Anal. Chem. 1980, 52. 1527-1529
1527
Absorption mode spectra can be obtained by convolutionbased phase correction as suggested by Marshall (11) and experimentally demonstrated in our laboratory (12). We believe the unique relationship between resolution and signal-to-noise in FT/MS will prove important in analytical applications of the method, particularly in gas chromatographic FT/MS experiments under way in our laboratory.
LITERATURE CITED
Figure 2. Simultaneous increase in resolution and signal-tenoise ratio with decreasing pressure for benzene at m l z 78. As benzene pressure was varied, electron beam emission current was adjusted to keep the number of ions in the cell constant for each spectrum. Spectra are
absorption mode, rather than magnitude mode displays (7). is half maximal. Therefore, the resolution increases linearly with magnetic field strength and varies inversely with mass ( 4 , 5 ) . Furthermore, t h e resolution increases in direct proportion to T, the time constant for decay. As the value of this parameter in increased, say by lowering t h e pressure in the analyzer cell, signal to noise ratio (Equation 5) and resolution (Equation 9) increase together if other parameters are held constant. Alternatively stated, the area under the peak remains constant as 7 is changed a n d other parameters are unchanged. T h e essential trade-off is resolution vs. mass range rather than resolution vs. signal-to-noise, as in other forms of mass spectrometry. T h e mass range trade-off is inherent in mixer-mode operation of the F T / M S , which has been explained elsewhere ( 3 ) . It should be noted that frequency swept FTICR absorption mode specta, as in our experimental examples, are in general most suitable for high resolution measurements.
(1) Comlsarow, M. B.; Marshall, A. G. J Chem. Phys. 1975, 62, 293. (2) Marshall, A. G.; Comlsarow, M. 8. Anal. Chem. 1975, 4 7 , 491A. (3) Ledford. E. B., Jr.; Ghaderl, S.;White, R. L.; Wllklns, C. L.; Gross, M. L. Anal. Chem. 1980, 52, 463. (4) Marshall, A. G. Anal. Chem. 1979, 51, 1710. (5) Comlsarow, M. 8.; Marshall, A. G. J Chem. Phys. 1976, 6 4 , 110. (6) Comlsarow, M. B. In "Transform Techniques In Chemistry", Grlffiths, P. R., Ed.; Plenum: New York. 1978; Chapter 10. (7) Marshall, A. G.; Comisarow, M. B.; Parisod, G. J . Chem. Phys. 1979, 7 1 , 4434. (8) McIver, R. T., Jr.; Ledford, E. B., Jr.; Hunter, R. L. J. Chem. Phys. 1979,
In press.
(9) Brlgham, E. Oran "The Fast Fourier Transform"; Prentice-Hall: Englewood Cliffs, N.J., 1974. (10) Comlsarow, M. B. J . Chem. Phys. 1978, 49, 4097. (11) Marshall, A. G. Chem. Phys. Lett. 1979, 63, 575. (12) Ledfwd, E. B., Jr.; WhRe, R. L.; Ghaderl, S.; Gross, M. L.; Wilklns, C. L. Anal. Chem., 1980, 52, 1090.
Robert L. White Edward B. Ledford, Jr. Sahba Ghaderi Charles L. Wilkins* Michael L. Gross* Department of Chemistry University of Nebraska-Lincoln Lincoln, Nebraska 68588
RECEIVED for review January 28,1980. Accepted May 2,1980. Support of this research by the Nat.iona1 Science Foundation (Grant No. CHE-77-03964) and a grant from the Gulf Research Foundation is gratefully acknowledged.
Breakdown of Methylmercury in Sodium Hydroxide Solution Sir: We have recently cited the advantages of alkaline digestion as a method for preparing biological samples for methylmercury extraction and subsequent analysis by gas chromatography ( I , 2 ) . Alkaline digestion, using 45% (w/v) sodium hydroxide, was initially developed for hair by Giovanoli-Jakubczak e t al. ( 3 ) . A rapid breakdown of methylmercury in the absence of hair (about 43%), resulting in a n increase of inorganic mercury, was observed during the digestion procedure. T o prevent this breakdown in hair, dilute (1'3'0) cysteine solution was added during digestion to complex the methylmercury liberated from the sample matrix. However, since cysteine is slowly oxidized in strong alkaline media, methylmercury in the hair digest began to break down a t a rate of 0.3% per day after 3 days. Sample storage conditions were not specified. Methylmercury breakdown in sodium hydroxide digests of biological samples can result in significant analytical errors over extended digest storage periods. Such errors can be critical, especially for samples being examined for possible environmental methylmercury contamination (e.g., human and animal tissue). Consequently, we have specified t h a t alkaline sample digests be analyzed immediately after preparation. T h e above findings have prompted us to investigate the extent of methylmercury conversion to inorganic 0003-2700/80/0352-1527$01.00/0
mercury in sodium hydroxide solution as influenced by storage time and temperature, and the presence of biological samples.
