Anal. Chem. 1988, 6 0 , 1084-1086
1084
Guggenheim ( 4 ) may be equal to or better than the result based on eq 2-5.
CONCLUSIONS The use of the algorithm described in ref 1 should not be advocated, since it does not converge to the global minimum as defined by the least-squares criterion. If for any reason finding the global minimum is thought to be superfluous, noniterative approaches, e.g. the method of Guggenheim ( 4 ) , will give results of comparable quality in still much less computing time. If the global minimum is wanted, and we surely support this approach, the elimination of linear parameters and the use of analytical instead of numerical derivatives is indeed the method of choice. An algorithm based on the correct normal equations (eq 2 , 3, and 6 in this comment) and otherwise following the ideas presented in ref 1 will find the global minimum in a one-dimensional parameter space. Elimination of linear parameters (5-7) and analytical derivatives ( 4 9 )have been suggested before, however, and, contrary to an explicit statement in ref 1, have been used in combination (8, 9). An algorithm based on the correct normal equations combined with the ideas presented in ref 1 then is rather close to the approach described in ref 9. As the main remaining difference the implicit changes of the best estimates for a and b as a function of k, i.e. the partial derivatives aalak and abldk are included in ref 9, but not in ref 1. As a consequence, the time needed per iteration is reduced, but the number of it-
erations is increased. More important, a much better initial estimate for k is needed using eq 2, 3, and 6 rather than following the ideas described in ref 5-9 where any estimate between lo* and 10 gave unproblematic convergence.
LITERATURE CITED Jericevic, Z.; Benson, D. M.; Bryan, J.: Smith, L. C. Anal. Chem. 1987, 5 9 , 658-662. Wentworth. W. E. J. Chem. Educ. 1965, 42, 96-103. Abramowltz, M.; Stegun, I. A. Haandbook of Mathematical Functions; NBS Appl. Math. Ser. 55; Natlonal Bureau of Standards: Washington, DC, 1964; p 952. Guggenhelm, E. A. Philos. Mag. 1926, 2, 538. Richards, F. S. G. J . R . Stat. Soc. 1961, 23, 469-475. Lawton, W. H.; Sylvestre, E. A. Technometrics 1871, 75, 461-467. Gampp, H.; Maeder, M.; Zuberbuhler, A. D. Talanta 1980, 2 7 , 1037-1045. Golub, G. H.; Pereyra, V. SIAM J. Numer. Anal. 1973. 10, 413-432. Gampp, H.; Maeder, M.; Meyer, C.: Zuberbuhler, A. D. Talanta 1985, 3 2 , 95-101.
Harald Gampp Marcel Maeder* Andreas D. Zuberbuhler* Institute of Inorganic Chemistry University of Basel CH-4056 Basel, Switzerland
RECEIVED for review April 14, 1987.
Accepted December 9, 1987. We thank one of the referees for the suggestion to specifically include 7-9 into this work. The work was supported by the Swiss National Science Foundation, Grant No. 2.851-0.85.
TECHNICAL NOTES Fluorlnation of Sulfur Tetrafluoride, Pentafluorosulfur Chloride, and Disulfur Decafluoride to Sulfur Hexafluoride for Mass Spectrometric Isotope Ratio Analysis Swroop K. Bains-Sahota and Mark H. Thiemens* Department of Chemistry, B-017, University of California-Sun Measurements of isotopic ratio variations have been employed to study a wide range of processes, e.g. paleoclimatology, rock formation temperatures, and quantum chemical mechanisms. Hulston and Thode ( I ) were the first to show how stable isotopic variations in meteoritic material could be used to distinguish nuclear and chemical processes. Since chemical processes were thought to obey mass-dependent fractionations, any departure from this fractionation law would then reflect a nuclear process such as nucleosynthesis, radiogenic decay, or spallation. A mass-dependent isotope effect between species of mass M and (M + 2 ) is approximately twice as large as the effect between species of mass M and (M + 1). Therefore, in order to distinguish between mass-dependent and mass-independent processes, at least three isotopes of the same element must be simultaneously measured. Clayton et al. ( 2 )observed a non-mass-dependent oxygen isotope distribution in the high-temperature minerals within the carbonaceous chondrite Allende. Since chemical processes were assumed to produce mass-dependent fractionations, the observed effect was attributed to a nuclear process. Since that time, mass-independent isotopic fractionations have been observed in chemical processes ( 3 ) . For oxygen a chemically produced mass-independent effect has been observed which
