Studies of Arenetrica rbonylch romium Derivatives by Gas-Liq uid Chromatography and Nuclear Magnetic Resonance Catherine Segard, Bernard Roques, Claude Pommier, and Georges Guiochon Department of Chemistry, Ecole Polytechnique, Paris, France Thirteen benzenetricarbonylchromiums have been prepared and analyzed by gas chromatography between 80 and 180 O C . Three derivatives are new: n-propyl-, n-butyl-, and isobutylbenzene complexes. No decomposition occurs in the chromatographic system as shown by the mass spectra of the eluted compounds. Apiezon L and SE-30 greases are suitable for these analyses; all the complexes can be resolved from each other except o-xylene and ethylbenzene derivatives. The NMR study shows that the temperature has an important effect on the conformational equilibria between the eclipsed and noneclipsed conformers. However, because of a sterical hindrance of rotation, the ferf-butylbenzene complex exists up to 35 O C in a single noneclipsed form. The spectra of polysubstituted complexes are in accordance with the electronic effects of the substituents.
THESE PAST
YEARS, organometallic compounds and particularly arene derivatives of metalcarbonyles have received great attention. Gas-liquid chromatography has been used recently for the analysis of some of these complexes ( I ) , and mass spectrometry provides a suitable method for the identification of the eluted compounds (2). Nuclear magnetic resonance spectra of these complexes have been investigated in several papers (3-8) in order to elucidate the structure and a controversy has opposed several authors regarding the restricted rotation in arenetricarbonylchromium derivatives ( 4 , 6-8). An equilibrium between two preferential conformers (I and 11) of the monosubstituted benzenetricarbonylchromiums is usually accepted.
Cb
I
II
For Jackson et al. ( 4 , 6) this equilibrium is temperature dependent and can be observed by NMR spectrometry. For Barbieri and Taddei (7), the change in NMR patterns with temperature cannot be related to an exchange between the two conformers. For other authors, no variation with temperature was observed in the proton chemical shifts of these complexes (8). (1) H. Veening, N. J. Graver, D. B. Clark, and B. R. Willeford, ANAL.CHEM.,41,1655 (1969). (2) W. J. A. Van den Heuvel, J. S. Keller, H. Veening, and B. R. Willeford, Anal. L e f t . ,3 , 279 (1970). (3) R. V. Emanuel and E. W. Randall, J. Chem. SOC.( A ) , 1969, 3002. (4) W. R. Jackson, W. B. Jennings, S. C . Rennison, and R. Spratt, J. Chem. SOC.( E ) ,1969,1214. (5) D. E. F. Gracey, W. R. Jackson, W. B. Jennings, S. C. Rennison, and R. Spratt, ibid.,p 1210. (6) W. R. Jackson, W. B. Jennings, and R. Spratt, Chem. Commun., 1970, 594. (7) G. Barbieri and F. Taddei, ibid.,p 312. (8) T. F. Jula and D. Seyferth, Znorg. Chem., 7, 1245 (1968). 1146
In this work, we shall describe the preparation of 13 derivatives of benzenetricarbonylchromium, their analysis by gas chromatography and mass spectrometry, and their NMR spectra run either isothermally at different temperatures or at variable temperature. These analylical data will then be used to discuss the structure of these compounds. EXPERIMENTAL The complexes have been prepared by the method described by Nicholls and Whiting (9): an excess of arene mixed with hexacarbonylchromium is refluxed in diglyme for several hours under nitrogen. The extraction of the organometallic compounds was made as described in (9) but we found that silica gel (70-325 mesh, Merck) gives better results than the proposed alumina for the chromatographic separation of the complex from the excess of free arene. Further purifications are obtained either by crystallization in hexane or by sublimation under vacuum. Melting points are measured in glass capillaries with a Biichi apparatus. The mass spectra were recorded on a Varian C H 7 spectrometer. The gas