EXPERIMENTAL Materials and Reagents. Sodium hydroxide solutions of methylmercury standards and tissue samples were prepared and stored in 20-mL glass scintillation vials (Packard Instrument Co.). Methylmercuric bromide was obtained from Pfaltz and Bauer. A stock aqueous solution containing 1.10 ppm MeHgBr was prepared in 0.05 N Na2C03. Cysteine hydrochloride (monohydrate) was obtained from Matheson, Coleman and Bell, the 1% aqueous solution being prepared fresh prior to use. Sample Preparation. Two milliliters of the MeHgBr stock aqueous solution (containing 2200 ng MeHgBr) were placed in a 20-mL glass vial. Two milliliters of 45% NaOH, 1.0 mL of 1% cysteine hydrochloride, and 5.0 mL of 1% NaCl were added. Final NaOH concentration was 7.0 N. Three MeHgBr solutions were prepared. One was stored in the dark at room temperature (23 "C), a second was kept refrigerated ( 3 "C), while the remaining solution was kept frozen (-20 "C) during storage. Accuratelyweighed and finely-chopped samples of human hair (4.10 mg) and fresh flounder fillet (1.0 g) were prepared in a similar manner, except that the sample was gently heated until dissolved and then allowed to cool before adding 1% NaCl. The tissue digests were stored at -20 "C in a freezer. 1980 American Chemical Society
1528
ANALYTICAL CHEMISTRY, VOL. 52, NO. 9, AUGUST 1980
Table I. Methylmercury Breakdown in Alkaline MeHgBr Solutions Storage a ng Hg/aliquot inorganic
storage time, days
A
methyl R
F
A
R
F
A
total
R
F
0 1 3 10 17 25
74.1 68.4 65.1 62.7 52.6 45.0
76.4 71.6 71.2 66.5 63.6 60.8
73.5 71.8 71.4 70.7 70.4 70.0
1.5 8.3 8.3 11.6 20.4 26.4
0.7 5.0 4.8 7.6 10.5 12.1
0.9 1.6 1.4 3.0 1.1 1.5
75.6 76.7 73.4 74.3 73.0 71.4
77.3 76.6 76.0 74.1 74.1 72.9
74.4 73.4 72.8 73.7 71.5 71.5
a A: room temperature (23 "C), R : refrigerator ( 3 "C), F: freezer (-20 "C). Values represent averages of duplicate Values represent the sum of the corresponding methyl- and inorganic mercury analyses on 0.5-mL sample aliquots. values.
Table 11. Methylmercury Breakdown in Alkaline Sample Digests sample hair, human
storagea fresh digest freezer, 7 days refrigerator, 10 days
ng Hg/mg hair, g muscle methyl inorganic 22.5 23.1 20.8
2.8 4.0 5.3
flounder muscle
a
fresh digest 120.5 65.6 freezer, 7 days 118.8 69.2 refrigerator, 1 0 days 113.9 71.4 Sample digests were initially stored 7 days in a freezer, then refrigerated for 10 days.
Analysis. Sample digests were analyzed in duplicate for methyl- and inorganic mercury by the gas chromatographic procedure of Cappon and Smith (I). All digests were analyzed immediately after preparation. For the MeHgBr solutions, 0.5-mL aliquots (representing 110 ng MeHgBr or 74.7 ng Hg) were taken for mercury analysis. The total mercury content of the digests was obtained by adding the corresponding average values for methyl- and inorganic mercury.