Diego, La Jolla, California 92093 produces an isotopic fractionation identical with that observed in Allende. The mass-independent effect has been suggested as involving isotopic symmetry effects in the production of ozone ( 4 ) . A mass-independent sulfur isotope effect has also been reported in the formation of S2FIo(5). The study of mass-independent fractionations involving symmetry and quantum effects is of considerable importance and necessitates multiisotopic measurements. Employment of multiisotopic measurements allows the detection of physical-chemical processes which might not be observed by other spectroscopic techniques, such as those reported for oxygen and sulfur (3-5). Development of the analytical capability to simultaneously measure 3aS/32S,33S/32S,at high precision, may also eventually permit measurement of these ratios in atmospheric samples such as sulfate and H,S. These gas-phase reactions may produce mass-independent isotopic fractionations which might ultimately be of interest in identifying specific atmospheric transformations. At present, only 34S/32S ratios have been measured in atmospheric sulfur species. Isotopic analyses of sulfur minerals and native sulfur were first performed by conversion to sulfur dioxide for mass spectrometric analysis. As discussed by Puchett et al. (6), sulfur hexafluoride is a superior gas for mass spectrometric
0003-2700/68/0360-1084$01.50/0 0 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 10. MAY 15, 1988
Flgue 1. Apparatus for fluwination of sulfur compounds wilh bromine pentafiuoride.
analysis. It is chemically inert, is insensitive to moisture, and does not adsorb on the walls of glass or metal vacuum systems. Fluorine has only one isotope, and no corrections are needed for isobaric interferences. SF, produces a simple mass spectrum on electron impact, and the major ion SF5+occurs in a mass region free of instrumental background. Hulston and Thode ( I ) employed SF, for 34/32 and 33/32 isotopic measurements by conversion of sulfur species with fluorine gas. T h e fluorination products were further purified by gas chromatographic separations. Due to the difficulty in handling F,, Puchett e t al. (6)used BrF, as the fluorinating agent. The fluorination products were scrubbed on a KOH trap and SF, was separated from other products on a 5-A molecular sieve column. Although BrF, is less dangerous than F, or BrF,, it is inconvenient to dispense into the nickel reaction tubes (6). Thode and Rees (7)extracted sulfur from sulfides by reaction with BrF,. The product SF, was subsequently cryogenically separated from unreacted BrF, and other products. The final cleanup was then achieved gas chromatographically with a molecular sieve (5 A) column. To obtain ultrapure samples, separation on a gas chromatographic column is essential. A column capable of separating SF, from other fluorinated compounds (SiF,, CF, and SF,) is required, since separation between these compounds on molecular sieve 5 A, as reported in previous work, is inadequate (8). In this paper, we report a procedure for the fluorination of gaseous sulfur compounds, which have previously not been studied, to SF, for sulfur isotopic measurement of both %/"S and %3/"S ratios. Since the fluorination process is phase and molecule specific ( 1 , 6 , 3, such results are important if different species are to he studied.
EXPERIMENTAL SECTION The apparatus for fluorination and purification is similar to that described by Clayton and Mayeda (9)and Puchett et al. (6). Fluorination. Figure 1is a diagram of the fluorination line used in the reported experiments. BrF, is the fluorinating agent (SCM Chemicals) since it can conveniently he used in a metal vacuum system (vapor pressure a t 298 K = 390 mm). The procedure is as follows: BrF5is aliquoted (301 ratio of BrF5-sample) into the expansion volume V (=lin. pressure on the Helicoid gauge) from the reservoir of BrF, in the Kel-F tube, and condensed into an all-nickel reaction tube a t 77 K for -20 min. Small amounts of residual noncondensahles are removed, and the sample to he fluorinated is condensed, along with BrF,. The reaction tube valve is closed and the reaction tube suhsequently heated to 400 "C for -15 h. After reaction, the nickel tuhe is chilled to 77 K far -20 min and the nancondensables are again removed. The fluorination products are transferred through the removable KOH trap (K) to the stainless steel U-trap which is maintained at liquid nitrogen temperature for approximately 1 h. The trap containing KOH pellets serves to adsorb HF, Br,, and other corrosive byproducts, resulting in a preliminary cleanup. The KOH pellets are replaced after -10 transfers to prevent clogging, presumably as a result of formation of solid phase KBr. The
1085
Flgure 2. Schematic of gas chromatographic injection and peak collection system.
