Iron. The oxidation of iron(I1) to iron(II1) is known to be reversible a t a mercury electrode (2). Slopes consistent with a reversible polarographic wave were found in this work. The reduction of iron(I1) occurs at about -1.3 V ( 4 ) and is not reversible (slope = 70 mV). In addition, the potential for the iron(I1) reduction is about the same as the reduction potential for the bilirubin wave, and thus cannot be studied. As a result, only the oxidation wave could be studied. Both iron(I1) and iron(II1) could form complexes with bilirubin. In reference ( I ) , the CM/CBR ratio varies from 0.3 to 6 with only two values greater than one. In addition, the reduction peak occurs at about -0.40 V vs. SCE, about 900 mV positive of other reported work. It is most probable that what was seen was the reduction of iron(II1) which was formed from the oxidation of iron(I1) while sitting at the initial potential before initiating the voltage scan (a hanging drop electrode was used). The data for the oxidation of iron(I1) as a function of the concentration of bilirubin are given in Table 11. The Ell2 values are independent of the concentration of bilirubin but are shifted to more positive potentials with an excess of bilirubin. This indicates that both iron(I1) and iron(II1) complex strongly with bilirubin. The shift in potential is equal to:
from the data and is equal to 175. The exact value of KF~(II) cannot be found unless CM is reduced significantly so that lower values of CBR can be used. But it can be estimated that KFe(I1) is greater than 1 X 104M-' and thus K F ~ ( I IisI ) greater than 2 X 1O6M-'.
CONCLUSION When the formation constants for bilirubin are performed under the conditions prescribed by the voltammetric theory, the values obtained are much less than originally measured. The exact causes of the shifts especially for cadmium are unknown but may be due to the use of the hanging mercury drop electrode. Filming and absorption can affect results dramatically, especially if the drop is not changed every time. No mention of whether the drop was changed is given. Polarographic determinations are usually simpler because of the fact that a new, clean drop is formed every time. In general, unless one is willing to go to considerable more work, formation constants can be accurately determined only for ions that are reduced reversibly and for conditions where the amount of ligand is much greater than the amount of metal. Deviations from these conditions lead to unreliable values of the formation constants. LITERATURE CITED
((1) J. D. Van Norman and M. M. Humans, Anal. Chem., 46,926 (1974). (2) M. Goto and K. B. Oldham, Anal. Chem., 45, 2043 (1973). (3) R . S. Nicholson, Anal. Chem., 37, 1351 (1965). (4) J. Heyrovsky and J. Kuta, "Principles of Polarography", Academic Press, K F ~ ( I I ) New York, 1966, p 537.
where KF~(III) is the Fe(II1)-BR formation constant, is the Fe(I1)-BR formation constant and CBR is the surface concentration of bilirubin. If KFe(1I)CBR >> 1 and KFe(1II)CBR >> 1, then AE1/2
-0.059 KFe(II1) = -log -
n
KFdII)
Michael D. Ryan Todd Wehr Chemistry Building Marquette University Milwaukee, Wis. 53233
(3)
Only the ratio of KF~(III)/KF~(II) can be found accurately
RECEIVEDfor review January 16, 1975. Accepted May 5 , 1975.
Comments on the Search for Amino Acids in Apollo 15, 16, and 17 Lunar Samples. A Study in Contamination Control Sir: Lunar samples from the Apollo 11-17 missions have been extensively examined for trace amounts of low molecular weight carbon compounds (mainly CH4, CO, and C o d , and to a lesser degree for traces of amino acids or their precursors. However, the presence and/or origin of amino acids in lunar samples is still controversial. The purpose of this correspondence is to try to point out some of the salient findings to hopefully minimize uncertainty and to invite comments on this subject. Amino acid analyses of samples returned during the Apollo 15, 16, and 17 missions will be discussed because the analytical techniques in these experiments were generally improved from experience gained by working with samples from earlier missions. I t is interesting to note that various investigators recovered very small but significantly different quantities (from 5 to 213 ng/g) of amino acids from lunar samples (2-3 and Table I). It was observed ( I ) that when 500 mg of an Apollo 15 sample was spiked with 100 ng of a mixture of amino acids, the recovery range of these compounds varied between 0 to 64% as determined by gas-liquid chromatography and ion exchange chromatography ( I ) . It was suggested ( 2 ) that glycine in an Apollo 17 SESC sample (Le., Surface Environmental Sample Container) was derived from 1718
ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
rocket exhaust. On the other hand, it was also suggested (3, 4 ) based on the examination of various Apollo samples that amino acids were derived from precursors in lunar fines and that the rocket exhaust of the Lunar (descent) Module did not cause (3) extensive synthesis of amino acid precursors. The analysis of the descent engine exhaust products of a Lunar Module ( 5 ) revealed no amino acids but showed the presence of at least 53 rocket exhaust products, some of which could be precursors of amino acids, e.g., HCN, CH3NH2, CHO, etc. A core sample brought back from the Moon during the Apollo 15 mission was found to be substantially contaminated with amino acids (6). Examination of the palmar tactile surfaces showed no amino acids while a connecting ring joint and the interior of an astronaut's training glove showed an amino acid distribution completely unlike that found in lunar samples but which was characteristic of hand contaminations ( 7 ) . The possibility has also been raised (among others, 4, 7 ) that the lunar amino acids may be related to solar wind implantation. Solar wind derived hydrogen in lunar samples is well documented. Solar wind derived carbon has also been established. CH4 is concentrated inside the surface layer of "non-mature" lunar soil grains (8).It was also suggested that solar wind
Table I. Amino Acids in Hydrolyzed Aqueous Extract of Apollo 15 and 17 Lunar Samples Apollo 15
S a m p l e No. Weight Vol. of water for e x t r a c t i o n A m i n o acids
Apollo 17
C o n t a m i n a t i o n s in astronaut's training glove
15012,4" 5.993g
15013,4* 6.0012g
15498,3' 5.3704g
70011,35d 9.0519g
78501,2ge 4.9518g
5 ml/g
5 m1,'g
5 ml/g
5 ml/g
5 ml/g
Units: ng l g
1 cm2 area, outside palmar s u r f a c e of glove'
Units: n g l g
Units: n g l g
Inside s u r f a c e of glove n e a r retaining r ingg Units: n o r m a l ized t o ornithine
17 0.5 n.d. 0.32 15 4 n.d. Aspartic acid 4 7 n.d. 2 2 n.d. 3.28 Threonine 9 27 n.d. n.d. 3.45 Serine n.d. n.d. 19 44 6 n.d. 2.87 5 7 G l u t a m i c acid 74 115 21 n.d. 6 18 4.88 Glycine 4 9 24 n.d. 2.21 1 6 Alanine n.d. 6 n.d. n.d. Valine 0.25 n.d. n.d. 12 8 n.d. n.d. 2 7 0.58 Leucine 5 3 n.d. n.d. Isoleucine n.d. n.d. 0.41 n.d. 5 n.d. n.d. n.d. n.d. Tyrosine 0.13 n.d. n.d. Phenylalanine n.d. n.d. 10 n.d. 0.31 13 n.d. n.d. n.d. Ethanolamine n.d. 7 0.32 4 n.d. n.d. Methylamine 1 3 n.d. n.d. n.d. 6 7 n.d. n.d. n.d. Lysine 0.29 Histidine n.d. n.d. n.d. n.d. n.d. n.d. 0.58 1 n.d. n.d. Ornithine n.d. 1 n.d. 1.oo Citrulline n.d. n.d. n.d. n.d. n.d. n.d. 0.22 Total 14 55 212 213 47 n.d. a Fines collected at Station 6; 5 km from LM. Surface Environmental Sample Container (SESC, i.e., sealed on the Moon and opened in an ultra-clean laboratory). * Fines collected beneath the Lunar Module (LM), SESC sample. Interior of a lunar rock (breccia with mare basalt clasts) collected a t Station 4;3.5 km from the LM. Pulverized in mullite mortar in the laboratory. Fines collected beneath the LM, SESC sample. e Fines collected a t Station 8; 2 km from the LM. 1 cmz area of glove washed with water (25 ml) which was then hydrolyzed and analyzed (13).g Glove stood in 200 ml of water up to the ring and maintained at 40 O C for 30 min. The water was centrifuged and 100 ~1 of it was then hydrolyzed and analyzed (13). Not detected.
nitrogen impinges in a chemically active form (plasma state) and probably reacts with hydrogen and other elements forming nitrogen compounds on lunar grain surfaces (9). Another problem with lunar amino acid studies is contamination during analysis. It was shown that amino acids, proteins, and bacterial contamination may be present in distilled water, NH40H ( I O ) and HC1 ( I O , I I ) as well as in glacial acetic acid (12). In view of the conflicting findings and experimental difficulties, the presence of amino acids in lunar samples becomes highly questionable.