chromatographic experiments have been carried out on a Girdel chromatograph (Giravions Dorand, 92Suresnes, France) equipped with a flame ionization detector. The two columns used (2 mm i.d., 1.5 m long) are filled with Chromosorb W, 80-100 mesh, acid washed, and coated with 10% SE-30 or Apiezon L grease. The weights of stationary phases contained in the columns are 0.290 gram and 0.300 gram of SE-30 and Apiezon L greases, respectively. The compounds are injected either pure, with a solid sampler (Hamilton) or as a benzenic solution (about 40 grams/]. of each solute) with a microsyringe. Each retention volume is calculated from the results of three or four experiments. NMR spectra were recorded at 60 MHz and 100 MHz with Varian A-60 and HA-100 spectrometers. Proton shifts in free arenes and complexes have been measured from solutions about 5 % w/w in CDCI3, with tetramethylsilane as internal reference and locking signal. Small signals separations were measured by the use of an expanded SCdk and a slow sweep rate. The probe temperature was calibrated using methanol sample in the temperature range -60 OC to f40 "C (IO). The variable temperature experiments have been performed with a Varian V-6040 controller. RESULTS AND DISCUSSION
Newly Synthesized Compounds. We have prepared the 13 arenetricarbonylchromium complexes reported in Table I. The values of the melting points of these compounds are in good agreement with the values found in the literature (4, 9, 10-13), except that we found no mention of three of these (9) B. Nicholls and M. C. Whiting, J . Chem. SOC.,1959, 551. (10) Varian Associates, Variable Temperature System Manual, V 4341, Palo Alto, Calif., 1969. (11) W. R. Jackson, B. Nicholls, and M. C. Whiting, J . Chem. SOC.,1960, 469. (12) E. 0. Fischer, K. Ofele, H. Essler, W. Frohlich, J. P. Mortensen and W. Semmlinger, Chem. Ber., 91,2763 (1958). (13) G. Natta, R. Ercoli, and F. Calderazzo, Chim. Znd. (Milan), 40, 287 (1958).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971
Table I. Specific Retention Volumes (V,) and Relative Retentions (CY)of Arenetricarbonylchromium Complexes on SE-30 and Apiezon L Greases SE-30 Apiezon L Peak Number v,,ml CY V,, ml CY 1 1 385 1 1150
Ligand Benzene Toluene p-Xy lene rn-Xylene o-Xylene Ethylbenzene Mesitylene (1,3,5trimethylbenzene) Pseudocumene (1,2,4trimethylbenzene) Cumene (isopropylbenzene) n-Propylbenzene tert-Butylbenzene Isobutylbenzene n-Butylbenzene
2 3 4 5 6
485 510 530 575 585
1.25 1.35 1.40 1.50 1.52
1450 1750 1900 2160 2270
1.25 1.50 1.65 1.90 2.00
7
605
1.57
2490
2.15
8
680
1.75
2640
2.30
9 10 11 12 13
720 845 960 1010 1290
1.85 2.20 2.50 2.65 3.35
2860 3380 3880 4280 5650
2.50 2.95 3.35 3.70 4.80
Table 11. Compositions and Melting Points of New Arenetricarbonylchromium Complexes Composition w/w) Calculated Found C H Cr C H Cr
(z
Ligand n-Propylbenzene n-Butylbenzene Isobutylbenzene
56.25 57.77 51.77
4.68 5.18 5.18
20.29 19.24 19.24
derivatives: n-propyl-, n-butyl- and isobutylbenzenetricarbonylchromium, the preparation of which seems to be described here for the first time. The theoretical and experimental elementary composition as well as the melting points of these compounds are given in Table 11. Otherwise, the new complexes have been identified by their mass spectra. The results agree with the known fragmentation of similar derivatives ( 1 4 ) : successive losses of C O groups give peaks such as [Arene-Cr-(CO),]+ (n = 0, 1, 2, 3). Fragmentations of the alkyl substituent give peaks such as [(p-(CH,),I+ and [(p-(CH,),-Cr]+ (rn = 0, 1, 2, 3 ) . As example, the mass spectrum of n-butylbenzenetricarbonylchromium is given in Figure 1. Gas Chromatographic Study. The gas chromatograph has been coupled with the mass spectrometer. The spectra of the eluted vapors recorded in such conditions show that no
(14) S. Pignataro and F. P. Losing, . I . Orgunornetul. Chem., 10, 531 (1967).
