Anal. Chem. 1985, 57,698-704
698
1,2,3,5-tetramethylbenzene, 527-53-7;1,2,4,5-tetramethylbenzene, 95-93-2; l-methyl-4-isopropylbenzene,99-87-6; pentamethylbenzene, 700-12-9;n-pentylbenzene, 538-68-1;n-hexylbenzene, 1077-16-3;n-diisopropylbenzene, 99-62-7;p-diisopropylbenzene, 100-18-5; 1,3,5-triethylbenzene, 102-25-0; hexamethylbenzene, 87-85-4;butylbenzene, 104-51-8;dimethylethylbenzene,29224-55-3; butylmethylbenzene,27458-20-4;ethylpropylbenzene,82162-13-8; diethylmethylbenzene, 25550-13-4; dimethylpropylbenzene, 82161-99-7;ethyltrimethylbenzene, 41903-41-7; methylpentylbenzene, 1320-01-0; butylethylbenzene, 82169-27-5; dipropylbenzene, 31621-49-5; ethylmethylpropylbenzene, 94278-81-6; ethyltetramethylbenzene, 94278-82-7;heptylbenzene, 1078-71-3; hexylmethylbenzene,27133-94-4;ethylpentylbenzene,94278-83-8; butylpropylbenzene, 94278-84-9; dimethylpentylbenzene, 94278-85-0; butylethylmethylbenzene, 94278-86-1; dipropylmethylbenzene, 42300-93-6; diethylpropylbenzene, 94278-87-2; butyltrimethylbenzene,94278-88-3;dimethylethylpropylbenzene, 94278-89-4; methyltriethylbenzene, 41903-42-8; propyltetramethylbenzene,94278-90-7;diethyltrimethylbenzene, 67143-86-6 n-octane, 111-65-9;n-decane, 124-18-5;n-undecane, 1120-21-4; n-dodecane, 112-40-3;n-tridecane, 629-50-5; n-tetradecane, 62959-4; methylpropylbenzene, 28729-54-6; butyldimethylbenzene, 82161-98-6;nonane, 111-84-2.
LITERATURE CITED (1) Daishima, S.;lida, Y. Shitsuryo Bunseki 1983, 37,73. (2) Munson, M. S. B.; Field, F. H. J. Am. Chem. SOC.1987, 89, 1047. (3) Harrlson, A. G.; Lln, P. H.; Leung, H. W. Adv. Mass Spectrom. 1978, 7 8 , 1394. (4) Daishima, S.;Iida, Y. Shitsuryo Bunsekl 1982, 30,249. (5) Daishima, S.; Iida, Y. Shitsuryo Bunsekl 1982, 30,61. (6) Iseda, K. Nagoya Kogyo Guutsu Shikensho Hokoku 1982, 37, 306. (7) Buchanan, M. V. Anal. Chem. 1982, 54, 570. (8) Lin, Y. Y.; Smith, L. L. Biomed. Mass Spectrom. 1979, 6 , 15. (9) Hunt, D. F.; McEwen, C. N.; Upham, R. A. Anal. Chem. 1972, 4 4 , 1292. (10) Buchanan, M. V. Anal. Chem. 1984, 56, 546. (11) Hunt, D. F.; Sethi, S. K. J. Am. Chem. SOC.1980, 102, 6953. (12) Freiser, E. S.; Woodin, R. L.; Beauchamp, J. L. J. Am. Chem. SOC. 1975, 97,6893. (13) Martinsen, D. P.; Buttrill, S. E., Jr. Org. Mass. Spectrum. 1976, 7 7 , 762. (14) Hawthorne, S. E.; Sievers, R. E. Envlron. Sci. Techno/. 1984, 78, 483.
RECEIVED for review October 9, 1984. Accepted November 16, 1984. This work was performed under Cooperative Agreement No. DE-FC21-83FE60181for the US.Department of Energy, Office of Fossil Energy.
