Table 111. Five-Day Study of Two P a i n t Samples Paint D , theoretical: 790 u g l g
Paint A , theoretical: 110 ug/g
Day
1 2 3 4 5
Size of group
5 5 5 5 5
Mean
95% Confidence interval
Day
118 109 115 116 110
*2.2% 18.1% i7.8% +6.7% *12.8%
2 3 4 5
1
Nominal added Me an Maximum Minimum Range Variance Standard deviation Standard e r r o r 95% Confidence interval
Paint D
110 114 125 96 29 52 7.2 1.4 i2.6%,
790 798 829 752 77 298 17.2 3.4 *l.O%
5 5 5 5 5
Mean
95% Confidence interval
802 791 801 804 790
12.5% *4.2% +1.3% +3.2% *1.5%
CONCLUSION
Table IV. Gross Statistical D a t a , 25 Determinations Paint 2
Size of group
An alternative to alkali fusion for the atomic absorption determination of total chromium has been presented. The method is rapid, accurate, and requires a minimum of attention by the analyst. Statistical data indicate an acceptable day-to-day reproducibility. Further evaluation of the procedure in this laboratory has indicated a similar degree of precision and accuracy in the determination of chromium present as lead chromate and strontium chromate. The method can easily be adapted to other applications, such as geological, metallurgical, and raw material analyses.
with the bomb technique are in good agreement with the nominal values, and no form of background correction is needed. Next, the precision of the method was determined. In this study, the chromium contents of two spiked paints were determined. Five determinations per day over five days were performed for each paint. Table I11 shows the mean and 95% confidence interval of both paints for each group of five determinations on a daily basis. In the case of both paints, analysis of variance indicated no statistical difference between days at the 0.05 level. Table IV presents the gross statistical data obtained from each group of 25 determinations. For a 1.0-gram sample and a final dilution volume of 100 ml, a detection limit of 1 pg/g chromium in the sample should be seen by most atomic absorption instruments using conventional flame techniques.
ACKNOWLEDGMENT The author thanks M. Jao and A. Widzisz for generating the fusion data, and J. Holyk for performing the statistical operations. LITERATURE C I T E D (1) B. Delaughter, At. Absorption Newslett., 4, 273 (1965). (2)R. E. Mansell and H. W. Emmel, At. Absorption Newslett., 4, 365 (1965). (3) M. R . Midgett and M. J. Fishman, At. Absorption Newslett., 6 (6),128 (1967). (4) T. R. Gilbert and A. M. Clay, Anal Chim. Acta, 67, 289 (1973). (5) B. Bemas, Anal. Chem., 40, 1682 (1968). (6)P. T. O'Shaughnessy, Anal. Chem., 45, 1946 (1973).
RECEIVEDfor review July 26, 1974. Accepted October 29, 1974.
Gas Chromatographic and Mass Spectral Analysis of a-Methyl Amino Acids Otto Grahl-Nielsen Department of Chemistry, University of Bergen, N-5000 Bergen, Norway
Einar Solheim Department of Pharmacology, University of Bergen, N-5000 Bergen, Norway
Because of the extensive research into extra-terrestrial and prebiotic synthesis of amino acids, an increasing number of non-protein amino acids have been discovered and identified. Among them are the a-methyl amino acids C Y amino isobutyric acid and isovaline ( I ). The method of choice for the analysis of mixtures of non-protein amino acids is gas chromatography. Sufficient volatility of the
amino acids has been obtained by esterification of the carboxyl group and acylation of the amino group. We have previously reported that the inner esters of acyl-amino acids, azlactones or oxazolin-5-ones, are alternatives to N acyl-amino acid esters as volatile derivatives for gas chromatographic analysis ( 2 ) .The present report deals with the analysis of a mixture of a-methyl amino acids as the corre-
ANALYTICAL CHEMISTRY, V O L . 47, NO. 2, FEBRUARY 1975
333
a'
9 W v)
z
: L
Flgure 2. N-Benzoylation of an a-methyl amino acid followed by formation of 2-phenyl-4-methyl-oxazolin-5-one
-
Table I. Relative Intensities of Characteristic Fragments in the Mass Spectra of 2-Phenyl-4-methyl-4-R-oxazolin-5-ones
1 I
T irne I
Temp100
I
I
I
L
8
12
1
I
130
160
- - I
6°1m~n
Figure 1. Chromatogram of a mixture of 2-phenyC4-methyC4-R-oxazolin-Sones from 12 a-methvl amino acids Column: 3% OV-17 on 100-120 mesh Gas-Chrom Q. 3-mm i.d. X 150 cm, glass. Carrier: helium at 1 2 mllmin. Initial temperature 100 O C , program rate 6'Imin. Detection:electron impact