56.43 57.52 57.63
4.59 5.39 5.60
20.41 19.08 19.18
Melting point, "C 42.5-43.5 18-19 40-40.5
decomposition occurs in the chromatographic system, so that the peaks observed do correspond to the elution of the compounds injected. When studying the chromatographic behavior of each pure complex we found that injections of solids lead to the appearance of memory effects on both columns used: when injecting a pure compound A, a peak corresponding to the compound B injected in the previous analysis appears. This effect was not observed with injections of the complexes dissolved in benzene. This procedure thus seems better, although a very slow decomposition of these solutions occurs. Table I gives the specific retention volumes of the arenetricarbonylchromium derivatives on SE-30 and Apiezon L, and their relative retentions to benzenetricarbonylchromium measured at 145 OC on both phases. At the same temperature, the retentions on Apiezon L are about 3 to 4 times greater than the retentions on SE-30 silicone gum. If the logarithm of the specific retention volume of nalkylbenzenetricarbonylchromiums is plotted os. the number of carbon atoms of the substituent group, a straight line is
ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, A U G U S T 1971
1147
9 1
120
110
130
Y
12
NO
150 tempemtun
(od
T
60
40
80
time (mi".)
Figure 5. Analysis of a benzenic solution of the 13 arenetricarbonylchromium complexes on the SE30 column Conditions; column temperature programmed from 80 to 150 "C at 0.8 "C/mn; injection and detection block temperature 220 "C; helium flow rate 30.6 cm3/min(for the peak numbers, see Table I) 102 0 (6)
C Number
, 1
2
(0
(E)
3 (NP)
-
4
(NB)
Figure 2. Specific retention volumes of nalkylbenzenetricarbonylchromium complexes measured on Apiezon L ( 0 ) and on SE-30 (0). The arenes are benzene (B), toluene (T), ethylbenzene (E), n-propylbenzene (NP), nbutylbenzene (NB) obtained (Figure 2). At 145 O C the increment ACH2 in log V , when the carbon number of the side chain is increased by one unit is 0.19 on SE-30 and 0.17 on Apiezon L. These values are closed to the values reported (15) for the logarithm of the relative retention of successive alkylferrocenes on Apiezon L at 150 and 125 OC: 0.15 and 0.18, respectively. (15) C. Pommier and G. Guiochon, Chrornatographiu, 2, 346
(1969).
At constant temperature on the SE-30column, the separation of para- and meta-xylenetricarbonylchromiumsis poor; the complexes of ortho-xylene and ethylbenzene are eluted in the same time and are not completely resolved from the mesitylene complex. The selectivity of the Apiezon L column is greater for the xylene derivatives and the separation of o-xylene and ethylbenzene complexes is improved but the resolution of mesitylene- and pseudocumene-tricarbonylchromiums is bad. Figure 3 shows a chromatogram of a benzenic solution of the 13 derivatives, obtained at 140 "C with SE-30column. As can be seen on Figures 4 and 5 , improved separations are obtained by programming the column temperature. In these conditions, all compounds can be separated from each other on one of the two columns except the o-xylene and ethylbenzene complexes. The analysis of the 13 derivatives on the Apiezon column (Figure 4) is time-consuming and this phase is more specially interesting for a better resolution of the first compounds of the series. However,
It
12
iu
10
4
Figure 3. Analysis of a benzenic solution of the 13 arenetricarbonylchromium complexes on the SE-30 column Conditions: column temperature 140 "C, injection and detection block temperature 220 "C, helium flow rate 30.6 cm3/min(for the peak numbers, see Table I) 9
150
90
163
qrn
iW 120
wmpwotwr 1%
)0(.
4L
c t i r n (min)
Figure 4. Analysis of a benzenic solution of the 13 arenetricarbonylchromium complexes on the Apiezon L column Conditions: column temperature programmed from 80 to 180 "C at 0.8 'C/mn, program started 3 min after injection; injection and detection block temperature 220 "C; helium flow rate 31 cm3/min(for the peak numbers, see Table I) 1148
ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971
~~
Table 111. Chemical Shifts of Monosubstituted Arenes Uncomplexed (6,) and Complexed (Ad, Signal Separation 8 and Relative Population in Conformer I (Conditions: 100 MHz, solvent CDCla, reference TMS, temperature +35 "C) ,.6 ppm 6,, ppm Ligand Protons in free arene in complex A6 = 6. - 6,, ppm 8, ppm PI, n-Propylbenzene CHI 0.91 0.94 -0.03 CHz 1.63 1.57 +0.06 CHr((o) 2.54 2.29 $0.25 5.37 $1.80 Ring protons m. 7.17 5.18 +1.99 0.19 71 . O 0.p. n-Buty lbenzene CHa 0.90 0.94 -0.04 CHz-CHt 1.52 1.57 +O. 05 CHt(Q) 2.58 2.29 +0.29 5.37 $1.80 Ring protons m. 7.17 5.17 +2.00 0.20 72.2 0.p. Isobutylbenzene CHI 0.88 0.92 -0.04 +O. 08 CH 1.84 1.76 CHz 2.46 2.18 +0.28 5.39 +1.78 Ring protons m. 7.17 0.24 76.2 0.p. 5.15 +2.02 1.30 1.27 +O. 03 tert-Butylbenzene CH3 0. 7.29 5.42 +1.87 Ring protonsa m. 7.16 5.14 +2.02 P. 7.03 5.31 $1.72 a At 220 MHz (4).