EXPERIMENTAL In an attempt to clarify the problem of the presence of amino acids in lunar samples, the following results may be considered. T h e data were obtained by techniques described previously (13). Due attention had been paid to the prevention of terrestrial amino acid contaminations, the sources of which have been well documented by ion exchange chromatography ( I O , 11, 14, 15) and by gas chromatography (16, 17). Table I shows the results of ion exchange chromatographic analyses of hydrolyzed aqueous extracts obtained from Apollo 15 and 17 samples. There are two aspects worth noting in this table. (a) There seems to be an indication of an abiologically synthesized suite of amino acids such as reported by Miller (18) and by Oro (19). Lemmon (20) has provided a very informative review of the subject. Generally, glycine was the most abundant, while alanine, glutamic acid, and other amino acids in lesser amounts have been reported. This assemblage seems to contain some biological (terrestrial) contaminations. (b) The Apollo 15 and 17 results seem to be contradictory. In the Apollo 15 samples, the amino acid content does not show a distinct relationship with the distance from the Lunar Module. In the Apollo 17 samples, the reverse may be the case. This relationship has been independently confirmed (21j,
CONCLUSIONS On the basis of available data, one can only say that if amino acids are present in lunar samples, the majority of them do not have the distribution pattern, type, or variety
usually associated with biological origin, i.e., they are not contaminations from terrestrial biological sources. It is the authors' opinion that it is not possible with the amounts of lunar samples that have been made available to date to determine if amino acids are indigenous to the Moon either in free, in complexed, or in precursor forms. The Apollo 17 and, to a lesser extent, the Apollo 15 results seem to suggest an origin from the rocket exhaust, but an origin due to solar wind implantation reactions, meteorite impacts, etc., cannot be ruled out until extensive simulation experiments are conducted in the laboratory.
LITERATURE CITED (1) C. Ponnamperuma, C. W. Gehrke, R. W. Zumwait. and C-N Cheng, "Lunar Science IV", J. W. Chamberlain and C. Watkins, Ed., The Lunar Science Institute. Houston, 1973, p 595. (2) C. W. Gehrke, R. Zumwalt, K. Kuo. C. Ponnamperuma, A. Shimoyama, M. Gay, R. Pal, and P. Buhl, "Lunar Science V", The Lunar Science institute, Houston, 1974, p 263. (3) S. W. Fox, K. Harada, and P. E. Hare, "The Apollo 15 Lunar Samples", J. W. Chamberlain and C. Watkins, Ed., The Lunar Science Institute, Houston, 1972, p 299. (4) S. W. Fox, K . Harada, and P. E. Hare, "Lunar Science iV", J. W. Chamberlain and C. Watkins, Ed., The Lunar Science Institute, Houston, 1972, p 260. (5) D. A. Fiory and B. R. Simoneit, Report, "For The Organic Gas Analysis
Group, The Action Committee on Organic Contamination and The Lunar Sample Analysis Planning Team (1969). I '
(6) K. Harada, P. E. Hare, and S.W. Fox, "Lunar Science IV", J. W. Chamberlain and C. Watkins, Ed., The Lunar Science Institute, Houston, 1973, p 333. (7) B. Nagy, P. B. Hamilton, and H. C. Urey, 15th COSPAR Meeting, Madrid, Spain (abstracts), paper k. 32, 252 (1972). (8) D. J. DesMarais, J. M. Hayes, and W. G. Meinschein, Nature (London), Phys. Sci.. 246, 65 (1973). (9) 0. Muller. "Analyse extraterrestrischen Materials", W. Kiesi and H. Malissa, Ed., Springer-Verlag, Wien, 1974, p 53. (10) P. B. Hamiiton and T. T. Myoda, Clin. Chem., 20, 687 (1974). (11) P. E. Hare, Ann. Rep. Dir. Geophys. Lab., Carnegie lnst., 232 (1964-
65).
(12) L. St. Onge and P. B. Hamilton, unpublished results, 1974. (13) V. E. Modzeleski, J. E. Modzeieski, M. A . J. Mohammed, L. A. Nagy, B.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975
1719
(14) (15) (16) (171 il8j (19) (20) (21)
Nagy, W. S . McEwan. H. C. Urey, and P. B. Hamilton, Nature (London), Phis. Sci., 242, 50 (1973). P. B. Hamilton, Nature (London), 205, 284 (1965). P. B. Hamilton and B. Nagy, Space Life Sci., 3, 432 (1972). J. J. Rash, C. W. Gehrke, R. W. Zumwalt, K. C. Kuo, K. A. Kvenvolden, and D. L. Stalling, J. Chrornatogr. Sci., I O , 444 (1972). C. W. Gehrke. Soece Life Sci.. 3. 342 11972). S. L. Miller, J . ' A h Chern. Soc, 77, 2351 (1955). J. Oro, Nature(London), 197, E02 (1963). R. M. Lemmon, Chern. Rev.. 70, 95 (1970). P. E. Hare, personal communication, 1974.
