affinity CE (VACE). M.A.H. Busch and co-workers at the University of Amsterdam (The Netherlands) review these methods and discuss their limitations. When the mobilities of a protein and its complex are equal, accurate binding parameters can be obtained by the FA, HD, and VP methods. However, when the mobilities differ, systematic deviation will occur in the constants if determined by those three methods; the ACE or VACE methods should be used instead. The ACE method can be performed only when the mobilities of the complex and protein differ. For a monovalent complex, all five methods work equally well; although FA is often favored because it is the simplest and most robust of the methods and requires the least amount of sample. When multiple equilibria are involved, the ACE method cannot be used. In addition, the VACE method is complicated by multiple equilibria and, unless the relationship between interacting species is well known, should not be performed. (J. Chromatosr. A 1197, ,77, 329-53)
was the arrangement of the detectors. The detectors are coaxial with holes in the center for ions to enter the flight tubes and need to be floated at high voltage to maintain a field-free region, while providing appropriate accelerating voltages for both ion polarities. The ion signal needed to be capacitively decoupled from the accelerating voltages, but that turned out to be a relatively minor problem, according to Prather. She says that through a major effort, everything just "came together", and the design was up and running in less than a year. It could have taken even less time if they hadn't been so "picky" about the signal she The instruments combine a conventional method of atmospheric particle analysis with mass spectrometric analysis.
The ATOFMS samples particles at the beach.
Before the particles enter the mass spectrometer, they travel through a particle sizing region, with two continuous-wave laser beams for aerodynamic sizing. The laser beams are directed to the system by fiber optics, eliminating the need for aligning the laser. On average, about 10% of the sized particles are chemically analyzed, or "hit". The histograms of hit versus missed particles are well matched in terms of aerodynamic diameter. Therefore, when the system is properly aligned, no size bias is apparent. Prather says that —90% of the sized particles are missed because the Nd:YAG laser used for desorption and ionization has relatively low power and a small probe volume. The instrument is being used with more established atmospheric monitoring techniques to determine scaling factors that can be used to convert the number distribution to a real distribution of what is present in the atmosphere. For the ATOFMS data to be used with historical
conventional data, the number distribution must be scaled to a mass distribution. Prather says, "As part of developing a new technique, you have to show that you can match existing technology before you go on." She comments that a strength of doing continuous monitoring in conjunction with conventional offline sampling is that the continuous monitoring allows more judicious offline sampling, saving both time and money. The instrument travels well. Allhough it hasn't actually been operated during transport, Prather says that because the instrument works before transport and can be fully operational within 10 min. of arrivall ,i is reasonable to assume that the instrument could work en route. She says that one of the goals is to use the instrument aboard aircraft to monitor air quality. In the recently published work, the performance of the ATOFMS was characterized with wood smoke particles, because they are easily generated and form a broad distribution centered at relatively small sizes. Now, however, the instruments are being used to study real atmospheric problems. The mass spectra are being used to broadly characterize particles according to origin, rather than to provide a definitive chemical identification. "We do really well at identifying the inorganic f ("in-
tent The organic content is more difficult because you can get fragmentation We would like to as far as the organic content goes have one or two oeaks stand out as tracer Deaks If we see that peak
Schematic of the particle sizing and mass spectrometer regions of the instruments.
ethyl sulfoxide (DMSO) to quantitatively generate a methyl radical. A fluorescaminederivatized nitroxide then reacts with the methyl radical, forming a stable product, Identifying free radicals is a difficult task, Omethylhydroxylamine, which is sepaparticularly in complicated biological and rated by reversed-phase HPLC and quantienvironmental matrices. In many cases, fied fluorometrically. The method detects radical concentrations are too low to be nanomolar levels of OH in complicated maquantified by traditional spin trapping trices. In contrast to aromatic hydroxylation methods. Neil Blough and co-workers at techniques involving benzoic acid or salithe University of Maryland-College Park cylic acid, this method is fairly innocuous to and the University of Maryland School of cells andtissues.Both methods require Medicine, Baltimore, have developed a high reagent concentrations to trap radicals new, highly sensitive method for detectlike OH quantitatively. With aromatic hying and quantifying trace levels of the hydroxyl radical (OH) in biological matrices. droxylation however you can run into solubility and toxicity problems says Blough The method which is described in this issue of Analytical Chemistry (p. 4295) in- High concentrations of DMSO do not pose much of a problem volves the reaction between OH and dim-
What's downstream?
combination of peaks, we can be reasonably sure that it came from a particular source or had a particular reaction occur on it. We're trying to learn about the chemistry of these particles as well as their origins." The next step for the instruments is more of what they've already been doing—monitoring atmospheric phenomena. Prather's group, in collaboration with Glen Cass's group at the California Institute of Technology, used the ATOFMS to study marine aerosol chemistry. She says, "The experiments went extremely well in that we directly monitored aerosol processes as they were occurring on the particles. We plan to perform similar field measurements as part of SCOS97. In addition, when the field studies are completed we plan to interface the ATOFMS instruments to reaction chambers so we can run laboratory studies on aerosol reactions to obtain complementary information on tropospheric processes" Celia Henry
According to Blough, the initial goal was to lower free radical detection limits to levels typically found in natural waters. Spin trapping with electron paramagnetic resonance (EPR) detection is not sufficiently sensitive or reliable to investigate radical processes in many environmental situations. Blough and his co-workers have been developing a more sensitive alternative to spin trapping for about 10 years. "Originally, the idea was to have a pure opttcal lensor," says Blough. When a niiroxide is covalently attached to a fluorophore, the fluorescence is efficiently quenched. However if the nitroxide reacts with a free radical the quenching is eliminated and the fluorescence increases thus providing a sensitive means for measuring radical scaveng-
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