z
Table IV. Chemical Shifts of Di- and Tri-Substituted Arenes Uncomplexed (6,) and Complexed (6,) (Conditions: 60 MHz, solvent CDCla, reference TMS, temperature +35 "C) L ppm ppm Ligand Protons in free arene in complex A6 = 6, - 6,, ppm Toluene CHa 2.28 2.13 +0.15 (as reference) 5.39 $1.78 Ring protons m. 7.17 5.12 +2.05 0.p. o-Xylene CH3 2.20 2.11 +o. 09 Ring protons 7.05 5.20 +1.85 p-Xylene CH3 2.23 2.08 +O. 15 Ring protons 7.02 5.20 +1.82 +o. 11 m-Xylene CH3 2.25 2.14 Ring proton 5 7.08 5.33 +1.75 2, 4, 6 6.95 4.95 +2.00 2.05 so.11 1,2,CTrimethylCH3 1 2.16 2.08 $0.08 benzene 2 4 2.21 2.13 +0.07 +1.82 Ring protons 3, 5 5.06 6.88 +1.60 5.28 6 +O. 07 2.13 CH3 1, 3, 5 2.21 1,3,5-Trimethyl+1.92 4.83 6.75 benzene Ring protons
it is worth noting that no decomposition seems to affect the peak shape of the more retained solutes. Nuclear Magnetic Resonance Study. We have recorded the NMR spectra of all compounds reported in Table I. The observed proton chemical shifts, when known, are in good agreement with the values given by previous authors (3, 4). We shall discuss the results obtained for the new complexes prepared in this work and for some other compounds in order to study the conformational equilibria; the main results are reported in Tables I11 and IV. In all cases, we observed a very important shielding of the aromatic protons of the complexes compared to the free arene ones (=2 ppm). This phenomenon has been attributed to two factors (3): a large reduction of the paramagnetic ring current produced by the involvement of the ?r electrons in the carbon metal bonds; and an important diamagnetic anisotropy of the Cr(CO)3 moiety increasing the resonance of all the aromatic protons. These two factors are large enough to reverse the deshielding effect resulting from the
reduction of the ?r electron density on the ring protons due to the complex formation. STUDYOF NMR SPECTRA RUNAT AMEIIENT TEMPERATURE. Monosubstituted Benzene Complexes. At 35 "C the spectra of n-propyl-, n-butyl-, and isobutylbenzene show a singlet somewhat broadened or a very compact multiplet corresponding to the ring protons. On the other hand the spectra of the corresponding tricarbonylchromium complexes show an AzBzCsystem for the same protons with a triplet (2 protons by integration) at lower field and a broadened doublet (3 protons by integration) at higher field. Many authors (3, 5 ) have shown from the study of the NMR spectra of arenechromiumtricarbonyl complexes that the protons eclipsed by a carbon monoxide group are relatively more deshielded than the other ring protons. So, we have assigned the lower field triplet to the protons in meta position and the doublet to the protons in ortho and para positions. Consequently, we observe that the n-propyl-, n-butyl- and isobutylbenzene complexes are preferably in the eclipsed
ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971
1149
is given by:
The proportions PI of conformer I in the three studied complexes at ambient temperature have been calculated using the value Omax = 0.45 ppm ( 4 ) and are reported in Table 111: the large excess of conformer I is significant of the stabilization of this conformer by electronic factors. Di- and Tri-Substituted Benzene Complexes. As expected from the symmetry of the ortho- and para-xylene complexes, all the aromatic protons and methyl groups show the same chemical shifts; moreover, the difference A6 between the aromatic proton chemical shifts 61 and 6 2 measured in the free arene and in the complex, is about the mean value of the same differences measured for ortho and para protons in free and complexed toluene rings (see Table IV). This is attributed to the equivalence of the two preferential eclipsed conformers. For electronic reasons, the m-xylene complex has a preferential conformation 111: the protons in position 2, 4, 6 are more shielded than the proton 5. Moreover, the methyl protons have the same chemical shift as those of the toluene complex for which the eclipsed conformer has been shown to be more stable.
