Chemical Mass Markers in Chemical Ionization Mass Spectrometry D. V. Bowen' and F. H. Field The Rockefeller University, New York, N. Y. 10021
This note describes a chemical mass marking procedure which has been developed and is used routinely for analytical chemical ionization (CI) mass spectrometry in our laboratory. The chemical mass markers are used to establish m/e values with confidence and also to improve the accuracy of mass identification that can be achieved by our data processing system. The problem of assigning m/e values to peaks in a mass spectrum is central to mass spectrometry. Several approaches have been used with magnetic deflection mass spectrometers. These include the manual interpretation of oscillographic recordings (I), data systems that employ a Hall probe ( 2 ) , and data systems in which the time at which a peak is detected is converted to an m/e value ( 3 ) . The use of Hall probes will not be considered here. The interpretation of oscillographic recordings and algorithms for converting time to mass have traditionally shared a requirement that the spectrum contain a high density of peaks. Both approaches rely on self-correcting extrapolation, in which it is usual to identify m/e 28 and 32 and then to proceed towards higher mass. This process can adequately cross gaps of 10 to 50 mass units between peaks. However, spectra occur, especially in CI, field ionization (FI), and field desorption (FD) mass spectrometry, with gaps of as much as 100 mass units in which even very low intensity peaks cannot be found. If the mass spectrometer scan function is not reproducible to better than 0.1%, errors in mass assignment can occur in the presence of such large gaps. These errors can be between 1 and 5 mass units and are therefore unacceptable. The addition of compounds with known mass spectra has been suggested as a means of surmounting this problem (1). However, this procedure is not routinely used in low resolution electron ionization (EI) mass spectrometry because the large number of peaks produced by most suitable compounds (e.g., perfluorokerosene, PFK, or perfluorotributyl amine, PFTBA) makes observation and interpretation of the spectrum of the compound of interest very difficult. We have taken advantage of the minimal fragmentation present in CI mass spectra to devise a technique in which chemical mass markers are routinely used for analytical CI mass spectrometry.
Previous experience in this laboratory with the Rockefeller Chemical Physics mass spectrometer had indicated that it was necessary to add reactant gas to the batch inlet system so that the total pressure of sample and reactant gas was as much as ten times the pressure in the source ( 4 ) . By contrast, it has never been necessary to add any extra pressure of reactant gas to the batch inlet system of the DuPont 21-492. We do not understand this difference. Table I1 summarizes typical scans, obtained a t source temperatures near 200 "C, for seven chemical mass markers. Intensities are reported as a percentage of the base peak intensity. The masses reported have been determined by a variety of techniques. These include comparison of the mass marker ions with ions of a reference compound such as PFK, using oscillographic recordings, the data systems, and peak matching. These comparisons were made with E1 spectra, methane CI spectra, and isobutane CI spectra. Additional experiments were performed to determine the masses of ions a t high m/e using the accelerating voltage switching technique of Plattner and Markey ( 5 ) . The E1 spectrum of compound IV has been reported (6).
