Mass Standards for Chemical Ionization Mass Spe,ctrometry Ismet Dzidic, D. M. Desiderio, M. S. Wilson, P. F. Crain, and James A. McCloskey Institute of Lipid Research and Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77025
wall, and force-fitted into a tubular Vespel (polyamide) insulator which provides insulation between accelerating potential and ground. The reagent gas was passed from the insulator into a coiled 1/16-in.i.d. stainless steel capillary which terminated in a ball joint, spring-loaded against the wall of the ion source block. A final, fixed constriction in the tip of the ball joint provided a pressure drop from approximately atmospheric to 1 mm H g pressure. The reservoir, variable leak, and inlet line were normally heated t o 220 "C. During high pressure operation, the reservoir diffusion pump was closed t o the reservoir by a Bendix (VCS-22A) valve. Perfluorinated hydrocarbons or other volatile samples were introduced into the reservoir with the variable leak open, the entire system under high vacuum, and the accelerating voltage power supply turned off. The reagent gas was introduced after the sample vapor had expanded through the lines to the ion source. Fluorocarbons were introduced both by direct probe and reservoir; tetracosane-d50 (solid) and the perdeuterated synthetic hydrocarbon mixture (viscous liquid) were introduced by probe. Spectra were acquired using methane o r isobutane as the reagent gas under the following conditions: accelerating potential 6 or 8 kV; electron energy 200 eV; repeller field 0-30 volts/cm; ion source temperatures 100-270 " C ; ionization chamber pressure 0.4 mm Hg, measured with a Baratron MKS capacitance manometer; analyzer pressure 4-6 X 10-7 mm Hg. High resolution chemical ionization spectra were recorded at resolution values of 15-20,000, using Ilford Q2 photographic plates developed for 15 min (20 "C) in Kodak Microdol-X.
AN IMPORTANT ELEMENT of mass spectrometry is the use of reference standards for the determination cf massyin particular when high resolving power is employed. With the recent advent of chemical ionization mass spectrometry ( I ) and the ability to produce high resolution chemical ionization mass spectra (2), the need arises for reference compounds which exhibit suitable spectra produced by chemical ionization. The problem of providing a reference mass scale for low resolution work is less severe than with conventional electron ionization methods because chemical ionization spectra frequently show peaks of low intensity (-0.05-0.5% relative intensity) a t essentially every nominal mass, even though the extent of molecular fragmentation is markedly reduced. However, for high resolution work and problems relating t o the determination of molecular structure, the measurement of exact mass requires the availability of internal standard peaks over a broad mass range. We have therefore examined the chemical ionization mass spectra of mixtures of perfluorinated and also perdeuterated hydrocarbons, a t both low and high resolving power, using methane or isobutane as reagent gases. EXPERIMENTAL
Perfluorinated hydrocarbons were purchased from Pierce Chemical Co., Rockford, Ill. (Perfluoroalkane-225, boiling range 225-250"). Tetracosane-djo and a mixture of perdeuterated alkanes ("perdeuterated synthetic hydrocarbon") were obtained from Merck, Sharp and Dohme of Canada, Ltd., Montreal. Mass spectra were determined with a CEC 21-llOB double focusing instrument, modified for high pressure operation based largely on the work of Futrell and Wojcik (2). The principal exception to their design was the method of reagent gas introduction. The reagent gas was passed through a conventional glass reservoir syqtem and variable leak (Varian 951-5100), which was maintained at approximately atmospheric pressure during operation t o avoid arc discharges through the inlet lines. The glass inlet line terminated at a l/,8-inS Swagelok fitting o n the exterior of the ion source housing. A metal capillary was passed through the housing
RESULTS AND DISCUSSION
Since mixtures of long chain fluorocarbons (commonly called perfluorokerosene or PFK) are the most commonly used mass standards in mass spectrometry, their behavior upon chemical ionization was examined. Figure 1 shows the spectrum of a high-boiling fraction of perfluorokerosene obtained from direct probe introduction a t 120 "C, using methane reagent gas. Qualitatively similar spectra resulted from introduction of the marker along with methane through the reservoir, but no spectrum was produced by isobutane reagent gas under similar conditions. With the exception of mle 57, derived from traces of butane in the reagent gas, the major peaks of the spectrum were determined t o have the
