Anal. Chem. 1907, 59, 1843-1846
found in the acetone eluate with the method described. The lineal correlation coefficient of added vs. found radioactivity was 0.9995 ( n = 12). The mean percent recovery of L-alanine radioactivity with the method described was 82.3 f 0.75 ( n = 12). No differences were observed between the recoveries of [14C]-~-alanine standard solutions and those of rat plasma samples with known amounts of [14C] alanine, irrespective of the presence of other labeled materials. The 17.7% loss of radioactivity in the whole derivatization/purification process seems due to partial spontaneus reduction of the pyruvic acid hydrazone at alkaline pH (15) accounting for incomplete formation of pyruvic acid 2,4-dinitrophenylhydrazonesince the pyruvic acid hydrazone formation from L-alanine is quantitative in these conditions (13) and pyruvic acid 2,4dinitrophenylhydrazoneis quantitatively trapped in Amberlite XAD-7 columns (11). The high repetition of this figure (17.7%) under the conditions described made superfluous the inclusion of known standard solutions to correct each batch of samples studied at the same time. However, the values obtained must be addressed taken into account the overall recovery of the process, Le., multiplying by a 1/0.823 factor. The recovery of other labeled materials is also shown in Table I. No interferences from D-glucose, glycerol, L-lactate, or the amino acids tested were observed using our conditions in accordance with both the specificity of L-alanine dehydrogenase for this substrate (13) and their null reactivity with the 2,4-dinitrophenylhydrazine(10,111. The specificity of this method is the same as the spectrophotometric enzymatic measurement of L-alanine (13), the contamination of [U-14C]-~-serine-a possible substrate of L-alanine dehydrogenase (13)-being irrelevant. Actually, only minor interferences could be expected because the procedure de-
1843
scribed ensures a high degree of specificity at the enzymatic level and eliminates other interferences through the use of its own blank. This method is applicable to large numbers of samples for the determination of L-alanine-specific radioactivity in tracer metabolic studies. Registry No. [U-“C]-~-Alanine,18875-37-1;L-alanine dehydrogenase, 9029-06-5;pyruvic acid 2,4-dinitrophenylhydrazone, 790-12-5.
LITERATURE CITED Alemany, M.; Palou, A.; Codina, J.; Herrera, E. Diabetes Metab. 1978, 4 , 181-186. Martlneau, A.; Lecavaiier, L.; Faiardeau, P.; Chiasson, J. L. Anal. Biothem. 1985. 151, 495-503. Squires, E. J.; Brosnan, J. T. Anal. Biochem. 1978, 8 4 , 473-478. Adam, W.; Slmpson, D. P. J . Clln. Invest. 1974, 54, 165-174. Macnicol, P. K. Anal. Biochem. 1978, 8 5 , 71-78. Bishop, R.; Siqs, A. P. Analyst (London) 1975, 100, 369-376. Pons, A.; Garcia. F. J.; Paiou. A.; Aiemany, M. J . Biochem. Biophys. Methods 1981, 5 , 153-156. Roca, P.; Palou, A.; Aiemany, M. J . Biochem. Biophys. Methods 1983. 8, 63-67. Dressier, M. J . Chromafogr. 1979. 165, 167-207. Gianottl, M.; Roca. P.; Paiou, A. J . Biochem. Biophys. Methods 1984, 10, 161-185. Roca, P.; Qianotti, M.; Paiou, A. Anal. Biochem. 1985, 148, 190-193. Moreno, P.; Snchez, E.; Pons, A.; Palou, A. Anal. Chem. 1988, 58, 585-587. Williamson, D. H. Methods of Enzymatic Analysis; Bergmeyer, H. U., Ed.; Academic: New York, 1974; Vol. 4, pp 1679-1685. Turner, J. C. Sampb Reparation for Liquid Scintilliation Counting; Amershamsearle: Arlington Heights, VA. 1973; pp 18-20. Griffin, R. W. Quimica Org6nica Moderns; Revert& S . A,, Ed.; Barcelona, 1981; pp 316-317.
RECEIVED for review December 29,1986. Accepted March 16, 1987. This work was supported ,by a grant from the “ComisiBn Asesora de InvestigaciBn Cientifica y TBcnica” (PB 85-0326) from the Government of Spain.