Laser Mass Spectra of Simple Aliphatic and Aromatic Amino Acids Cass D. Parker and David M. Hercules* Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
Mass spectra of amlno aclds have been studied using the LAMMA laser microprobe. Quasi-molecular Ions, (M H)’ and (M H)-, are produced In hlgh yields. The malor neutral fragment loss from (M H)’ corresponds to HCOOH. Loss of other acids Is related to the amlno group postllon. For alkyl amino acids, add loss plus formation of imino Ions ( m / z 30) are the only slgnlficant fragments at threshold power densltles. Fragmentatlon Is llmlted In the negatlve Ion spectra; (M H)- and CN- are the only Ions observed at low power densltles. Fragmentatlon In both the posltlve and negative Ion spectra Is Influenced by substltuents on the alkyl or aryl chalns; the electron denslty of an aryl group has a signlflcant effect. I n a-@flsslon, the phenylalanine quasl-molecular Ion gives the a-fragment, but no a-fragment Is observed for tryptophan. Slgnlflcant thermal decomposltlon of amlno acids does not occur In laser mass spectrometry; spectra are comparable to chemical ionlzatlon using reagent gases havlng hlgh proton afflnltles.
-
+
+
-
The use of mass spectrometry to study nonvolatile organic compounds has been expanded in recent years, particularly for biomolecules which require derivatization for conventional chemical ionization (CI) or electron impact (EI) methods. Introduction of fast-heating direct insertion probes (1) and new ionization sources such as field desorption (FD) ( 2 ) , secondary ion mass spectrometry (SIMS) ( 3 ) , fast atom bombardment mass spectrometry (FAB) ( 4 ) ,plasma desorption mass spectrometry (PDMS) ( 5 ) , and laser mass spec-
trometry (LMS) (6)make derivatization no longer necessity for many compounds. Mass spectra of the amino acids have been reported with several ionization sources: electron impact (EI) (7), FD (8), CI (9),SIMS (3),FAB ( l o ) ,and LMS (11). Mass spectra of the amino acids obtained by these techniques show similarities; for example, all show quasi-molecular (M + H)’ ions (except E1 which shows M+3 and peaks corresponding to loss of formic acid from the quasi-molecular ion. Negative ion spectra have been reported by SIMS and low energy electron impact (LEEI) (12). SIMS reported detection of (M - H)- quasimolecular ions, while LEEI reported (M - H)-, with fragmentation arising from M-e; losses included NH2, HzO, C02H, and the alkyl chain. We report here results obtained for amino acids using laser mass spectrometry (LMS). The positive-ion spectra resemble those obtained by using other techniques. Of particular interest is the similarity to CI mass spectra obtained by using reagent gases having relatively high proton affinities. This attests to the “softness” of LMS as a technique for obtaining spectra of organic compounds. Fragmentation is not extensive at threshold power densities, particularly in negative-ion LMS.
EXPERIMENTAL SECTION Laser mass spectra were obtained with commercially available instrumentation: the Leybold-Heraeus LAMMA-500and LAMMA-1000 laser microprobes. Mass spectra of DOPA, phenylalanine methyl ester hydrochloride, and tryptophan were obtained only on the LAMMA-1000; other samples were run on both instruments; fragmentation patterns did not differ significantly. The LAMMAdOO is described in detail elsewhere (13). The major difference between the two instruments is that the laser beam
0003-2700/85/0357-0698$01.50/00 1985 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 3, MARCH 1985
699
Table I. Positive Ion Laser Mass Spectra of Amino Acids, Intensities Releative to Base Peak (100%)
compound
(M + H)+
(M + H - COZHZ)"
glycine (75) alanine (89) &alanine (89) valine (117) leucine (131) isoleucine (131) proline (115) 3-aminobutyric acid (103) 4-aminobutyric acid (103) 6-aminocapric acid (131) histidine (156) 3-methylhistidine (170) phenylalanine (165) tyrosine (181) tryptophan (204) tryptophan (204)j DOPA (197) phenylalanine methyl ester (179)' proline methyl ester (129) glycine ethyl ester (103) N,N-dimethylglycine
100 100
89 (mlz 30)
100
100 100 64 40 100 95 100
other
92
317
5 23 43
58" 36 12
5
100 100 11
0113 13/25
100 100 100 75 7 21
85 ( m / z 44)" 11 (mlz 44)"
lOOb
3
63
99 80
19 12
100 45
20
22" lOOd lOOd lOOd
17
9 51 1If 54 488 ( m / z 30)
100 100 100 100
6
m / z 30