RESULTS AND DISCUSSION The CI spectra of seven mass markers, obtained with isobutane and methane as reactant gases, are reported in Table 11. The isobutane and methane CI spectra are similar. All of the s-triazines (compounds I-IV) give (M 1)+ as the base peak. The fragment ions produced, although 1)+ions, can still be useful mass weaker than the (M markers. These fragment ions include ions a t M 1 - 20, corresponding to protonation and loss of HF, and ions at M 1 - 18 or M + 1- 38 (loss of 18 from M 1 - 20), which may result from impurities caused by incomplete fluorination. These ions may also result from rearrangement of the collision complex, as shown in Reaction 1:
+
+
+
+
+
+
[R-F-C~HS]
-+
[R-H.H]+
+ C4H7F
Similar ions have been observed with lanthanide complexes (7). The other ions observed, for example m/e 190 (C4H6N2) in compound I and mle 164 (CsH6N) in compound 11, are probably fragmentation products. We do not report the complete spectra of the two high molecular weight triazines (compounds I11 and IV) because we have used these only for mass identification above mle 1000. Of the compounds reported, perfluorotributylamine (PFTBA)
EXPERIMENTAL The compounds used (Table I) are all available from PCR, Inc., Gainesville, Fla. The spectra reported were obtained with a DuPont 21-492 double focusing mass spectrometer modified for chemical ionization in our own laboratories under license from Scientific Research Instrument Corp., Baltimore, Md. The mass spectra were recorded on oscillograph paper, or through an AEI DS-30 data system to which our own programs for chemical ionization mass identification had been added, or via a VG-2040 data system. In a typical experiment, between 0.25 and 2 ~1 each of one or more mass markers are injected into a batch inlet system. From this heated volume, commonly a t a temperature of 200 "C, the mass marker passes through a gold foil leak to the source of the mass spectrometer. The mass marker, reactant gas, and sample are present simultaneously in the source, and the spectra obtained are the combined CI spectra of the sample and the mass marker. Author to whom correspondence should be addressed.
Table I. Compounds Used for D a t a in Table II R R I. R = CF, 11. R = C,F, - " 111. R = C;F,, IV. R = CgFIg
c I
I R
Mol wt Mol w t Mol w t Mol ~t ~
= 285 = 435 = 1185 = 1485
0
II
V. C-F,i-C-O-CH, Mol w t = 428 VI. (HCF,CF2CF2CF2CH20),P0 Mol a t = 740 Mol w t = 671 VII. (C,F,),S
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
2289
Table 11. CI Spectra of Some Chemical Mass Markers Reactant, rel. int.
mle
i-C4H10
Reactant, rel. int. i-C4Hl0
mle
CH4
I, mol wt 285
287 286 285 267 266 190 121
8.6 100
7.4 100 2.8 1.5 22.6 2.5 2.5
7.5 16.9
430 429 409 407 381 331 314 300
11, m o l wt 435
437 436 43 5 418 416 366 164 119 76 69
6.6 100
19.2 27.1
5.5 100 1.9 10.1
9.4 100 2.7 1.5 2.7 1.9 3 .O
VI, mol w t 740
6.8 100 0.7 1.7 4.9 2.3 4.3 2.2 2.8 3.4
14 .O 100
742 74 1 722 721 642 64 1 539 527 509 443
111, mol wt 1185
1188 1187 1186 1168 1167 1166 1150 1149 1148 1130 1110 866
CH4
V, mol w t 428
18.5 100 4.4 23.5 2.5 16.7 8.2
2.8 2 1 .o 6.1 1.7
13.2 16.1
VII, mol w t 671
5.3 37.6 100 3 .O 5.1 12.8 3.8 7.5 24.1 7.7 8.4
653 652 6 15 614 596 576 558 538 502 464 434 414 396 376 264 220 219 135 131 119 107 106 105 100 69
100
9.4
8.7
21.0
a IV, m o l w t 1485
1.9 15.7 4.9 37.0 2.6 18.0 1.3 5.7 3.7 1.5 2.8 100 2 .o 3 .O 1.9 2.1 51.9 1.4 4.4 1.2 17.0 1.4 1.7 1.6 12.4
1488 7.5 7.1 43.4 36.1 1487 1486 100 100 1469 5.7 12.5 1468 17.0 1467 6.3 1466 12.5 18.2 15.8 1450 16.4 1449 8.2 1448 26.4 10.8 1432 11.9 1431 8.8 1430 14.5 6.7 b a Spectra were not examined below m / e 1000 for i-C4H10 nor below m / e 850 for CHI. Spectra of IV were not examined below m / e 1400.
