SCIENCE
Fourier-Transform Mass Spec Joins Analytical Repertoire Scientists modify FTMS, use new ionization techniques, and show high-resolution spectra over wide mass ranges with exact-mass measurements Stephen C. Stinson, C&EN New York
Fourier-transform mass spectrometry (FTMS) has graduated from an esoteric specialty to an important adjunct to analytical chemistry. This is the theme that emerged from papers presented by three research groups to technical sessions at this year's Pittsburgh Conference & Exposition on Analytical Chemistry & Applied Spectroscopy, held late last month in New Orleans. For example, analytical chemistry professor Charles L. Wilkins and his coworkers at the University of California, Riverside, showed highspeed, high-resolution spectra over wide mass ranges with exact-mass measurements. This achievement lets chemists use MS to analyze complex mixtures with efficiencies that match separation speeds of modern capillary gas chromatography (GC). To keep pressures in the ion trap in the 10~ 8 -torr range needed for high resolution, the Riverside group
used a splitter to reject 99% of GC eluates, restriction tubing, and a pulsed valve to introduce chemical ionization reagent gas. In another approach to the same problem, analytical chemistry professor Robert L. Settine of the University of Alabama, Birmingham, demonstrated use of an ion trap divided into two compartments. Ions generated in the relatively high-pressure, 10~ 6 -torr side were trapped and analyzed at lower pressures in the second compartment. The California team also has developed laser desorption as a "soft i o n i z a t i o n " t e c h n i q u e for h i g h molecular-weight, nonvolatile compounds of biological and industrial importance. And analytical chemistry professor David H. Russell of Texas A&M University described use of a cesium ion gun that achieves similar good results. Laser beams and cesium ions give strong peaks related to the parent ion as well as rich fragmentation patterns as aids to structural studies of complex species. Russell and Wilkins have modified the amplifiers of their respective standard FTMS instruments to work at lower frequencies, which translate into detectability of higher-mass ions. For example, Russell showed spectra with cesium iodide
Oligosaccharide ions fragment to characteristic products
(Reducing end)
(Nonreducing end) K -Cleavage M-Cleavage
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March 18, 1985 C&EN
X-Cleavage
ion clusters in the 12,000-atomic mass unit (amu) range. For their part, the California chemists detected an ion of 6841 amu in the spectrum of polyhexafluoropropylene oxide. Russell says such successes point the way to studies of ions whose masses extend toward the upper, 150,000-amu, theoretical limit of the FTMS technique. The research that Wilkins of Riverside described in New Orleans was done with graduate students Mild r e d L. Coates, Carl F. Ijames, Carolyn L. Johlman, and Cathy L. C. Yang, and postdoctoral fellows Robert S. Brown, Charles C. Koch, David A. Laude Jr., and David A. Weil, supported by the National Institutes of Health and National Science Foundation. Russell's work with graduate student Mauro E. Castro at Texas A&M was supported by NIH. Settine is on leave from Alabama to Nicolet I n s t r u m e n t Corp., Madison, Wis., w h e r e he worked with research managers Sahba Ghaderi and Duane P. Littlejohn. One barrier to exact-mass measurement in analysis of mixtures by GC-FT-MS over a wide range of ion masses is the need for a calibration substance. But inclusion of a calibrant in the sample may yield calibrant peaks that overlap those of the sample. The calibrant also can raise pressures in the ion trap, degrading resolution. And subtraction of background may be difficult, because calibrant ions may influence frequencies of sample ions. A second barrier to exact-mass measurement that some workers have feared is the high computer memory requirement. Resolution depends partly on the number of data points per peak. In her paper, Johlman of Riverside described leakage of perfluoro-
FTMS manipulates ions in cubical cavity •Transmitter
-Receiver Trap^. Trap Collector -Ion source
Receiver
Transmitter