-/Hi
93d 11'
100
4h
(M + H - H3CC02H)+. (M + H - H8CCH2CO2H)+.a fragment at m/z 74, H2Nt=CHCOOH. (M + H - H2NCHzCO2H)+.e a fragment at m/z 88, H2N=CHCOOCH3. f(M + H - HC02CH3)+.g(M + H - HCOZCHzCH3)+.h(M + H - HCOZCH3)+.'(M + H HXOH)'. j LAMMA-1000 data numbers in Darentheses for all comDounds are formula weights. irradiates the sample by transmission in the LAMMA-500 and by front-surface in the LAMMA-1000. The output of a frequency quadrupled Q-switched Nd-YAG laser, 265 nm, 15 ns pulse width, is focused on the sample through a microscope objective. Spectra obtained from the LAMMA-500 used a 32X objective. In the LAMMA-500 changes in laser spot size had little effect on the spectra. The laser energy was varied with a set of neutral density filtersto give the lowest power density needed to obtain a mass spectrum, los W/cm2. Ions formed were accelerated to 3 kV into the drift tube of a time of flight mass spectrometer having first-order correction for ion kinetic energy spread. The output from a 17-stage electron multiplier was coupled to a Biomation transient recorder after appropriate amplification. The transient recorder acted as a storage buffer for selected mass ranges. The timing sequence was triggered by the laser pulse. Data were transferred directly to a strip chart recorder or to a HP-1000 computer system for data processing. The mass scale of the TOF is linear with time. Calibration of the positive and negative ion mass scales were done daily. This involved calculation of the TOF time constant, k (rn - &'I2), using mass markers for both positive and negative ion spectra. The compound selected as a mass marker was tris(ethy1enediamine)cobalt(III) iodide, Co(en)&. This complex gives a characteristic pattern due to ion/molecule reaction products of the form (CO,I,-~)+and (CO,CNI,-~)+in its positive ion spectra, and (Co,,I,+J-, (Co,I,lCN); and (CO,I~,+~)in ita negative ion spectra. The ion/molecule reactions yield ions up to rn/z 1300. The cobalt-amine complex does not have the inherent matrix problems associated with CsI, which yields a scale accurate only to rn/z *2 with changing thickness or laser power. All amino acids were obtained from Sigma Chemical Co. and were run without further purification. Powdered samples were dissolved or suspended in methanol or ethanol and evaporated onto polymer coated Cu SEM grids. Suspension or dissolution had little effect on the spectra. A major effect can be correlated with crystal size in the LAMMA-500. Very small crystals gave the most consistentand reproducible spectra. Water was not used as a solvent due to sodium and/or potassium contamination which leads to extensive cationization.
Positive I o n L M S ~M+H)+ ;"I I
+NH,
LEUCINE M W
131 M + t i - C H ~ Oj * ~
86
-
RESULTS AND DISCUSSION Table I shows the results obtained for the positive ion LMS of the aliphatic and aromatic amino acids studied. Figure 1 shows the positive ion LMS of leucine, which is typical of the
132
H3C-CH-CH2-CH-COO-
i
HZC= NH2
30 ,
20
IO
30
40
50
,
.
.
.
6 0 7 0 BO 90 100
.
I
120
140
Figure 1. Positive ion LMS of leucine, single trace.
Scheme I. Ion Formation in LMS of Amino Acids R-CH-COO-
Pair t
+A J '
R-HC=~+HCOOH
alkyl-substituted amino acids. The major ions observed for leucine are the quasi-molecular ion (M H)+at m/z 132, and the fragment ions (M + H - C02H2)+,m / z 86, and H2C= NH2+,rn/z 30. The aromatic acids exhibit additional fragment ions in their positive-ion LMS; e.g., a-/3 fission results in either an ion at m / z 74 H2N+=CHCOOH or an aryl moiety corresponding to (M + H - H2CNH2COOH)+.The LMS of substituted amino acids, e.g., hydroxy, sulfhydryl, guanidino, etc., will be discussed in a separate communication (14). As seen in Table I, the quasi-molecular ion (M + H)+ is an intense peak in the spectra of most amino acids. The (M + H)+ ion results from intermolecular proton transfer between the amino and acid groups of neighboring amino acids ( I d ) , as shown in Scheme I. Proton transfer is the major mechanism for (M + H)+ and (M - H)- ion production, not only at threshold power densities but even at power levels up to 100
+
700
ANALYTICAL CHEMISTRY, VOL. 57, NO. 3, MARCH 1985 IO0 (M+H)+ Q-Alonine
Yo
+
L
90
H2C-tH2-c=0 I ' 0 p 3 0
H2C-NH2 30
1
50 -
Phenylolonine Mefhyl E s t e r . HCl
I
I
I.
I
NO+ ( M + H - N H I+~
K+
23
39
( M+H - ti,,