sponding 2-phenyl-oxazolin-5-ones, obtained by N-benzoylation of the amino groups followed by inner ester formation by dicyclohexylcarbodiimide. EXPERIMENTAL Twelve a-methyl amino acids were synthesized by the Strecker synthesis applied on a n equimolar mixture of the corresponding ketones ( 3 ) .The amino acids were alanines with the following substituents in a-position: methyl, ethyl, n -propyl, isopropyl, n -butyl, isobutyl, tert -butyl, n -pentyl, isopentyl, sec -pentyl, n -hexyl, and n -heptyl. After the synthesis was finished, the amino acid hydrochlorides were extracted from the dried salt mixture with absolute ethanol which afterward was removed under reduced pressure. A small amount of the mixture of the amino acid hydrochlorides was dissolved in water, and the solution made slightly alkaline by addition of NaOH. Benzoyl chloride was added under vigorous stirring, and the solution was kept alkaline by addition of NaOH. After 15 minutes, the excess benzoyl chloride was removed by extraction with diethyl ether. The reaction mixture was acidified with HCl, and the N-benzoyl-amino acids were extracted into ethyl acetate which was dried over sodium sulfate. Dicyclohexylcarbodiimide was added to the solution and, after proper mixing, a 1 - ~aliquot 1 was injected into the gas chromatograph of a Varian MAT 111 GC-MS. The glass column (3 mm i.d. X 150 cm) was packed with 3% OV-17 on Gas-Chrom Q 100-120 mesh. Helium was used as carrier gas a t 12 ml/min. The resulting gas chromatogram is shown in Figure 1. The mass spectra were taken a t 80 eV a t a scan speed of 100 massedsec. The yield of the inner ester formation was tested as follows: Different amounts of N -benzoyl-a-amino isobutyric acid [synthesized and purified as described by Steiger ( 4 ) ] in ethyl acetate were treated with a slight excess of dicyclohexylcarbodiimide and chromatographed. Similarly, different amounts of 2-phenyl-4,4-dimethyl-oxazolin-5-one [synthesized by the action of dicyclohexylcarbodiimide on N -benzoyl-a-amino isobutyric acid in tetrahydrofuran, and purified by distillation under reduced pressure (5 )] were chromatographed. The peak areas were determined by cutting and weighing. 334
R
mol wt
?d ( M -
Methyl Ethyl a-Propyl Isopropyl n-Butyl Isobutyl tevf-Butyl n-Pentyl Isopentyl sec-Pentyl n-Hexyl n-Heptyl
189 203 217 217 231 23 1 231 245 24 5 245 2 59 273
10
17"
4
13b 9
28):
4 4 2 3
mfe 175
m le
161
105
77
17"
100
42 43 39 62 39 45 85 45 36 45 46 46
13b 10 65 6 18
47 31
10
60
9
57
100
22
100 1 1
13 15
m le
w ' e
100 100 100 100 100 100 100
100
74 85
100 100
a T h e (M - 28).+ fragment is identical with the m / e 161 fragment. * T h e (M - 28).f fragment is identical with the m / e 175 fragment.
RESULTS A N D DISCUSSION The derivatization reactions are outlined in Figure 2. Benzoylation of amino acids by the action of benzoyl chloride in aqueous solution in the presence of alkali has been shown to be a smooth and rapid reaction of high yield ( 4 ) . The only possible byproduct is benzoic acid which will not interfere with the next reaction. Addition of excess alkali was avoided by checking the pH during the reaction. Although no detailed kinetic study was undertaken, it was obvious that increased branching of the amino acid side chain decreased the rate of reaction. Isolation of the N-benzoyl-amino acids was not necessary, they were extracted into ethyl acetate after acidification of the reaction mixture and, after drying shortly with sodium sulfate, dicyclohexylcarbodiimide was added to the solution. As experienced earlier (6 1, the inner ester formation was very rapid: the reaction mixture could be injected immediately after proper mixing of the reactants. Influence of the steric effects of the side chain on the reaction was not experienced. The test described above revealed that the yield of the inner ester formation in the case of N-benzoyl-a-amino-isobutyric acid was quantitative. The marked variations in peak areas in Figure 1 were most likely due to differences in yields of the Strecker synthesis of the a-methyl amino acids. The peaks in the gas chromatogram in Figure 1 were identified by comparison of the retention times under standard conditions with those of individually synthesized, derivatized, and chromatographed amino acids. The elution of the derivatives roughly followed the molecular weights. For
ANALYTICAL CHEMISTRY, VOL. 47, NO. 2, FEBRUARY 1975
the propyl and butyl derivates, branching of the chain increased the volatility. The isopentyl derivative was eluted before the n -pentyl, analogously with the butyl derivatives; however, the sec -pentyl derivative was eluted after the n pentyl derivative. Good separation was obtained except for the propyl derivatives where isopropyl emerged as a shoulder on the n-propyl peak. The see-pentyl derivative was not properly separated from the n -hexyl derivative. The oxazolin-5-ones could also be identified by their mass spectra which were fairly simple. The most prominent ions were rnle 175, 161, 105, and 7 7 . The relative intensities of the fragments are given in Table I. Only the two lowest homologs had molecular ions. The size of the higher homologs could be determined from the (M - 28)-+ fragment which resulted from loss of the carbonyl group. This fragment was further cleaved between the first and second carbon of the side chain to give the very abundant rnle 161 fragment. The compounds with branching a t the first carbon of the side chain did not give the M - 28 fragmentation, and they did also lack the rnle 161 fragment. The ( M - 28).