1
co
m 5,e
40
5.6
5,4
$2
s (Ppm)
Figure 6. Variation with the temperature of the NMR pattern of the isobutylbenzenetricarbonylchromium in the aromatic protons range Conditions: 100 MHz, solvent CD3COCD3
conformation (I) at ambient temperature, despite the adverse interactions between the alkyl chain and the superimposed carbonyl group. The stabilization of the eclipsed conformer is due to electronic factors: the greatest ?r electron density on the ortho and para positions permits a maximum overlap between the filled 7r orbital of the arene and the metal free orbitals. If an equilibrium fast on the NMR time scale exists between the two conformers (I) and (11) of relative populations PI and PII(PI PIX= l), this equilibrium can be represented by the signal separation 8 measured on the NMR spectrum between the triplet and the doublet centers. From the treatment developed by Jackson et al. ( 4 ) for the toluenetricarbonylchromium, the population PI is related to 8 and Om,, the signal separation for the single conformer I at -273 "C, by:
+
and the equilibrium constant of the exchange equilibrium:
I Ft: I1 1150
(2)
E
P
Similar argumentations of symmetry and electronic effects show that the 1,3,5-trimethylbenzene complex (mesitylene complex) has a preferential conformation (IV) in which all the methyl groups are eclipsed and that the 1,2,4-trimethylbenzene complex (pseudo-cumene complex) is more stable in conformation (V) because of the conjugation of the inductive effects of the 2 and 4 methyl groups on the proton 3. STUDYOF NMR SPECTRA RUNAT VARIABLE TEMPERATURE. In order to clarify the controversy between Jackson et al. (6) and other authors (7, 8) regarding the equilibrium between conformers (I) and (11) and the influence of the temperature on this equilibrium, we have recorded the spectra of the isobutylbenzene and tert-butylbenzene complexes dissolved in CDaCOCD3in the temperature range -60, +35 "C. The influence of the temperature on the NMR spectra of the iso-butylbenzene complex is illustrated in Figure 6. In the same temperature range the spectrum of the iso-butylbenzene shows no significant change. Applying the treatment mentioned above ( 4 ) and using the same value, Om,, = 0.45 ppm, we have calculated the equilibrium constant and the thermodynamic functions of the reaction 2 for isobutylbenzenetricarbonylchromium (Equation 3). The entropy and the enthalpy of the reaction calculated from K values (Table V) are about 0 and -0.54 kcal/mole, respectively. This enthalpy is slightly lower than the values given for methyl-, ethyl- and isopropylbenzene complexes (-0.9 to -0.75 kcal/mole) ( 4 ) , in accordance with the lower inductive effect of the isobutyl group compared to the effects of methyl-, ethyl-, and isopropyl substituents.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 10, AUGUST 1971
Table V. Signal Variation (e) and Equilibrium Constant ( K ) of Isobutylbenzenetricarbonylchromium at Various Temperature (Conditions: 100 MHz, solvent, CDaCOCDs) 253 228 213 308 278 T, "K 0.239 0.253 0.266 0,202 0.223 8 , ppm 3.26 3.57 3.89 2.62 2.96 K
Thus, the unavoidable conclusion, as recognized by Jackson et a!. (4) for the isopropylbenzene complex and in opposition to the conclusions of Barbieri and Taddei (7) and a fortiori to those of Jula and Seyferth (8), is that the modification of the spectra of some arene tricarbonylchromiums must be attributed to a conformational exchange I F? 11. This has been clearly observed here in the case of isobutylbenzene complex. CONCLUSION
The entropy effect AS E-O-isdifferent from the values found for ethyl- and isopropylbenzene complexes but similar to those given for toluenetricarbonylchromium ( 4 ) . This result means probably that the rotation of the isobutyl group is free, just as is the rotation of the methyl group in the toluene complex: it is possible that arrangements of the lateral chain and the larger distance between the ring and the tertiary carbon atom of the side chain minimize the interactions with the superimposed carbonyl group. The spectrum of the tert-butylbenzene complex shows no significant change in the same temperature range -60, +35 "C. This can be explained by a severe restriction of the rotation around the chromium-arene bond: the difference in the para proton chemical shifts measured in the free arene and in the complex at ambient temperature is 1.72 ppm (from chemical shifts in tert-butylbenzene measured at 220 MHz, ref. 4), very close to the value calculated at -273 OC for the difference in chemical shift of the meta proton in uncomplexed and complexed toluene rings (1.75 ppm) (from computed values of 6 relative to toluene, ref. 3). Therefore, the tert-butylbenzenetricarbonylchromium exists only in the conformation I1 at all investigated temperatures.
