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Table I. Atomic Absorption Analysis of Light and Heavy Waters Reportedas pg/mLa Ni Zn cu sample Cr Mn Fe co 0.0336 0.07 5 -0.003 1. town tap water 0.000 0.001 0.024 0.001 c 0.001 f 0.001 i 0.0007 i 0.002 i 0.0002 i- 0.0002 ?: 0.0001 0.003 -0.008 -0.002 2. house deionized water 0.000 0.000 -0.005 0.002 f 0.0003 i 0.0006 + 0.0000 i 0.001 i 0.0005 i 0.001 i 0.0001 - 0.0048 - 0.003 0.004 3. laboratory Milli-Q water -0.001 0.000 0.000 0.002 f 0.0007 f 0.0002 t 0.001 i 0.005 f 0.0002 t 0.0001 i 0.0000 0.0066 0.001 -0.002 4.Merck, Sharp and Dohme, Inc., 0.000 0.004 0.006 0.001 f 0.002 99.7% D D,O, cat. + 0.0002 t 0.0002 i 0.002 0.0002 f 0.0005 i 0.001 MD-175, lot B-343 0.0437 0.002 0.000 5. Aldrich Chem. Co., 100.0% D D,O 0.000 0.001 0.002 0.001 (low Daramagnetic immrities). c 0.0001 i 0.0002 0.002 2 0.0001 i 0.0009 f 0.0006 I 0.0004 iot 021967 D A 6. Stohler Isotopes, Inc., 100% D D,O 0.002 0.006 0.016 0.007 0.0097 0.018 0.2644b (low paramagnetic impurities) i 0.001 f- 0.0003 i 0.003 i 0.002 i 0.002 i 0.0003 i- 0.01 cat. +SD-18, lot k l 2O-bL samples in triplicate. Linear calibration assumed with standards dissolved as nitrate or chloride salts in 2% vlv InjecHNO, (G, F. Smith Chemical Co., catalogue -1621 double distilled from Vycor) and blanked against 2% v/v ”0,. ted as a 5-pL sample. _+
_+
Zn content of three commercial heavy waters and three different samples of light waters at sensitivity levels (amount of metal in a 20-pL sample which gives an absorbance of 0.2) from 0.07 pg/mL (Ni) to 0.004 pg/mL (Zn). Table I gives the data obtained. The samples were stored in identical glass containers and injected using polypropylene pipet tips and a “Pipetman” adjustable pipet. The fresh tips were rinsed 3 times in each sample before injecting that sample. The conclusions are: sample 6 is contaminated with Fe, Cu, and Zn (and to a lesser, but still detectable level, with Ni); sample 5 is contaminated with Zn; sample 4 (ordinary quality D20) is uncontaminated by any of these metal ions. The highest paramagnetic ion contamination in any of the D 2 0 samples is a t the 0.3-pM level and thus under the levels which have been reported to cause experimental difficulties ( 3 , 5 ) in some NMR work. However, levels of this order of magnitude have been reported to cause problems in T I investigations of molecules which complex metal ions strongly, e.g., imidazole residues in peptides and proteins (6). Furthermore, in exchange procedures involving lyophilization from large volumes of D20, it is obvious that significant increases in the paramagnetic ion content of the product are incurred. It is doubtful that effects mentioned in the literature concerning much larger amounts of paramagnetic impurities in D 2 0 than we find are due to the existence of the ions in the solvent before their containers are opened in individual laboratories. Certainly, the use of “low paramagnetic impurities” grade D,O by NMR workers on grounds of lowered
paramagnetic ion content is without justification. Furthermore, researchers in biochemical areas who use D,O for any purpose should be aware of the relatively high zinc ion contamination of some heavy waters since this ion forms complexes with many important biopolymeric species. Finally we have found (in unpublished work) that a major source of paramagnetic ion contamination of some species under NMR investigation comes from lyophilization or crystallization of samples from large volumes of solvents such as reagent grade glacial acetic acid.