( I ) F. H.Field, Accormts C / ~ IRes.. . 1, 42 (1968). (2) J. H. Ftitrell and L. H. Wojcik. Rer. Sci. Imfrum., 42, 244 ( 1 97 I ) . I
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Figure 1. Chemical ionization mass spectrum of Perfluoroalkane-225, obtained using methane reagent gas
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ANALYTICAL CHEMISTRY, VOL. 43, NO. 13, NOVEMBER 1971
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i
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Figure 3. Selected section of a photographically recorded high resolution chemical ionization mass spectrum of N6,NG-dimethyladenosine, with perdeuterated hydrocarbonsas internal mass standards a 0 Q
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same elemental compositions and exact masses as those resulting from electron ionization (3). The principal difference is the substantially greater abundance of high mass ions, t o 881 (C1#35), a region most notably from mje 643 (C14Fg5) where PFK produces ions of very low abundance by electron ionization ( 4 ) . The numerous minor peaks of low intensity which fall between the recurring major fluorocarbon peaks have low fractional mass values-/.e., above 0.96 and below 0.02 amu. Essentially all of these corresponded in mass either to the minor fluorocarbon ions tabulated by Beynon (3), or to hydrogen-containing species, e.g., mje 401.001 1, CllH3F14. We note however, that in either case it is impossible t o distinguish between c8 and HF5, a difference of 0.0002 mass unit. These results indicate that the usual standard mass ions for perfluorokerosene can be used without change for chemical ionization work involving methane, and further suggest that mixtures of perfluorinated alkanes having a broad range of boiling points could be used to generate standard mass fluorocarbon peaks of similar intensity over a very wide mass range. The early work of Field showed that the methane chemical ionization spectra of n-alkanes exhibit a regular, low intensity pattern of C,Hr,+, ions and a very abundant M-H ion ( 4 ) . This led us to believe that perdeuterated alkanes would have useful properties as mass standards for chemical ionization work involving methane. Indeed, the spectrum of tetracosane-& (mol wt 388) shows the expected pattern of ions, the largest in both mass and abundance being mje 386 (M-D). As an extension, we examined the spectrum (Figure 2 ) of a mixture of perdeuterated hydrocarbons containing components up t o Cb0. Since the material was a viscous liquid of somewhat lower volatility than high-boiling fractions of perfluorokerosene, it was introduced by direct probe (simultaneously with sample for high resolution work). At lower probe temperatures such as 120 O C , lower weight components (Cg0)were observed, while at the higher temperature represented in Figure 2 , a range of components up to Cto was ob(3) J. H. Beynon, “Mass Spectrometry and Its Applications to Organic Chemistry.” Elsevier, New York, N. Y., 1960. Appendix 6. (4) F. H. Field. M. S. B. Munson, and D. A. Becker, Adcatr. Clrem. .!?e?., 58, 167 (1966).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 13, NOVEMBER 1 9 7 1
served. The repeating pattern of 16 mass units for C,DZ,+I ions, as well as the abundant high mass peaks available a t higher temperatures, makes this material useful as a general purpose internal standard. Isobutane reagent gas produced a spectrum from the mixture of perdeuterated hydrocarbons which was similar t o that shown in Figure 2, with the exception that peaks corresponding t o C,DZ, or C,D2n-lH2 were of increasing intensity in the lower mass region, and were greater than CnDzn+lbelow C I ~ . The high fractional mass of deuterium (2.01410) results in very high fractional mass values of deuterioalkane ions (upper abscissa, Figure 2), which increase a t the rate of approximately 1.78 millimass units per amu of alkane mass. The large, favorable mass difference between marker and sample ions is illustrated in Figure 3, which shows a small portion of the high resolution spectrum of the nucleoside N6,Nf-dimethyladenosine.The ion of mje 178.1092 (5.3 relative intensity) is an adduct representing the base moiety plus a rearranged hydrogen from ribose and a methyl group from the reagent gas (5). The substantial mass difference between CBHI2NS and CllDZ3, even in this low mass region of the spectrum, assures that for virtually all organic ions there is no danger of overlap with deuterioalkane ions. Also, the lower groups of ions, such as CllHD22, CllD22-CllH2D21 (difference = 0.0015 mass unit), and CllD21, are potentially useful as additional standard mass peaks. (Lists of standard masses for the series CnD2n+l, and for the minor ions C,D,,H and C,D2, - to 4, are available from the authors upon request.) The use of perdeuterioalkanes as reference standards is ~