Paul B. Hamilton Alfred I. du Pont Institute Box 269, Wilmington, Del. 19899
Bartholomew Nagy Laboratory of Organic Geochemistry Department of Geosciences The University of Arizona Tucson, Ariz. 85721
RECEIVEDfor review February 11, 1975. Accepted May 15, 1975. The analytical work reported in Table I was supported by NASA Grant NGR 03-002-237 and by the A. I. du Pont Institute.
I AIDS FOR ANALYTICAL CHEMISTS Simple Interface for Automated Mass Fragmentography with a Standard Laboratory Data System John M. Strong' and Arthur J. Atkinson, Jr. Clinical Pharmacology Unit, Departments of Pharmacology and Medicine, Northwestern University Medical School, Chicago, 111. 606 7 7
Robert J. Ferguson Hewlett-Packard, Skokie, Ill. 60076
Several data systems are now commercially available to process the large amount of qualitative analytical information that can be generated rapidly with a combination gas chromatograph-mass spectrometer (GC-MS). These expensive systems are also capable of selectively monitoring only a few mass spectral ions during a gas chromatographic run, thus permitting quantitative as well as qualitative analysis by the technique termed mass fragmentography (1). However, the data bandling requirements for conventional GC-MS and mass fragmentography are quite different. Particularly when a low-cost GC-MS is committed primarily to quantitative analysis, it may be advantageous to analyze the results with a correspondingly inexpensive, general purpose laboratory data system. This report describes a simple device for interfacing a Finnigan Model 3000 GC-MS, equipped with a modified Finnigan Model 240-01 Automatic Peak Selector (APS), to a Hewiett-Packard Model 3352 Laboratory Data System. The resulting GC-MS-APS-Data System is well suited for the repetitive operations involved in analyzing a large number of samples for a given compound, or several compounds, on a semi-routine basis. This system offers the added advantage of a greater degree of automation in data handling than is provided by current commercial GC-MSData Systems.
EXPERIMENTAL T h e operation of the GC-MS and APS for quantitative mass fragmentography has been described previously (2). As shown in Figure 1, the interface feeds signals from the APS outputs to four individual single channel analog-to-digital (A/D) converters (Hewlett-Packard Model 18652 A). Each channel of the APS can be focused on a separate mass spectral ion during chromatography and a visual display is presented hy a four-pen recorder (Model KA-40, Rikadenki Kogyo Co., Tokyo, Japan) connected to the interface. The interface consists of four channels, each containing the cirAuthor to whom correspondence should be addressed a t 303 E. Superior St., Chicago, Ill. 60611. 1720
ANALYTICAL CHEMISTRY, VOL. 4 7 , NO. 9, AUGUST 1975
cuits shown in Figure 2. A dc bucking voltage is applied to the signal from the APS to zero the background output from each APS channel. A ten-turn potentiometer serves as the bucking control by varying this voltage continuously over the range of 0 to 6 volts. The signal is then divided and fed in parallel, through filters that minimize channel switching noise, to separate output jacks for the computer and recorder. A 1O:l voltage divider a t the computer output jack matches the output voltage of the APS to the input specifications of the A/D converter. T h e 3352 B Laboratory Data System prints out the mass fragmentographic peak retention times and peak areas from each APS channel on a Hewlett-Packard 2752 A Teleprinter. Teleprinter maintenance has been reduced by installing a Model 312 A 0541 Dual-input Idle Line Motor Control (United Data Services Co., Phoenix, Ariz.) to switch off the teleprinter automatically in the intervals when no data are being printed. Qualitative identification of each compound of interest is confirmed by its printed chromatographic retention time and the relative intensity of two mass spectral ions characteristic for that compound, as indicated by the computed area ratio of the two mass fragmentographic peaks. T h e area of the base peak from each compound being measured is compared with t h a t of a reference compound added to the sample a t the start of the analysis for quantitation by the internal standard method.
RESULTS AND DISCUSSION The operation of the combined GC-MS-APS-Data System was evaluated by analyzing lidocaine samples by a mass fragmentographic method that has been described previously (2).Lidocaine samples ranging from 6 to 300 ng were injected on the gas chromatographic column together with 300 ng of trimecaine as the internal standard. Since the base peak of both the trimecaine and lidocaine mass spectra occurs a t mle 86 and both compounds have mle 120 as a fragment ion, these ions were monitored by the APS. For each sample, the precision of the lidocaine content estimated by the data system print-out of peak area was compared with that obtained from the same chromatographic runs by manually measuring the height of the mass fragmentographic peaks displayed by the recorder. Manual measurements gave a precision of f3.97% (& std dev) for