Ion-Pair Chromatographic Determination of Anions Using an Ultraviolet-Absorbing Co-Ion in the Mobile Phase Brian A. Bidlingmeyer,* Carmen T. Santasania, and F. Vincent Warren, Jr. Waters Chromatography Division of Millipore Corporation, 34 Maple Street, Milford, Massachusetts 01 757
UV-visuallzatlon LC Is applied to the determination of elght common Inorganic anions. Chromatographic separation and detectlon are accomplished by using a UV-transparent, lonpair reagent (tetrabutylammonlum Ion) and a UV-absorbing co-ion (saikyiate ion) of the ion-pair reagent. The method, which uses a standard reverse-phase column and a UV spectrophotometer, is capable of separating and detecting a wide range of ions including those that are normally non-UVabsorbing. Spectrophotometric detectlon limits are in the low nanogram range and compare favorably to those obtained by using conductometric detection. Quantitation with either detector Is possible over several orders of magnitude.
Ion analysis by liquid chromatography (LC) has been evolving as a significant technique in analytical chemistry. There are two main LC approaches which are being used. One approach uses a fixed-site, ion exchange column; the other uses 0003-2700/S7/0359-1843$01.50/0
a reverse-phase column with an eluent containing an ion-pair reagent. For organic ions the most common approach is ion pairing as it has the advantage of retention control through both electrostatic and lipophilic interactions (1). This leads to a broad flexibility which is difficult to match with the fixed-site approach. For inorganic ions the use of fixed-site ion exchangers is popular (2). However, a number of ion-pair applications have appeared (3-8). In the case of inorganic ion analysis the most popular detection mode has been conductance because of ita wide applicability. A specialized mode of ion pairing is the technique of UVvisualization (1,9, IO),in which a UV-absorbing lipophilic ion serves as the ion-pair reagent. This reagent is adsorbed onto the stationary phase in a dynamic equilibrium and retention is due to a combination of lipophilic and electrostatic forces. The lipophilic, UV-absorbing ion coelutes with the non-UVabsorbing analyk molecule, thus facilitating its detection. The prediction of W visualization (9) and the explanation of the effect (1, 10, 11) have been based upon the ion-interaction @ 1967 American Chemical Society
1844
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987
model (8). Support for the hypotheses of the ion-interaction model has been offered by a number of investigators (3,12-15). Most recently, Purdy and Bedard described the quantitative thermodynamics of the model (16,17). The UV-visualization technique has been applied to the analysis of a wide variety of difficult to detect compounds such as alkyl sulfonates (10, 18),aliphatic acids (19), esters and alcohols (20-24), choline esters (25,26),drugs (27,28),and inorganic anions (29-31). For most of these reports, the quantitative aspects of the approach have only been demonstrated for a well-defined system, which often involves a narrow linear range (10, 18, 19, 29). In this study we apply a modified UV-visualization approach to the determination of inorganic ions, in which the lipophilic ion which is of opposite charge to the analyte ion is UV-transparent, while the co-ion of the lipophilic ion-pair reagent is UV-absorbing. This is opposite to what has been typically reported (10,19, 32) but agrees with the approach taken by Druex (3). Using the modified reverse-phase, ion-pair system, we are able to analyze inorganic anions using either spectrophotometric or conductometric detection. We have found that by using UV visualization, spectrophotometric detection of the otherwise non-UV-absorbing inorganic anions is comparable to conductometric detection. The useful linear range for the spectrophotometric detection spans several decades, and peak height and area do not depend upon the levels of other ions present in the sample as previously noted (19). UV-visualization for inorganic ion determination is a convenient approach using standard LC hardware.