LITERATURE CITED (1) Cutneil, J. D.; Glasel, Jay A.; Hruby, V. J. Org. Magn. Reson. 1975, 7, 256. (2) Bleich, H. E.; Cutnell, J. D.; Glasel, Jay A. Biochemistry 1976, 15, 2455. (3) Pearson, H.; Gust, D.; Armitage, I . M.; Huber, H.; Roberts, J. D.; Stark, R. E.;Vold, R . R.; Vold, R. L. Proc. NaN. Acad. Sci., U . S . A . 1975, 72, 1599. (4) Hilton, B. D.; Bryant, R. G. J . Magn. Reson. 1976, 21, 105. (5) Cohen, J. S.; Bradley, R. B.; Clem, T. R. J. Am. Cbem. SOC. 1975, 97, 908. (6) Wasylishen, R. E.: Cohen, J . S . Nature (London) 1974, 249, 847.
Jay A. Glasel
Department of Biochemistry University of Connecticut Health Center Farmington, Connecticut 06032 for review April 2, 1979. Accepted May 21, 1979. This work was supported by NSF grant PCM 78-08543. RECEIVED
Chemical Ionization Mass Spectrometry of Nonvolatile Organic Compounds Sir: One of the barriers to wider use of bioanalytical methods based on mass spectrometry is the widespread occurrence of compounds of biologic importance which are nonvolatile. These compounds usually have one or more of the following characteristics: (a) an ionic functional group (or groups) that prevents volatilization without decomposition or structural alteration, (b) numerous nonionic functional groups leading to intermolecular hydrogen bonding of sufficient strength so that functional group elimination or decomposition occurs before volatilization when the compound is heated, and (c) a molecular size that leads to bond cleavage, rather than vaporization, as a consequence of heating. Typical 0003-2700/79/0351-1858$01.00/0
compounds of type a contain an amino group(s) and a strong acid group, or a quaternary amino group with an associated inorganic negative ion. The conversion of a compound containing a protonated amino or a quaternary amino group to a gas phase positive ion might occur through reaction a t the gas-solid interface with appropriate gas phase positive ions-not by altering the ionic structure of the amino group, but by reaction with the negative ion in the molecular structure. The ionization of a type b highly hydrogen bonded neutral compound to a positive ion, by reaction with an appropriate gas phase ion, might also occur; the reaction should result in formation of a product ion with an intact C 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 11, SEPTEMBER 1979
100
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Figure 1. Mass spectrum (ammonia)of taurine (2-aminoethanesulfonic acid). Glass probe extension,with SE-30 film, solid sample in source, source temperature 260-310 O C . Ions at m l z 126 and 251 correspond to MH' and M,H+
CI
200
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250
300
Figure 2. Mass sepctrum (ammonia)of betaine hydrochloride (M'CI-). Glass probe extension, with SE-30 film, solid sample in source, source temperature 200-270 O C . The ion at m l z 118 corresponds to M;' the ion at m l z 104 corresponds to MH+ for N,Ndimethylglycine;and t h e ion at m l z 132 is due to the methyl ester as an M' ion
molecular structure, or in ions showing functional group elimination (dehydration, for example). It is unlikely, however, that compounds of type c would yield gas phase ions even if positive ions were formed on the surface of the solid as a result of reaction a t the gas-solid interface. The purpose of this report is to describe a technique for obtaining mass spectra of nonvolatiles which does not require use of an emitter probe or physical alteration of the source, and which can be used for solids without the possibility of sample loss due to lack of adhesion to a Teflon, glass, or metal surface. The results shown in Figures 1-3, and previous literature reports (1-7), indicate that compounds of types a and b will give mass spectra as a result of plasma reaction a t the sample interface. Figure 1 shows the mass spectrum of taurine (2-aminoethanesulfonic acid) generated by reaction with gas phase ammonium ions. The major product ion, a t m / z 126, corresponds to a protonated molecule. A dimer ion, a t m / z 251, is also present; this corresponds t o an ion containing two molecules of taurine and one proton. Figure 2 shows the mass spectrum of betaine hydrochloride (trimethylcarboxymethyl ammonium chloride). Two types of ion products are formed. The ion a t m / z 118 is the quaternary ammonium ion corresponding to the molecular structure without the chloride ion. T h e second, at m / z 104, is a protonated ion derived from N,N-dimethylglycine (formed from betaine by thermal N-demethylation). An ion due to the methyl ester (a product of the thermal reaction), is also present a t m / z 132. Figure 3 shows the mass spectrum of sucrose, a compound of type b, with isobutane as the reagent gas. The ions a t m / z 343,325,307,289, and 271 correspond to MH' and successive losses of water. In these studies, different types of probe tips were employed with the same results. Both glass and copper rod extensions were made to fit the tip of a standard heated probe for a Finnigan 1015 quadrupole mass spectrometer with a chemical SUCROSE