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( 5 ) M. S. Wilson, I. Dzidic, and J. A. McCloskey, Biocliim. Binplrys. Acta, 240, 623 (1971). _ _ _ _ _ _ _ ~ ~ ~
clearly not required in many instances, and unlabeled hydrocarbons may be equally satisfactory. However, since the mass difference between CnHln+land most organic ions is less than in the case of CnDln+l,resolution requirements will in general be higher. In addition, if CnH2n+lions are produced by the sample under investigation, they cannot be resolved from reference ions of the same composition arising from the hydrocarbon standard. As shown in the upper abscissa in Figure 2, the fractional mass of C,D2,+1ions reaches 1.0 amu a t approximately m/e 560. Therefore the region mje 650-730, which corresponds t o the fractional mass range 0.15 t o 0.30, becomes opaque with respect t o possible overlap between sample and reference peaks for many organic ions. The hydrogen content of the molecule and the resolving power employed then become important considerations, should perdeuterioalkanes be used in that region of the spectrum. ACKNOWLEDGMENT
The authors thank F. E. Montgomery for his assistance in the conversion of the instrument for chemical ionization; Mrs. N. R . Earle far calculation of standard mass values; and Drs. J. H. Futrell, M. S. B. Munson, a n d F . H. Field for their comments and discussions regarding instrument modifications for high pressure work. We thank Dr. Wojcik for a copy of his manuscript prior to publication.
RECEIVED for review June 1, 1971. Accepted July 19, 1971. This work was supported by the Robert A. Welch Foundation _ (4-125), the National Institutes of Health (GM-13901, N I H 69-2161, GM-02055) and computer facilities through NIH grant F R 259. ~
~
Pulse Polarography of Halide Ions in Molten Nitrates William O’Deen and R. A. Osteryoung Department of Chemistry, Colorado Stute Unicersity, Fort Collins, Colo. 80521
DEPOLARIZATION OF MERCURY by halide ions has been studied in both aqueous and fused salt solvents. Kolthoff and Miller found a two-electron reversible formation of mercurous halide if halide concentrations were kept below millimolar level in aqueous solutions ( I ) . They employed conventional dc polarography. Biegler employed both ac and dc polarography on this system and found evidence for an initial oneelectron faradaic step (2). A “halidomercury” product was also postulated as an intermediate occurring in calomel formation by Hills and Ives (.?). Bewick, Fleischmann, and Thirsk have concluded that calomel formation is a crystal growth process most likely involving a one-electron oxidation of mercury ( 4 ) . Similar studies have been carried out in molten nitrates. Swofford and Holifield employed conventional polarography
and found the halide depolarization of mercury t o be an irreversible two-electron process (5). However, by using linear sweep voltammetry, Francini, Martini, and Monfrini found the process t o occur via a one-electron reversible step (6). We have extended these studies in nitrate melts using integral (normal) pulse polarography in an attempt t o clarify these conflicting results (7). In integral (or normal) pulse polarography (I.P.P.), pulses of successively increasing amplitude are applied t o the electrode from a fixed base potential. With a dropping mercury electrode (DME), the fixed initial potential may be maintained for the life of the drop prior t o the time of the pulse application. In the case of a DME, the pulse is applied once during the life of a drop and is synchronized so that each pulse is applied t o a drop of the same area. Current is
( I ) I. M. Kolthoff and C . S . Miller, J. Amer. Clirrn. Sor., 63, 1405 ( 194 I). (2) T. Biegler, J. Elecirouiiol. Cliem., 6 , 357, 365, 373 (1963). (3) G. J. Hills and D. J. G. Ives, J. Clirm. Soc., (Ln/rdo/i)1951, 311. ~. (4) A. Bewick, M. Fleischmann, and H. R. Soc., 58, 2200 (1962).
( 5 ) H. S . Swofford, Jr., and Charles L. Holifield, ANAL. CHEM., 37, 1513 (1965). (6) M. Francini. S. Martini, and C. Monfrini. EIectrochin~.Metal. 2 (3), 325 (1967). (7) E. P. Parry and R . A. Osteryoung, ANAL. CHEM.,37, 1634 (1965) (and references therein).
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