tributylamine calibrant into the ion source and pumping it away before running the GC sample. Ionization was by electron impact (EI) or chemical ionization (CI) with methane r e a g e n t gas. With Wilkins a n d Laude, she constructed a calibration table of exact masses of the five calibrant peaks from 69 to 264 amu. Computer programing subtracted data that were not part of peaks, filled in those data with zeros, and allotted 64 kilobytes of memory per peak. The results were average errors as low as 2.8 ppm for exact masses of ions from a seven-component mixture and 4.5 ppm for 10 components. An advantage of FTMS for any analysis is that computer programing of instrument functions allows fast s w i t c h i n g a m o n g different modes. For example, Johlman described a simple sequence of quenching ions from the preceding analysis, EI, and then excitation and detection of ions, interspersed with appropriate delays. For CI, they programed an additional delay to pump away reagent gas before detection. The Riverside researchers ran an EI plus a CI analysis on each GC peak in less than two seconds total, well within the five- to 10-second spaces between peaks in modern GC. From research by the California group on higher-molecular-weight compounds, David Weil described work with glucose oligomers. Using tri-, tetra-, penta-, and heptaa-D-glucoses, laser desorption, and doping with potassium bromide,
Ions enter cubical cavity (exploded view shown) from source through hole in trap plate Magnetic field in direction of ion path causes ions to orbit around it (orbit of positive ions shown) Potential applied to trap plates keeps ions in cavity Electric field alternating at radiofrequency ranges applied through transmitter plates accelerates ions to characteristic orbits according to mass-charge ratios of ions Orbiting ions generate characteristic ac currents detected by receiver plates Detected currents are digitized and subjected to Fourier transform to yield mass spectra Collector plate traps electrons from ion source to complete ion current circuit
they have identified characteristic patterns of ion cleavages. Interestingly, cleavage occurs not at glycosidic linkages but within glucose moieties. Potassium bromide increases sensitivity and results in such ions as [M + K] + . Weil outlined a new classification system for these cleavages. The Riverside researchers use JA, A, and K to denote cleavage types. Their notations for ions are A if they came from the nonreducing end and Z from the reducing end of the chain. Arabic numerals denote the glucose unit involved. For example, Z6/c describes an ion arising from K-type cleavage in the sixth glucose unit from the reducing end. Weil cautions that this highly characteristic cleavage pattern is valid for this oligosaccharide series only. He calls for further work with other series to bear out whether such a diagnostic system can be used for structural studies generally. Demonstrating the power of laser desorption FTMS for other polysaccharides, Weil showed spectra of starch. Starch consists of 25% straightchain amylose and 75% branchedchain amylopectin. The interesting feature of starch spectra is what Weil calls a " s h a d o w i n g " of A/x-type peaks by other peaks that are 16 amu lower. Weil suggests that these s h a d o w i n g peaks are related to branching segments of amylopectins. In Russell's work on cesium ion desorption, he used a cesium aluminosilicate glass filament heated
to 1000 °C as the ion source. He has shown that he can aim the ions into the source region along the direction of the magnetic field applied to the instrument. Cesium ions are so massive, he points out, that there is no distortion of the ion beam by the field. To eliminate noise in the delicate detection of low frequencies for high ion masses, Russell positions the gun 24 cm from the sample to avoid noise from the gun operation. He also shields the electronics from outside interferences. In such compounds as massive as /?-cyclodextrin, Russell and Castro find that the sensitivity is about 100 times greater than one would expect. Russell suggests that the ionization really occurs via impact of cesium ions on sodium ion-molecule clusters that exist owing to sodium impurities in the samples. This leads to very sensitive detection of such ion types as [M + Na] + and [M + xNa - (x - 1)H]+. Presence of sodium ions also yields fragmentation chemistry that others might find useful to sequence peptides. In the spectrum of hippurylhistidylleucine, for example, the Texas group found no parent ion but only masses corresponding to parent plus sodium or parent minus leucine. Russell suggests that sodium complexes with histidine, leaving leucine in an exposed conformation and leading to preferential loss of leucine. This might find applications in studies on peptides to locate the position of histidine units. • March 18, 1985 C&EN
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