EXPERIMENTAL SECTION Apparatus. The Waters chromatographic system (Milford, MA) consisted of a Model 6000A solvent delivery system with its internal high sensitivity noise fiiter in line, a Model 710B WISP automatic injector, and a Model 481 variable wavelength spectrophotometer (at 288 nm) in series with a Model 430 conductometric detector. Automated runs and data collection were accomplishedwith a Model 840 data and chromatography control station (Waters). When appropriate, the analog output leads of the UV detector were reversed for the quantitation of negative peaks at 288 nm. A Resolve C18 Radial-PAK cartridge, 5 pm (8 mm X 10 cm), held in an RCM-100 radial compression module (Waters)was used exclusively throughout this study. Before equilibration with the final mobile phase, the cartridge was rinsed with 100 mL of acetonitrile and then with 50 mL of a 50/50 (v/v) mixture of acetonitrile and water. A flow rate of 2 mL/min was used for all analyses. Reagents and Mobile Phases. Salicyclic acid (SAL) (Gold Label grade) was obtained from Aldrich Chemical Co. (Milwaukee, WI). Tetrabutylammonium ion (TBA) was obtained as the hydroxide in a 1.5 M solution in water from Fluka Chemical Co. (Ronkonkoma, NY). Inorganic anions (phosphate, chloride, nitrate, nitrite, bromide, sulfate, iodide, and thiosulfate) used in the test mixture were in the sodium or potassium form and were obtained from a variety of suppliers as their reagent grades. All chemicals were used as received without further purification. TBA and SAL were mixed in water to form a 0.4 mM solution of each. The pH of this solution was 4.62. Individual standards and mixtures were prepared in water. Samples used for the lowest amount of anions detected were serially diluted and a 50-pL aliquot was injected into the LC. Purified water (18 MR) was obtained from a Milli-Q System (Millipore Corp., Bedford, MA) and used for the preparation of all samples and mobile phases. All mobile phases were filtered through a 0.45-pm cellulose acetate filter (HATF 04700) (Millipore Corp.) before use. Samples were filtered through 0.45-pm Millex-HA filters (SLHA 02505) (Millipore Corp.) prior to injection. RESULTS AND DISCUSSION Chromatographic Analysis. Eight inorganic anions (phosphate, chloride, nitrate, nitrite, bromide, sulfate, iodide, and thiosulfate) were combined into a test mixture for this
A.
21
I
I/ O o
3t
IO
15
20
25
15
20
25
MINUTES
B.
2t I\”.5
IO MINUTES
Figure 1. Chromatograms of inorganic anions obtained by UV-visualization chromatography: detection, (A) spectrophotometric at 288 nm, (B) conductometric; anion amounts, 100 ng (2 ppm) of CI-, 200 ng (4 ppm) each of H2P0,-, NO,-, Br-, NO,-, and I-, and 300 ng (6 ppm) each of SO,,- and S2OS2-.
study. These ions were chosen to cover the range of anions commonly analyzed in published applications. The separation of the eight anions is shown in Figure 1 using the UV-visualization system with spectrophotometric and conductometric detection. Figure 1A for the spectrophotometer has the ordinate labeled as negative detector response because the anion peaks are due to decreases in absorbance a t 288 nm. Figure 1B shows the chromatographic trace obtained by using the conductivity detector which was in series with the spectrophotometer. Control of retention is possible by varying the mobile phase composition. Retention of the anions decreases if the TBA-SAL concentration is increased or if an organic modifier is added to the eluent. The conductometric monitoring of the separation shown in Figure 1B is similar to one previously reported that used a “dynamically coated” reverse-phase column with a tetrabutylammonium salicylate (TBA-SAL) eluent (33). In that work, cetylpyridinium chloride was used to “coat” the bonded phase column prior to using the low conductivity, aqueous eluent of 0.4 mM TBA-SAL. The similiarity of our work to the previous separation stimulated our interest in evaluating the need to “precoat” the bonded phase column prior to using an ion-pair reagent. Therefore, a column was prepared according to the reported coating procedure (33) prior to use of a low conductivity eluent of 0.4 mM TBA-SAL, and the resulting separation was compared to Figure 1. After the initial coating process, the TBA-SAL eluent was introduced onto the column and a “breakthrough” of increased
ANALYTICAL CHEMISTRY, VOL. 59,