150
I00
ionization source. The surface of the extended tip was coated with a film of SE-30 (a 2% solution in isooctane was used), and the sample was placed on the surface of the siloxane film. The solid sample (single crystal or powder) was approximately centered in the ion source, with the probe entering from the side. Siloxane films were shown in an earlier study (8) to provide a surface free from adsor-ption effects for polar substances. Baldwin and McLafferty ( I ) obtained chemical ionization spectra of oligopeptides by direct exposure of the sample in the ion source. Holland, Soltmann, and Sweeley (2) and Soltmann, Sweeley, and Holland (3) found that an electrical field was not necessary to ionize samples from a field desorption emitter under field desorption/electron impact conditions. Hunt and his colleagues ( 4 ) used a field desorption emitter in this way under methane chemical ionization conditions, with direct insertion of the emitter in a modified source, and obtained positive ions with intact molecular structures for a number of nonvolatile compounds. In a related study with several nonvolatile compounds, Hansen and Munson ( 5 ) employed a Teflon probe tip, inserted into a CI source, with isopentane, methane, and nitrogen as reagent gases. Cotter and Fenselau (6) and Cotter (7) used tungsten and Vespel probe tips. I t was pointed out by Hansen and Munson ( 5 ) that an observed mass spectrum will vary with the conditions when thermal degradation is a competing reaction. In this study, the conversion of betaine t o N,Ndimethylglycine and to betaine methyl ester were competing thermal reactions (a similar effect was noted by Hunt ( 4 ) for choline chloride). These effects may or may not lead t o difficulties in quantitative analyses, depending upon the choice of internal standard. Our initial studies indicated that the composition of the probe extension was not a critical matter, as is suggested by
1
( I S O B U T A N E ) 161111 COPPER PROBE EXTENSION
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Figure 3. Mass spectrum (isobutane)of sucrose. Copper probe extension, with SE-30 film, solid sample in source, source temperature 220-260 OC. Ions at m l z 343, 325, 307, 289, 271 correspond to MH', MH' - H 2 0 , MH' - 2H20, MH' - 3H,O and MH' - 4H20
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 1 1 , SEPTEMBER 1979
the materials that have been used, but at times erratic results were obtained with all probe extensions. This effect was considered to be due to localized charges on films on the extension. A metal probe tip was then employed, with source and probe circuitry changes so that the ion source voltage was applied to the probe extension (9). No further difficulties were encountered. The currently preferred method of operation is to use an unheated source with a gold or copper probe extension for both thermal and electrical conduction, and to heat the sample by probe heating. The source may be heated if desired. I t should be noted that the controversy over field desorption mass spectra obtained without an applied field is not related to plasma desorption ionization (IO),although both techniques are used for studies of nonvolatile compounds. Compounds of type c will probably not yield ionic products under our conditions. The high energy conditions of Macfarlane ( 1 1 ) or the laser ionization techniques described by Meuzelaar and his colleagues (12) may be required in these instances. In this work, and in earlier related studies, it has been considered that ionization occurs by reaction a t a gas-solid or gas-liquid interface. The establishment of methods for the ionization of compounds that are nonvolatile or that undergo partial or total decomposition on heating should significantly extend the range of mass spectrometric bioanalytical applications. I t should be possible to carry out the analysis of liquid chromatographic effluent streams without pre- or post-column derivative formation. Further, if samples need not be volatilized in order to obtain CI spectra, new approaches can be made to many bioanalytical problems. The nature of the supporting surface is important in some instances; this is suggested by this work and by the studies of Friedman and his colleagues (13, 14) and Munson (5) on Teflon surfaces. A siloxane film has the advantage of providing an adhesive ill hold solids but that will not lead to adsorption surface that w losses of polar compounds.