U-trap is then isolated from the KOH trap and the sample condensed at I7 K into an evacuated sample tube (S,) as shown in Figure 1. Finally, the sample tube is transferred to the gas chromatograph for final purification. Gas Chromatography. The injection port of the gas chromatograph (HP5890A) was modified by incorporating a Valco six-port external volume gas sampling valve (Model 9105) between the gas chromatograph and the vacuum line. The detector vent was connected to a three-way valve which allowed collection of the eluted fractions. The gas chromatographic injection and collection system is shown schematically in Figure 2. A temperature program is used to obtain the desired separation between peaks. The gas chromatograph was equipped with a thermal conductivity detector maintained at 150 "C. A helium carrier gas (flow rate 30 mL/min) was used through a 12 ft, 'Ia in. o.d., Porpak QS (8W100mesh) column. The column temperature was initially held a t 27 O C for 5 min and then heated at a rate of 30 "C/min to a final temperature of a 100 "C. The total separation time is less than 30 min. The sample to he separated is cryogenically transferred to the sample loop of the sampling valve by condensing a t 77 K, isolating the loop with the Nupro valve (N),and suhsequently warming the loop to room temperature. The sample is injected by diverting the flow of the He carrier gas through the loop and to the column. When the eluted SF, is detected on the integrator (HP3390A), the output flow of He + SF, is diverted through the glass spiral traps (S) at 77 K, to collect the SF, After the SF, passes, the He flow is again diverted so that the other components are vented as waste. With the spiral traps (S) at liquid nitrogen temperature, the He is slowly evacuated. The traps are then allowed to come to room temperature and the SF, suhsequently transferred to a sample tube for mass spectrometric analysis. Sample sizes in the range 0.1-30 pmol exhibited good separation. Larger sized samples overloaded the column and the separation between peaks was poor. The gas chromatographic separations should he reproducible and minimize fractionation. The possible extent of isotopic fractionation on the gas Chromatographic separation system was determined hy control experiments. A sample of pure, isotopically predetermined SF, was injected onto the column and collected as described. The collected sample was analyzed mass spectrometrically and its isotopic composition compared to the starting SF,. Samples of various sizes were used to determine the effect of size on fractionation in the chromatographicseparation process. Mass Spectrometer. Isotopic analyses were performed on a Finnigan MAT 251 mass spectrometer which employs a triple ion beam collectors for simultaneous measurement of m / e 127, 128, and 129 for "SF5+, %F,+, and "SF,+, respectively. Isotopic ratios are reported in the conventional delta notation where
Similarly, P S ( % o ) expresses the 3sS/32Sisotope ratio (in per mil) with respect to a standard. The isotopic uncertainty in the measurements (6% ^I 633S^I 0.03%) was determined by repeat analysis of standards. Various sized samples were analyzed for
Anal. Chern. 1988, 60, 1086-1088
1086
Table I. Results of Control Experiments To Determine Extent of Fractionation of SF, on the Gas Chromatographic Column expt no. 1 2 3 4
sample size, pmol
633S.%O
634S,k
~[34s(0.505) - 33S),%O
6
-0.22
4.41
0.01
8
-0.17
0.04
10 19
-0.16
-0.25 -0.37 -0.26
-0.18
0.02 0.05
Table 11. Isotopic Results of Fluorination of Various Sulfur Species
molecule
sample size, pmol
634S,"%o
633S/634S
10.01 9.82 10.32 10.50
0.514
SF, SF4 SF, SF4
20 10
5.15 5.08 5.26 5.39
S2Fl"
25 30
10.30 10.52
20.07
S2FIO
20.51
0.513 0.513
SFSCI SF6CI
25 30
8.21 8.47
16.03 16.48
0.512 0.514
a
15
633S;%o
5
Against standard SFRdefined as 634S= 6%
0.518
0.510 0.513
= 0.00%0).
reproducibility of the mass spectrometer. Fractionated SFs samples were prepared, ranging from +5%0 to +40%0. These fractionated samples then define the isotopic mass fractionation line.