absorbance and increased conductance occurred which signified the equilibration of the eluent with the dynamically coated column. After the column was coated and equilibrated with the TBA-SAL eluent, anions were separated and monitored by both conductance and UV absorbance a t 288 nm. We obtained similar conductance profiles to the original report. Since the original report used only conductivity detection, there was no comparison of the UV response. In the initial separation obtained with the precoated column, retention times were longer than those observed in Figure 1. The separation was maintained for a few days, at which time another upset in the base line was observed. This upset in the base line was apparent in traces from both the conductometric and spectrophotometric detectors. Following the second upset, separation of anions was still achieved and this separation was identical with that which we observed in Figure 1. It was surmised that the initial "coating" of cetylpyridinium ion had been removed, leaving only the adsorbed coating of TBA. Therefore, the precoating was abandoned, and only the TBA-SAL eluent was used in the remaining work. Three items should be emphasized concerning Figure 1. First, the resolution of the anions using the ion-pair system is comparable to those reported using a conventional ion exchange column (34). Second, the W-visualization technique enables the spectrophotometer to be used as an alternative to the conductivity detector for otherwise non-UV-absorbing anions. This is especially relevant since the spectrophotometer is the most common LC detector. The third point is an apparent absence of the so-called "system peak". This characteristic of many ion exchange and ion-pairing techniques is due to the equilibrium process taking place in the system. The system peak in the present study elutes at approximately 250 min. This long elution time serves the analyst in that a large number of analyses may be done before the first system peak elutes. The chromatograph may then be run overnightto clear the column of the system peaks. If this approach is followed, no base line upsets from the elution of the system peaks will affect the anion separations. Even when samples are analyzed over a 24-h period, it is our experience that the system peaks result in only a slight base-line wander as the sharp analyte peaks elute. Comparison of Detector Response. The ability to quantitate low levels of inorganic anions is of significant practical concern for the proposed method. Literature reports (4,6,29)indicate that the ability to routinely work at the low nanogram level is desired. Inspection of Figure 1 indicates that the relative peak heights are similar for the conductometric and spectrophotometric detection modes, so either mode is usable for anions. It should be noted that quantitation problems might be observed for phosphate regardless of the detection mode, because this peak occurs as a rider on the trailing edge of the void volume peak. Therefore, if phosphate analysis is desired, the retention time for that anion should be adjusted to longer values. To investigate the capability of each detection mode for the quantitation of low levels of anions, the responses for various injected amounts of chloride, bromide, nitrite, and nitrate were studied. These anions are sufficiently retained to avoid the base-line problems noted for phosphate. For each of the four anions, progressively lower amounts were individually injected until a signal to noise ratio of 5 was observed. This injected amount is termed the "lowest amount detected" and is reported in Table I for the four anions studied. Table I indicates that either detection mode offers quantitation capability in the low nanogram range, as desired. Effect of Interfering Sample Components on Detector Response. In a previous report (19),it was demonstrated that
NO. 14, JULY
15, 1987
1845
Table I. Comparison of Detector Responses
anion chloride bromide nitrite nitrate
lowest amount detected: ng tppb) absorbance conductivity 6 (120) 62 (1240) 14 (280)
6 51 14 35
37 (740)
(120) (1020)
(280) (700)
"Lowest amount detected is reported at a signal-to-noiseratio of 5.
Table 11. Coefficients of Calibration Curve y = mx
+b
slope intercept ion
bromide
bromide + 100 ppm nitrate bromide
bromide + 100 ppm nitrate
detection
(m)
(b)"
nb
r
absorbance absorbance
225 230
673
15 0.9997 15 0.9997
conductivity conductivity
192
-2369
15 0.9997
196
-2685
15 0.9999
702
Area units in p V s. bFive levels measured in triplicate.