LITERATURE CITED 61) Baldwin. M. A.: McLaffertv. F. W. Ora. Mass Soecbom. 1973. 7. 1353-1356 (2) Holland, J F , Soltmann, B Sweeley, C C B~omedMass Specfrom 1976, 3, 340-345 (3) Soltmann, B , Sweeley, C C , Holland, J F Anal Chem 1977, 49, 1164-1166. (4) Hunt, D. F.; Shabanowitz, J.; Botz, F. K.; Brent, D. A. Anal. Chem. 1977, 49, 1160-1163. (5) Hansen, G.; Munson, €3. Anal. Chem. 1978, 50, 1130-1 134. (6) Cotter, R. J.; Fenselau, C. C. American Society for Mass Spectrometry, 26th Annual Conference, St. Louis, Mo., May 28-June 2, 1978; Abstracts, p 672. (7) Cotter, R. J. Anal. Chem. 1979, 5 1 , 317-318. (8) Thenot, J-P.; Nowlin, J.; Carroll, D. I.; Montgomery, F.; Horning, E. C. Anal. Chem., 1979, 5 1 , 1101-1104. (9) Carroll, D. I.; Nowlin, J. G.; Montgomery, F. E.;Stillwell, R. N.; Horning. E. C.American Society for Mass Spectrometry, 27th Annual Conference, Seattle, Wash., June 3-8, 1979. (IO) Beckey, H.D.;Rollgen, F . W. Org. Mass Specfrom. 1979, 14, 188-190. (11) Macfarlane, R. D.;Torgerson, D. F. Science 1976, 191, 920-925. (12) Posthumus, M. A.; Kistemaker, P. G.; Meuzelaar, H. L. C.: Ten Noever de Brauw, M. C. Anal. Chem. 1978, 50, 985-991 (13) Beuhler, R. J.: Flaniqan, E.; Green, L. J.; Friedman, L. Biochemistw1974. 13, 5060-5068 (14) Beuhler, R J , Flanigan, E , Green, L J , Friedman L J Am Chem SOC 1974, 96, 3990-3999
D. I. Carroll I. Dzidic M. G. Horning F. E. Montgomery J. G . Nowlin R. N. Stillwell J-P. Thenot E. C. H o m i n g * Institute for Lipid Research Raylor College of Medicine Houston, Texas 77030 Received for review December 15, 1978. Accepted June 20, 1979. This work was supported by Grants GM 13901 and GM 24092 of the National Institute for General Medical Sciences, and by Grant Q-125 of the Robert A. Welch Foundation.
AIDS FOR ANALYTICAL CHEMISTS Representation of Extraction Efficiencies Winston K. Robbins Exxon Research and Engineering Company, P.O. Box 121, Linden, New Jersey 07036
Extractions are widely used for analytical sample preparation. This paper presents a simple technique which can aid the analyst in the selection of extraction conditions. Theoretically, the best way to compare different extraction systems is through the use of distribution coefficients (KD) of the compound of interest. In a liquid-liquid extraction, a solute is distributed between two immiscible phases-the solvent (S)and the extractant ( E ) . The equilibrium may be expressed mathematically by the Nernst distribution law:
where K D is the distribution coefficient for the solute and CE and Cs are the concentrations in the two phases. The efficiency for an extraction is measured by E , the fraction extracted. From separations theory, E is related to K D by the equation 0003-2700/79/0351-1860$01 OO/O
where VE equals volume of the extractant, Vs equals volume of the solvent, and n equals the number of times the solvent phase is equilibrated against fresh extractant. Thus, a knowledge of the distribution coefficients allows comparison of different extraction procedures. Distribution coefficients for many aqueous systems have been tabulated in an extensive review by Leo ( I ) . The K D values for other systems may be conveniently determined by the techniques of Bowman and Beroza (2-4) or Berezkin and co-workers ( 5 ) . Since the relationship between K D and % E is cumbersome and difficult to visualize, most extraction conditions are 1979 American Chemical Society