25.3 27.1 26.1
7.6
186.1 188.1 185.3
1.4 5.5
40'
r';
RESULTS AND DISCUSSION Data on methylmercury and inorganic mercury content of the alkaline MeHgBr solutions during a 25-day storage period are presented in Table I. T h e extent of methylmercury breakdown as a function of storage time and temperature is shown in Figure 1. T h e influence of storage temperature is quite evident. Overall breakdown was greatest for the solution stored at room temperature, averaging 39.3% after 25 days. Corresponding values for the refrigerated and frozen solutions were 20.4 and 4.8%, respectively. For all solutions, there was a sharp increase in the breakdown during the first day of storage. This was followed by a more gradual increase, especially for the refrigerated and frozen solutions. Formation of MeHg(OH)2- in concentrated NaOH solution may have prevented quantitative methylmercury complexation by cysteine. This would make methylmercury more available for conversion t o inorganic mercury, which is also quantitatively complexed by cysteine. Subsequent methylmercury breakdown was largely due to gradual oxidation of cysteine, being t h e greatest at room temperature. T h e notable initial breakdown for the frozen solution probably resulted from the time lag in the complete freezing of the strong alkaline solution. Alkaline digests of human hair and fish muscle samples showed less methylmercury breakdown under similar storage conditions (Table 11). Digest storage a t -20 "C resulted in no significant breakdown ( < Z % ) even after 2 weeks. Subsequent refrigerated storage for 10 days resulted in notable methylmercury losses of 5.5 and 7.6% for the fish and hair digest, respectively. However, these losses were about one half that observed for the refrigerated MeHgBr solution after the
% MeHg breakdown
total
0
5
10
15
lo
15
S T O R A O I T I M E , DAYS
Figure 1. Breakdown of MeHgBr in 7.0 N NaOH solution. Values are based on [MeHgBr] for day 0. (A)storage at -20 OC,(0)storage at 3 OC, (0)storage at 23 OC
same storage period (13.0%). Apparently, some of the methylmercury in the sample digest still remains tightly bound to sulfhydryl-containing amino acid and peptide residues even after alkaline digestion, and will be less susceptible to demethylation by NaOH than unbound methylmercury. T h e mechanism of chemical demethylation of methylmercury in aqueous solution is uncertain. Zepp et al. ( 4 ) demonstrated t h a t photodecomposition of sulfur-bonded methylmercury complexes (including MeHg-cysteine) by sunlight is a possible pathway for inorganic mercury formation
Anal. Chem. 1980, 52, 1529-1532
in aquatic environments. This phenomenon probably played a minor role, if at all,in the present study, as all sample digests were stored in the dark. Other possible causes of methylmercury loss from aqueous solution are absorption on sample container walls (5-7) and volatilization of the intact molecule (6-8). The present results indicate that chemical breakdown to inorganic mercury is the main factor controlling methylmercury loss in 7.0 N NaOH solution. For all samples, t h e decrease in methylmercury content resulted in a corresponding increase in inorganic mercury (Table I). Formation of MeHg(OHI2- and complexation by cysteine would prevent methylmercury volatilization. However, mercury absorption on the walls of the glass vial may have occurred to a small extent, as the total mercury content of each MeHgBr solution slowly decreased ( - 5 % ) over t h e 25-day storage period. CONCLUSIONS The present study clearly demonstrates that methylmercury breakdown to inorganic mercury occurs to a significant extent in alkaline sample digests and can result in erroneous analytical results for this organomercury species. Therefore, sample digests must be analyzed immediately after preparation. If the digest has to be re-analyzed a t a later time, it may be stored in a freezer for up to one month without experiencing
1529
significant methylmercury breakdown. However, preparation of a fresh sample digest is highly advisable. LITERATURE CITED (1) Cappon, C. J.; Smith, J. C. Anal. Chem. 1977, 49, 365-369. (2) Cappon. C. J.; Smith, J. C. Bull. Environ. Contam. Toxlcol. 1978, 19. 600-607. (3) GiovanoliJakubczak, T.; Greenwood, M. R.; Smith, J. C.; Clarkson, T . W. Clin. Chem. 1974, 2 0 , 222-229. (4) ZeDD, R. G.; Bauahrnan, G. L.; Gordon, J. A,; Wolfe, N. L.; Cline, D. M. Environ. Left. 1$74, 6 , 117-127. (5) Burrows, W. D.; Krenkel, P. A. Environ. Sci. Techno/. 1973, 7. 1 127-1 131. (6) BuFrows,W. D.; Krenkel, P. A. Anal. Chern. 1974, 46. 1613. (7) Stoeppler, M.; Matthes, W. Anal. Chim. Acta 1978, 9 8 , 389-392. (8) Olson, K. R. Anal. Chem. 1977, 49, 23-25. '
'
Present address: Kettering Laboratory, Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, Ohio 45267.