in the use of the conventional UV-visualization approach, the response for a given ion depended upon the levels of other ions present in the sample. Since the presence or absence of other ionic species in real samples is a variable that cannot be controlled, it was necessary to determine if such a situation existed for the analysis of inorganic anions by the method discussed here. To investigate the possible influence of interfering anions on quantitation, calibration curves were prepared with bromide alone and then with bromide plus a constant amount (100 ppm) of nitrate as an "interfering" ion. This high level of nitrate was kept constant throughout this part of the study. Calibration data for both detection modes are shown in Table 11. Both detection modes show linearity over nearly 3 orders of magnitude with a calibration curve passing essentially through zero. A very slight increase in the response slope is seen in both the conductometric and spectrophotometric responses for bromide when nitrate is present. This very slight change in the magnitude of the slope is in the opposite direction to what was observed previously (19). It is important for the chromatographer to understand the practical impact of the observed slope differences, since this may influence quantitation. In the present case, a relatively large amount of nitrate has been added to the sample, leading to an observed 2% increase in calibration curve slopes. If, for example, a peak area of 150000 is measured for bromide, the calculated amount would be 660 ng if the bromide-alone calibration curve is used. If, however, the sample also contained 100 ppm nitrate, the correct amount would be 648 ng. The 2% difference in the slopes of the calibration curves is significantly less than had been previously reported (19). In order to determine whether the variability in the slopes of the calibration curves was due to the method or to random experimental error, Student t tests were performed on the slopes of the calibration curves. The t test results indicate the slopes to be statistically different. Reasons for the observed difference may stem from changes of the column during use, from ambient temperature fluctuation, or from the normal day to day variation in this system's reproducibility. From a practical viewpoint, in many applications the small difference in calibration slopes will have an insignificant impact on quantitation. The modified UV-visualization approach described here, in which the co-ion of the lipophilic ion-pair reagent ion serves
1846
Anal. Chem. 1087. 59. 1846-1849
as the visualizing agent, appears to offer certain advantages for the analysis of inorganic anions. The chromatographic run is completed within 25 min (Figure 1)with good resolution and detection at low nanogram levels via spectrophotometric or conductometricdetection. Compared to previous work with organic anions (19), the influence of interfering sample components on peak height and area is minimal for the modified UV-visualizationsystem. Work is continuing in our laboratory to determine if using this same approach will result in a more rugged LC method for the analysis of non-UV-absorbing organic ions which were previously determined by using the ion-pair reagent as the visualizing entity.
ACKNOWLEDGMENT The authors wish to thank J. Newman and M. Ciak for assistance in preparing the manuscript, B. Marshall for help with the statistical analyses, and J. Oberholtzer for helpful discussions. Registry No. H2P0;, 14066-20-7; C1-, 16887-00-6; NO3-, 14797-55-8;NOz-, 14797-65-0;Br-, 24959-67-9;SO4-, 14808-79-8; I-, 20461-54-5; S203", 14383-50-7;TBA-SAL, 22307-72-8;SAL-, 63-36-5. LITERATURE CITED Bidlingmeyer, B. A. J. Chromatogr. Sci. 1980, 18, 525. Small, H.; Stevens, T. S.; Bauman, W. C. Anal. Chem. 1975, 4 7 , 1801. Dreur, M.; LaFosse, M.; Pequignot, M. Chromatographia 1982, 75, 653. Molnar, I.; Knauer, H.; Wiik, D. J. Chromatogr. 1980, 207, 225. KruU, I . S.; Panaro, K. W.; Gershman, L. L. J . Chromatogr. Scl. 1983, 2 7 , 460. Skelly. N. E. Anal. Chem. 1981, 54,712. Schedt, G.;Rossnen, 6.; Schmuckler, G. J . Chromatogr. 1984, 302, 15.