is the only one for which (M + 1)+is not the base peak. In our hands, PFTBA has given weak and indeterminant mass spectra with isobutane reactant gas. Presumably this is a result of the low proton affinity of PFTBA. With methane as reactant gas, PFTBA gives no (M 1)+ and gives a large number of fragment ions. It is, however, still useful as a mass marker under some circumstances. Long chain perfluoro- or perdeuteroalkanes have been used as mass standards for high resolution methane CI (8). The typical scans reported were obtained with the source about 200 "C. Spectra obtained a t lower source tempera-
+
2290
tures often have lower intensities for the fragment ions and may show additive ions corresponding to addition of a reactant gas ion to the mass marker. Typical masses for additive ions are M 39, M 41,M 43,and M 57 for isobutane or M 17, M 29 and M 41 for methane. These additive ions generally have relative intensity less than 1% of the base peak. As the source temperature is raised, the relative intensity of fragment ions increases for these mass markers. In spite of this, the mass markers retain their utility a t source temperatures a t least as high as 350 O C since the total number of ions does not increase significantly and
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13. NOVEMBER 1975
+
+
+
+
+ +
+
+
the base peak remains the (M 1)+ion. As mentioned above, compound VI1 is an exception to this. Although variations in the spectra of the mass markers with reactant gas or temperature are small, we believe that it is good practice to obtain a spectrum with the mass marker alone at a temperature near that which will be used for obtaining spectra of the unknown compound. Such background spectra, which include ions from the reactant gas, the mass marker, and any contamination of the mass spectrometer, can be important in correctly interpreting the spectrum of an unknown compound. The utility of these background spectra is perhaps most obvious in the case of GC-MS experiments where it may be necessary to obtain spectra characteristic of GC column bleed for subtraction from spectra of the unknown sample. The subtraction of background spectra is just as important with probe distillations or with samples admitted via a batch inlet system. All of the compounds used routinely as mass markers are perfluorinated and are therefore mass deficient. The difference in mass between the mass marker ion and a sample ion of the same nominal mass may be as great as 0.1 to 0.5 mass unit. As a result, mass marker ions can be resolved from sample ions of the same nominal mass a t resolving powers as low as 1000. The mass deficiency of the mass marker ions allows them to be used even when ions in the spectrum of the unknown have the same nominal mass as ions in the mass marker, and also facilitates attempts to identify the mass marker ions in an unknown spectrum which has not been correctly mass identified. The compounds used most routinely are I, V, and VI for experiments below rnle 1000 in which the data system is used to identify masses, and I11 and IV for experiments above mle 1000 in which oscillographic recordings or peak matching are used. The simplest and most routine use made of these chemical mass markers is to improve the confidence of all CI mass assignments. In this use, the masses in a spectrum are assigned either by the data system or by counting on oscillographic recordings, and the positions of the chemical mass markers are noted. Then, provided all ions of the unknown compound fall between two ions from chemical mass markers, the masses assigned to the unknown compound can be verified and confidently accepted. An extension of this technique was made with the development of an algorithm for identifying masses in chemical ionization mass spectra using the data system. A new algorithm was developed because the existing data system relied on identification of mle 28 and 32 followed by a selfcorrecting extrapolation towards higher mle values. Because of the very small number of ions present in many CI spectra, a nonextrapolatory algorithm was found to be necessary. The scan function of our mass spectrometer is approximately of the form: In m = A - B t
(1)
the plot of In m vs. t exhibits some curvature when t and m cover a wide range. The actual scan function is reproduced to an acceptable degree by applying Equation 1 to a set of segments of equal time; typically 5-10 segments are used. Thus we have a set of functions In m = Ai
- Bit
(2)
for ti-1 < t < t i where Ai and Bi are the intercept and slope of the scan function for the i t h segment. The values of the slopes, Bi, are obtained from a calibration spectrum which contains a large number of peaks of known mle values and known times.