lection processes is negligible, and the column is the major source of sample fractionation. Although the gas chromatographic separations can introduce scatter of up to f 0 . 4 k , in 6%, the separation is essential for obtaining pure samples for mass spectrometric analysis. The gas chromatogram is useful in that it provides an indication of other possible sulfur species which may be present due to incompleteness of the fluorination process. Isotopic composition data for the fluorination of SF4,SF5C1, and S2Fl0are shown in Table 11. The sample sizes and 633S/6"4Sratios are included. The 633S/634Sratio for SF4, SF5C1,and S2FIofalls within the range 6% = (0.505 f 0.01)634S as predicted. The average value of the sulfur isotopic composition for SF4 is 634S= 10.2 f 0 . 2 7 ~ S2FIo ~ 634S= 20.3 f 0.2%, SF5Cl 634S= 16.2 = 0 . 2 % ~The reproducibility of the entire fluorination and separation, therefore, is f0.2%0 for 634S,similar to that obtained by Puchett et al. (6) for meteoritic mineral fluorination and purification. The chromatographic procedure reported in this paper differs from ref 6 in the use of Porpak QS as the column packing material which is required for these particular separations involving different S,-F, molecules from SF6and to permit determination of the fluorination yields. A molecular sieve column would be more suitable for fluorination and quantitative separation of sulfur-bearing minerals. The technique described here is preferred for fluorination of gaseous sulfurfluorine molecules and their subsequent purification and mass spectrometric analysis. Registry No. SF,, 1783-60-0; SF5C1, 13780-57-9; S2Flo, 5114-22-1;BrF5, 7789-30-2;33S,14257-58-0;34S,13965-97-4;32S, 13981-57-2.
-
RESULTS AND DISCUSSION The data for the control gas chromatographic fractionation experiments are given in Table I. There is a slight isotopic fractionation which occurs on the chromatograph and is consistent with the relationship 6?3 = 0.505(6%), as derived for SF, by Hulston and Thode (1). This fractionation ratio for 633S/634S depends on the specific molecule and its mass. The ratio derives from the dependency of the molecular partition function ratios upon the normal vibrational frequencies which are a function (in part) of the molecular mass (10). For both diatomic and polyatomic sulfur molecules, the relationship 633S= (0.505 f 0.01)634S holds, provided the extent of fractionation is not large (1). The magnitude of fractionation, due to the entire transfer and collection process on the fluorination line and gas chromatographic separation, was determined by a control experiment. The control SF6 sample yielded 633S= -0.2017~ and 634S= -0.305%0,which is similar to the fractionation on the column. This implies that the isotopic fractionation due to the transfer and col-
LITERATURE CITED Hulston, J. R.; Thode, H. G. J . Geoohvs. Res. 1965, 70, 3475-3484. Clayton, R. N.; Grossman, L.; Mayeda. T. K. Science (Washington, D . C . ) 1973, 182, 485-468.
Thiemens, M. H.; HeMenreich, J. E. Science 1983, 219, 1073-1075. Heidenreich, J. E.; Thiemens, M. H. J . Chem. Phys. 1985, 8 4 , 2 129-2-136. -~ ~
.
Bains-Sahota, S.K.; Thiemens, M. H. Meteoritics 1987, 22(4), 7. Puchett, H.; Sabels, B. R.; Hoering, T. Geochim. Cosmocbim. Acta 1973. 35. 625-628. Thode, H.'G.;Rees, C. E. Lunar Planet. Sci. 1979, 10, 1629-1636. Janssen, F. J. J. G. K e r n Sci. Tech. Rep. 1984, 2(2), 9-18. Clayton, R. N.; Mayeda, T. K. Geochim. Cosmocbim. Acta 1983, 2 7 , 43-52. Matsuhisa, Y.; Goldsmlth, J. R.; Clayton, R. N. Geochim. Cosmocbim. Acta 1978, 4 2 , 173-162.
RECEIVED for review September 25,1987. Accepted January 14,1988. NASA (Swroop Bains-GSRP#23376) and the donors of the Petroleum Research Fund, administered by the American Chemical Society, (18189-AC2,5) are thanked for financial support.
Plasma Desorption Mass Spectrometry of Peptides Adsorbed on Nitrocellulose from a Glutathione Matrix Ian Jardine,* Gale F. Scanlan, and Anthony Tsarbopoulos Department of Pharmacology, Mayo Clinic, Rochester, Minnesota 55905
Daniel J. Liberato Department of Drug Metabolism, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110 Two significant advances in peptide sample preparation for analysis by plasma desorption time-of-flight mass spectrometry (PDMS) (2-3) were recently reported (4, 5).
In the first advance, nitrocellulose films were used as sample stage backings to adsorb peptides and proteins for subsequent PDMS analysis. This method conveniently allows the ap-
0003-2700/88/0360-1086501.50/00 1988 American Chemical Society