(8) Bidiingmeyer, B. A.; Deming, S. N.; Price, W. P., Jr.; Sachok, 6.; Petrusek, M. J. Chromatogr. 1979, 786. 419. (9) Bidlingmeyer, B. A.; Demlng, S. N.;Price, W. P., Jr.; Sachok. 6.; Petrusek. M. Paper presented at Advances in Chromatography 14th International Symposium, Lausanne, Switzerland, Sept 24-28, 1979. (10) Billngmeyer, 8. A.; Warren, F. V., Jr. Anal. Chem. 1982, 5 4 , 2351. (11) Bidlingmeyer, B. A.; Deming, S. N.; Sachok, B. Poster presented at 13th International Symposium on Chromatography, Cannes. France, July 2, 1980. (12) Deming, S. N.; Stranahan, J. J. Anal. Chem. 1982, 54, 1540. (13) Deming, S. N.: Stranahan, J. J. Anal. Chem. 1982, 5 4 , 2251. (14) Deming, S. N.; Stranahan, J. J.; Lin, W.; Tang, M. Anal. Chem. 1983. 5 5 , 1872. (15) Vara-Avila, L. E.; Coudy, M.; Rosset, R. Analusis 1982, 10, 36. (16) Bedard, P. R.; Purdy, W. C. J. Liq. Chromatogr. 1985, 8 , 2417. (17) Bedard. P. R.; Purdy, W. C. J. Liq. Chromatogr. 1985, 8 , 2445. (18) Sachok, 6.; Deming, S. N.; Bidlingmeyer. B. A. J. Li9. Chromatogr. 1982, 5 . 398. (19) Bilingmeyer, B. A.; Warren, F. V., Jr. Anal. Chem. 1984, 5 6 , 487. (20) Parkin, J. E. J. Chromatogr. 1984, 287, 457. (21) Parkin, J. E. J. Chromatogr. 1988, 351, 532. (22) Schill, G.; Hackzell, L. Chromatograph/a 1982, 75,437. (23) Crmmen, J.; Herne, P.; Renson, M. Chromtographia 1984, 79, 274. (24) Parkin, J. E. J. Chromatogr. 1984, 303, 436. (25) Raghuveeran, C. D. J. Liq:ChrOmetgr. 1985. 8 , 537. (26) Jones, R. S.; Stutte, C. A. J. Chromatogr. 1985, 319, 454. (27) Crommen, J.; Herne, P. J. phsrm. Biomed. Anal. 1984, 2 , 241. (28) Collins, A. J. J. Chromatogr. 1988, 354, 459. (29) Carr, P. W.; Barber. W. E. J. Chromatogr. 1983, 260, 89. (30) Kang, S. W. J. Korean Chem. SOC. 1985, 2 9 , 365. (31) Dreux, M.; LaFosse. M.; Agbo-Haroume, P.; Chaabane-Doumandji, 6.: Gibert, M.; Levl, Y. J. Chromatogr. 1988, 354, 119. (32) Denkert. M.; Hackzell, L.; Schill, G.; Sjogren, E. J. Chromatogr. 1981, 218, 31. (33) Cassidy, R. M.; Elchuk, S. J. Chromatogr. 1984, 262, 311. (34) Haddad, P. R.; Heckenberg. A. L. J. Chromatogr. 1984, 300, 357.
RECEIVED for review December 19,1986. Accepted March 30, 1987. This work was presented, in part, at the 189th National Meeting of the American Chemical Society, Miami Beach, FL, April 28-May 3, 1985.
Modified Forms of Differential Pulse Polarography for Improved Sensitivity When Kinetically Controlled Processes Overlap Alan M. Bond,* Gerard Heneghan, and Daryl J. Tucker Division of Chemical and Physical Sciences, Deakin University, Waurn Ponds 3217, Victoria, Australia Donald E. Rivett Division of Protein Chemistry, CSIRO, Parkuille, Victoria 3052,Australia
Conventkmal differential pulse and square wave pohogaphlc methods are completely Inadequate for the determination of 9-acetamldoacrylic acid (N-acetyklehydroalanlne). The negatlve potential required for reductlon of the compound, comblned wRh the need for hlghly acidic condltlons, Introduces a resoMlon problem that lhnns the detectlon to concentratlons above lo4 M. A detalled lnvestlgatlon of alternatlve dmeremtlal puke polarographic modes shows that a 3 orders of magnitude Improvement In the Umtt of detectbn can be achieved by use of the technlqw called dlfferentlsl pulse reversbk polarography. TMS technlque explolts the dmerent tlme dependencles of the kinetically controlled reductlve process for 2-acetamkloacrylic acld to that for the lnlerlerlng hydrogen ion reductlon wave to mlnlmlze the Influence of the latter on the measured response. Thls rubstantlal improvement In detection Umlt is required for the development of a technlque for the monitoring of wool degradatlon. When proteins are exposed to heat, light, or alkali, 20003-2700/67/0359-1846$01.50/0
aminoacrylic acid, commonly known as dehydroalanine, or related compounds are produced as the result of p-elimination at cystine and serine residues (1-3). It has been proposed that the amount of dehydroalanine (DHA) could be representative of the degree of environmental damage to wool fibers. In the free state DHA is unstable and decomposes to form ammonia and pyruvic acid ( 4 ) . Thus the free amino acid cannot be isolated and quantified. Although DHA can be determined indirectly in wool hydrolysates by assaying for pyruvic acid produced during acid hydrolysis, the procedure is lengthy and is subject to errors due to side reactions ( 4 , 5). The need for a specific and sensitive analytical method for the determination of derivatives of DHA formed during the course of wool processing has led us to investigate the possibility of developing a method based on the electroanalytical technique of polarography. It is known that a,p unsaturated compounds, -C=C-C-, usually can be reduced chemically, without skeletal rearrangement, under the reduction conditions suggested by Birch (6) (alkali metal reduction in liquid ammonia in the presence of a proton donor). One possible exception of the requirement of a proton donor involves re0 1987 American Chemical Society