The values of Ai are determined with an algorithm that fits Equation 2 to a given experimental scan in a manner that takes into account time variations between scans. Shifts of times a t all masses occur between different scans. These shifts arise from the inability in practical operation to start each scan under exactly the same conditions. The effects of these shifts are compensated by a one-point timemass identification. The proper mle value is assigned by the operator to one time in the experimental scan, and using this mle value, this time, and the known values of Bi, the proper Ai values for the scan are calculated from Equation 2. It has been found experimentally that if the single point used to correct the calibration table for the unknown spectrum is at high mle, the errors in the final mass identification are lower than if the single point entered is a t low mle. In identifying a typical experimental scan, with the fixed point a t rnle 57, an error of 2 mass units was observed a t mle 429. By contrast, if the fixed point was a t mle 429, all masses between rnle 500 and rnle 57 were identified to within 0.2 mass unit. This experimental finding can be explained by the following calculation. Consider the quantity dmldt as derived from the scan function of Equation 1: =
eA-Bt
dmldt = -BeA/eBt
(3) (4)
From Equation 4, one sees that the absolute value of dmldt becomes smaller as t becomes larger, i.e., as m becomes smaller. For example, in a typical experiment, the time interval between rnle 429 and mle 430 was 780 units. The time interval between mle 57 and mle 58 was 5919 units. Therefore any time shift or error in the time shift compensation will have a greater relative effect a t high mass than a t low mass. The masses calculated from Equation 2 will always be somewhat in error, and the magnitude of the error may be expected to increase with the distance between the calculated mass and the one-point time-mass identification. Since the intrinsic value of dmldt is larger a t higher mle values, it is better to have the time-mass identification point a t higher mass. This causes the largest errors in time to occur a t low mle values where they will have the least effect on the calculated mass. For these reasons, it is now routine in our laboratory to use a high mle ion from one of the chemical mass markers as the time-mass identification point for unknown spectra. This nonextrapolatory technique has made it possible to use the mle 741 ion of compound VI with a slope-calibration scan as much as one month old to identify masses between mle 850 and mle 50 with errors no more than 0.3 mass unit. Computer programs suitable for use with the A.E.I. DS-30 are available from the authors. This capability is a standard part of the VG 2040 software. In summary, chemical mass markers can be used to improve the confidence of mass assignments in spectra which are not dense in ions, particularly CI spectra. Similar considerations will probably apply to FI and FD spectra.
ACKNOWLEDGMENT It is a pleasure to acknowledge the skilled technical assistance of Jeffrey Shabanowitz and Gemma Vella. LITERATURE CITED ( 1 ) F. W. McLafferty, "Interpretation of Mass Spectra", 2nd ed., W. A. Benjamin, Inc., Reading, Mass. 1973, pp 12-14.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
2291
(2) “Biochemical Applications of Mass Spectrometry”, G. R. Waller, Ed., Wiley-Interscience, New York, 1972, pp 51-132. (3) R. A. Hites and K. Biernann, Anal. Chem., 39,965-970 (1967). (4) M. S. B. Munson and F. H. Field, J. Am. Chem. SOC., 88, 2821-2630 (1966). (5) J. R. Planner and S. P. Markey, org. Mass. Spectrom, 5, 463-471 (1971). (6)1.Aczei, Anal. Chem., 40, 1917-1918 (1968). (7) 1.H. Risby, P. C. Jurs, F. W. Larnpe, and A. L. Yergey, Anal. Chem., 46, 726-728 (1974).
(8) I. Dzidic, D. M. Desiderio, M. S.Wilson, P. F. Crain, and J. A. McCioskey, Anal. Chem., 43, 1877-1879 (1971).
RECEIVEDfor review April 28, 1975. Accepted August 11, 1975. This work was supported in part by Grant No. RR o08G2 from the National Institutes Of Division of Research Resources.