+ fragment of the ethyl derivative had a mass of 175. Since the (M - 28).+ ion in all the other derivatives was accompanied with the rnle 161 fragment but not here, it is most likely that the (M - 28).+ fragment in this ~ the case was not due to loss of CO but to loss of C S H from side chain. The cleavage of the side chain as an olefin fragment very likely occurred via a McLafferty rearrangement ( 7 ) . Thus, all homologs with a hydrogen on the second carbon of the side chain had abundant fragments of m/e 175. The only exception was the tert-butyl derivative where
sterical factors probably hindered this fragmentation. The oxazolin-5-ones with secondary carbons a t the first position of the side chain, i.e. isopropyl and sec-pentyl, gave rnle 175 fragments of higher intensity than the unbranched homologs, i.e., 65% for isopropyl us. 10% for n-propyl, and 100% for see-pentyl us. 10% for n-pentyl. Branching a t the second carbon of the side chain also gave differences in the intensity of the rnle 175 fragment, i.e., 18%for isobutyl us. 6% for n-butyl. This was the only significant distinction between the mass spectra of these two derivatives. Branching further out in the side chain did not affect the McLafferty cleavage, i . e . , the isopentyl derivative had 9% intensity of the mle 175 fragment and the n -pentyl derivative 10%. In fact, it was impossible to distinguish the mass spectra of those two pentyl derivatives. The benzoyl fragment with rnle 105 was the base peak in all spectra. The phenyl ion of rnle 77 also was abundant in all spectra.
LITERATURE CITED (1) Y. Wolman, W. J. Haverland, and S. L. Miller, Proc. Nat. Acad. Sci. USA, 69, 809 (1972). (2) 0. Grahl-Nielsen and E. Solheim, Chern. Cornrnun., 1972, 1093. (3) P. A. Levene and R. E. Steiger, J. B i d Chem., 76, 299 (1928). (4) R. E. Steiger, J. Org. Chem., 9, 396 (1944). (5) H. Rodriguez, C. Chuaqui, S. Atala, and A. Marquez, Tetrahedron, 27, 2425 (1971). (6) 0. Grahl-Nielsen, J. Chrornatogr., 93, 229 (1974). (7) D. G. I. Kingston, J. T. Bursey, and M. M. Bursey, Chem. Rev., 74, 215 (1974).
RECEIVEDfor review July 15, 1974. Accepted September 30, 1974.
Characterization of Oil Shales by Laser Pyrolysis-Gas Chromatography Ray
L. Hanson and N. E. Vanderborgh
Department of Chemistry, The University of New Mexico, Albuquerque, N.M. 8713 1
Douglas G. Brookins Department of Geology, The University of New Mexico, Albuquerque, N.M. 8713 1
Oil shales occur in large quantities in the North American continent. They represent a large proportion of the known hydrocarbon reserves ( I , 2 ) . Currently work is being done on these materials to find new methods for their rapid characterization. Here we report initial studies on laser pyrolysis monitored by gas chromatography. Shales, like coal where considerably more analytical information exists, contain complex mixtures of high molecu‘lar weight hydrocarbons. It is of economic interest to know the amount of hydrocarbon in the rock; likewise moisture, ash, and particular elements have been determined in shales (3-5). Certainly much work will be done to improve these analyses in the immediate future. The standard method for oil shale characterization centers around combustion in oxygen. The evolved C 0 2 is measured volumetrically (6 1. Inorganic carbon (C03’-) is determined by an acidimetric procedure ( 7 ) . Other methods include the Fisher assays where shale samples are heated in a standard retort; the oil and other hydrocarbons
given off are collected and,weighed (8). This method has been used for years and most of the existing analytical data result from studies of this sort. More recent studies have looked a t the individual constituents obtained with solvent extraction followed by separation and identification (9, 1 0 ) . A. Giraud applied pyrolysis-gas chromatography to the geochemical characterization of kerogen in sedimentary rock. Low temperature cracking patterns showed products rich in aromatics. The mass of low molecular weight gases can be estimated from the C1-C3 hydrocarbons evolved while the C4-Cll alkanes serve as a reliable measure of the oil that can be produced. High temperature pyrolysis (here 500 “C) shows much more extensive cracking. Results indicate a series of cracking processes with the time dependence that one might predict for this type of analysis. Giraud indicated that pyrolysis must be of brief duration with minimal secondary reactions ( I 1 ). These requirements lead to the consideration of laser py-
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