C h r i s J. C a p p o n * J. C r i s p i n S m i t h ' Environmental Health Sciences Center and Department of Pharmacology and Toxicology University of Rochester School of Medicine and Dentistry Rochester, New York 14642 RECEIVED for review January 10, 1980. Accepted April 21, 1980.
Solvent Swelling for Enhancement of Carbon- 13 Nuclear Magnetic Resonance Spectral Information from Insoluble Polymers: Chloromethylation Levels in Cross-Linked Polystyrenes Sir: T h e cross-linked polystyrene system has been the support most commonly employed in recently developed solid-phase synthetic methods (1-3). Numerous high resolution NMR studies of linear polystyrenes, which form true solutions, have been reported (4-61, but there is a paucity of N M R studies on cross-linked, insoluble polystyrenes ( 7 ) . Important problems t h a t have been recognized with the widespread use of cross-linked polystyrene materials in solid-phase syntheses are t h e need for rapid, accurate, a n d nondestructive procedures of analyses for their functionality, understanding of the dispersion in reactivities and mobilities of appended groups, and information on t h e distribution of functionalities at the molecular level (8). Recently reported results using special techniques and special molecular properties have shown what significant structural information can be obtained by N M R studies of insoluble, bulk materials. These studies have involved multiple pulse techniques (9, IO), magic-angle spinning (11-13), solid plant materials (14-16), plastic crystals (17-19), synthetic polymer gels (20,21), biopolymers swollen by water or polar organic solvents (22-26), and solid rubbers a n d amorphorous polymers (27-31). We report here N M R analytical results on solvent swollen, cross-linked polystyrenes which demonstrate a new approach for obtaining analytical and structural information on functionalized polystyrenes that are so important as the backbone for many solid-state syntheses and reagents. This approach should also have applicability for studies of other synthetic and natural polymeric materials t h a t can be swollen by solvents. EXPERIMENTAL Linear polystyrenes molecular weight 25 000, 35 000, 100 000 and 250000 were experimental samples provided by John Ingham of our laboratory. The cross-linked polystyrenes were purchased 0003-2700/80/0352-1529$01 .OO/O
from Bio-Rad and Polysciences, Inc. Commercial chloromethylated, cross-linked polystyrenes were purchased from Bio-Rad, Eastman Organic Chemicals, Polysciences, Inc., and Pierce Chemical Company. The sample of 0.095% cross-linked polystyrene was a gift from Dow Chemical Company. Stannic chloride was purchased from MC/B Manufacturing Chemists, Inc. Chloromethylmethyl ether was purchased from Aldrich Chemical Company. The various polymers were chloromethylated by a procedure that has been described previously (16). By varying the stannic chloride concentration, polymers with degrees of substitution from 0.3 to 3.7 mequiv/g were produced. Most 13C spectra were obtained with a Jeol FX60 at 15.1 MHz (typical conditions: 8 K FT; 8 ps, 45" pulse at 0.6-s repetition; CDC1, solution in 10-mm 0.d. sample tubes; proton noise decoupled; internal deuterium lock); other spectra were obtained on a Varian XL-100 at 25.1 MHz and a Bruker WH-180 at 45.2 MHz (typical conditions: 8 K F T ; 85 ps, 25" pulse every 0.819 s; CHC13solvent in 25-mm 0.d. sample tubes; proton noise decoupled; internal deuterium lock). Available evidence indicates that T1's in polystyrenes for aliphatic -13CHz- and -I3CH< are in the range of 0.044.09 s and the aromatic C-H ones in the range of 0.04-0.1 s so the delays between pulses were at least 5 T t . In several cases, it was observed that going to delays about a fourth of those mentioned above, resulted in significant changes in relative peak areas for the 1%H2C1-, -13CHz- and --l%H< carbons. Assignments were based on previous work ( 4 4 ) ,NMR spectra of various model resins and gated-decoupled spectra in which directly bonded carbon-proton couplings could be observed. Proton spectra were obtained a t 100 MHz on a digital sweep Varian HA-100. The degree of chloromethylation in some of these resins and some of the commercial ones was determined by Volhard titration (32). However, we encountered difficulties in obtaining reproducible results by this analysis. It appeared in our hands that the liberation of the chloride from the resins requires 2 to 10 times longer than has been recommended previously (32). In addition, we encountered fading end points when enough acid was not present to tie up completely all the pyridine as the pyridinium salt. In
t Z 1980 American
Chemical Society