Deviation from Beer’s Law Caused by Change in Bathochromic Shift of Absorption Maximum F. W. E. Strelow’ and C. H. S. W. Weinert National Chemical Research Laboratory, P.O. Box 395, Pretoria 000 7, Republic of South Africa
Metallochromic indicators such as Xylenol Orange, Catechol Violet, or Chromeazurol S exhibit a bathochromic shift often accompanied by a considerable increase in sensitivity when used together with micelle-forming reagents such as cetylpyridinium bromide and cetyltrimethylammonium bromide or chloride for the determination of metals (2-3). Attempting to determine beryllium by the method described by Nishida ( 4 ) using Chromeazurol S as color reagent, but substituting benzyldimethylhexadecylammonium chloride for zephiramine (benzyldimethyltetradecylammonium chloride), because the first was available as a chemically pure reagent, we observed a reproducible change in the slope of the standard working curve a t 610 nm. In an attempt to discover the origin of this change, we prepared absorption spectra of the color complex using varying beryllium concentrations. The results of this investigation showed that the bathochromic shift caused by the presence of benzyldimethylhexadecylammonium chloride varies with beryllium concentration.
within 6.65 f 0.05. The solutions then were transferred into volumetric flasks of 50-ml volume and made up to volume with deionized water. After about 2 hours, the absorbance of the solutions was measured vs. a reagent blank. The results are presented in Figure 1. Figure 2 shows two standard working curves, one a t 620 nm, which was the wavelength of maximum absorbance of the standard with the highest beryllium concentration, the other one at 611 nm, the wavelength which on inspection of Figure 1seemed to give the best approximation to a straight line.
DISCUSSION Figure 1 shows a very definite change in the wavelength of the position of the absorbance maximum with decreasing POSITION OF M A X I M U M
EXPERIMENTAL Reagents. Beryllium standard: A solution containing 1gg of beryllium per ml was always freshly prepared by appropriate dilution of a more concentrated stock solution which had been obtained by dissolving beryllium nitrate (Merck extra pure) in deionized water containing 0.1M hydrochloric acid and standardizing gravimetrically as beryllium cupferate. Other reagents used are as follows: Chromeazurol S: A 0.06% solution of the reagent (Merck) in deionized water. Benzyldimethylhexadecylammonium chloride (BDHA): A lOmM solution of the reagent (Fluka purum) in deionized water. Hexamethylene tetramine (HMT): A 1.OM solution of the reagent in deionized water. Ca-EDTA: A solution 50mM in Ca-EDTA (Merck, Calcium-Titriplex 111, rein) and lOmM in CaC12 (Merck “pro analysi”) in deionized water. All common reagents were of analytical reagent grade purity. Dilutions were carried out with distilled water further purified by passing through an Elgastat deionizer. Glassware was cleaned by boiling in 1:l hydrochloric acid and rinsing with deionized water. Apparatus. A Metrohm E300 pH meter calibrated at pH 7.00 with a guaranteed buffer was used for pH measurements. The calibration was checked after each series of measurements. A Zeiss PMQII spectrophotometer connected to a stabilized power supply was used for spectrophotometric measurements. Absorption Spectra for Various Amounts of Beryllium. Amounts of the beryllium standard solution containing from 0.5 to 5.0 pg of beryllium were measured out accurately and added to 5 ml 1 M ammonium chloride solution in deionized water. Five ml of the Ca-EDTA solution were added followed by 5 ml of the BDHA and 5 ml of the chromeazurol S solution. The solutions were mixed thoroughly after each addition. Finally 5 ml of HMT solution were added and the pH was measured with a pH meter. It always was Author to whom correspondence should be addressed. 2292
I/
1
600
1
I
-
610 620 X,nm
I
I
630
Figure 1. Absorption spectra for various amounts of beryllium with Chromeazurol S (0.1 mM), BDHA (1.0 mM), hexamethylene tetramine (0.1 M), and ammonium chloride (0.1 M) at pH 6.65 & 0.05
ANALYTICAL CHEMISTRY, VOL. 47, NO. 13, NOVEMBER 1975