These preliminary results show that the association of physicochemical techniques can be very useful in the study of organometallic compounds: mass spectrometry and gas chromatography for identification and separation of the synthetized derivatives and nuclear magnetic resonance for the elucidation of their structure. It is remarkable that gas chromatography enables one to separate very complex mixtures and even to resolve closely related isomers. Kinetic studies and preparative scale separations by gas chromatography on various arenetricarbonylchromium derivatives are provided. The results of NMR experiments at variable temperature seem to solve the controversial problem of the eventual influence of the temperature on conformational exchanges. Further work in this field on complexes containing arenes substituted by groups with different electronic effects is in progress.
ACKNOWLEDGMENT We thank Mrs. N. Sellier and S. Combrisson for recording mass and NMR spectra. RECEIVED February 1 , 1971. Accepted March 29, 1971.
Identification of New Steranes, Terpanes, and Branched Paraffins in Green River Shale by Combined Capillary Gas Chromatography and Mass Spectrometry E. J. Gallegos Chevron Research Company, Richmond, Calf. 94802 Combined gas chromatography-mass spectrometry, GC-MS, of the branched-cyclic hydrocarbon fraction of Green River shale is used in a provisional identification of the carbon skeletons of two perhydro-p-carotenes; five pentacyclic triterpanes, one of which is gammacerane; two tetracyclic trk-homoditerpanes; 11 tricyclic terpanes; seven tetracyclic steranes, 5-0- and 5-p-cholestane1 5-a- and 5-p-ergostane1 5-0- and 5-p-stigmastane, and 5-a-pregnane1 and finally nine branched paraffins. Twenty-two of these components are reported here for the first time. Presented is a detailed GC-MS analysis of the branched-cyclic hydrocarbon fraction from Green River shale. This analysis constitutes an extension of work already reported on this shale fraction by several other investigators. The analytical identification of 36 individual components in the saturate fraction of Green River shale further demonstrates the importance of combined GC-MS. The stereochemical importance of the ratio of intensities of the m / e 149 to m / e 151 fragment ions in the identification by mass spectra of 5-a- or 5-p-steranes is discussed.
MUCHWORK has been done recently on the isolation and identification of individual components in the branchedcyclic hydrocarbon extract from Green River shale. Cummins and Robinson (I) reported the uneven distribution of n-alkanes and the presence of large proportions of CX-, CIS-, Clg-(pristane), and Czo-(phytane) isoprenoid alkanes. Burlingame et al. (2) reported the analytical isolation and identification of CZ7-,CZ8-,and G9-steranes and a pentacyclic triterpane, C30Hs2.Eglington et al. (3) have identified cholestane C2?HdS;ergostane, C Z ~ H ~ sitostane, O; CZ~H~Z; and perhydro-@-carotene,c4OH7S. Robinson and Cummins isolated a crystalline solid from their .benzene extract of (1) J. J. Cummins and W. E. Robinson, J. Chem. Eng. Dafa, 9,
304 (1964). (2) A. L. Burlingame, P. Haug, T. Belsky, and M. Calvin, Proc. Nut. Acad. Sci., 54, 1406 (1965). (3) Sister Mary Murphy, T. J., A. McCormick, and G. Eglinton, Science, 157, 1040 (1967).
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