6502
J. Phys. Chem. 1987, 91, 6502-6506
the high-angle region (highly polar forms), 18% a t 176' (n = 2), 21% at 172O-178' (n = 3), and 13% at 175O-176O (n = 4). The odd-even trend is weak in this series, but still definite. The averaged values (cos O ) , which are related to the dipole moment through eq 8, are estimated to be -0.0057 (n = 2), -0.0391 (n = 3), and 0.1 153 (n = 4). The present analysis provides the reason why the odd-even effect in the dipole moment is moderate and attenuates rapidly with n in the a,w-diester series. Finally, we would like to comment on the experimental observations of Riande et a1.6 for temperature coefficient of the unperturbed dimension of poly(oxyneopentyleneoxyadipoy1). The present investigation suggests that the E,, value of the adipate residue (Ecain Riande et a1.k notation) is about 0.8 kcal mol-', while this parameter has been assumed to be zero in Riande et
al.'s calculation. Use of E , = 0.8 kcal mol-l instead of zero gives K-l, the other rise to an increase of d In (?)o/dT by ca. 0.5 X parameter being fixed at given values. This revision raises the range of ECeC,required to reproduce experimental values of d In (?)o/dT ((0.2 to 0.6) X lo-' K-1).6 Although adoption of ECeCm = 1.0 kcal mol-' still predicts a slightly negative value of d In (r2),,/dT,an intermediate value (Ecsc, = ca. 0.5 kcal mol-') is compatible with experimental observations. With the conformational energy parameters thus established, various configurational properties of polyesters can be estimated. Results of these studies will be reported elsewhere. Registry No. CH,0C(0)(CH,)2C(O)OCH3, 106-65-0;C H 3 0 C (O)(CH2)3C(O)OCH3, 1119-40-0; CH,OC(O)(CH,),C(O)OCH3,62793-0.
Excimer Laser Desorption Mass Spectrometry of Biomolecules at 248 and 193 nm B. Spengler,* M. Karas, U. Bahr, and F. Hillenkamp Institut fur Medizinische Physik, Universitat Munster, 4400 Munster, FRG (Received: January 28, 1987; In Final Form: May 27, 1987)
Laser desorption mass spectrometry (LD-MS) of various peptides has been performed with excimer laser nanosecond pulses of 248- and 193-nmwavelengths. Whereas the spectra obtained with 248 nm resemble those obtained earlier with quadrupled Nd:YAG lasers at 266 nm, the peptides show a quite different fragmentation and association behavior at 193 nm. These observations can help in understanding the desorption and ion formation process. They indicate a wavelength-dependent competition between photochemical ion formation and ion generation via a collective process of the strongly perturbed solid. They can also help to further optimize the analysis of given organic molecules by LD-MS.
Introduction The formation of quasimolecular (=M + H', M - H-, M alkali+) and fragment ions in laser desorption mass spectrometry (LD-MS) is not nearly fully understood.'S2 In particular, there seems to be no general answer to the question of whether or not ion desorption is a thermal process in which ion emission commences after thermodynamic equilibrium has been attained in the condensed-phase system. Emission of neutrals is known to be a thermal process in CW CO, laser desorption." The desorption of large, fragile, and nonvolatile organic ions with other lasers, or UV lasers,10*' is taken particularly short-pulsed C02 to indicate a nonequilibrium process. This as well as other experimental observations seems to exclude a uniform theory for all laser desorption techniques independent of the greatly varying
+
(1)Hillenkamp, F. Springer Ser. Chem. Phys. 1983,25, 190. (2) Hillenkamp, F.Springer Ser. Chem. Phys. 1986,44, 471. Haverkamp, J.; Kistemaker, P. G. Int. J. Mass (3)Van der Peyl, G. J. Q.; Spectrom. Ion Phys. 1982,42, 125. (4)Van der Peyl, G. J. Q.; Isa, K.; Haverkamp, J.; Kistemaker, P. G. Org. Mass Spectrom. 1981,16, 416. (5)Van der Peyl, G. J. Q.;Isa, K.; Haverkamp, J.; Kistemaker, P. G. Nucl. Instrum. Methods Phys. Res. 1982,198, 125. (6)Stoll, R.; Rallgen, F. W. 2. Naturforsch., A: Phys., Phys. Chem., Kosmophys. 1982,37A, 9. (7)Van Breemen, R. B.; Snow, R. B.; Cotter, R. Int. J. Mass Spectrom. Ion Phys. 1983,49, 35. (8)Tabet, J. C.; Cotter, R. J. Anal. Chem. 1984,56, 1662. (9)Hardin, E.D.; Fan, T. P.; Blakley, C. R.; Vestal, M. L. Anal. Chem. 1984,56, 2. (10) Karas, M.;Bachmann, D.; Hillenkamp, F. Proceedings of the Tenth International Mass Spectrometry Conference; Todd, J. F. J., Ed.; Wiley: Chichester, U.K., 1986;pp 969-970. (1 1) Seydel, U.; Lindner, B.; Zihringer, U.; Rietschel, E. T.; Kusumoto Shiba, T. Biomed. Mass Spectrom. 1984,1 1 , 132.
0022-3654/87/2091-6502!§01 S O / O
TABLE I: Experimental Parameters of the Laser Desorption System wavelength, excimer quantum pulse focus nm nas enernv. eV duration, ns diam. um 248 KrF 5.0 13 42 193 ArF 6.4 13 48
pulse width, irradiances, and wavelengths. Until very recently, the influence of the wavelength on thermal and nonthermal desorption processes has been considered to be In a recent study it has, however, of only minor been shown that resonant absorption in the UV at the laser wavelength used facilitates a soft i ~ n i z a t i o n . ' ~This concept of resonant absorption has been extended to that of a matrix-assisted desorption of quasimolecular ions of nonabsorbing solutes, dissolved in absorbing solvents.10,16 Whether ion formation takes place in the condensed phase or in the gas phase after desorption is also an open question. Parker and Hercules" have suggested the formation of pairs of protonated and deprotonated ions in the gas phase. Cationization is another feature typical for laser desorption. Whereas cationization of desorbed neutrals as an equilibrium gas-phase reaction has been shown to dominate C W COz laser d e ~ o r p t i o n ,there ~ * ~ is good experimental evidence that this does (12)Hercules, D.M.;Day, R. J.; Balasanmugam, K.; Dang, T. A,; Li, C.
P.Anal. Chem. 1982,54, 280A. (13) Conzemius, R. J.; Capellen, J. M. Int. J . Mass Spectrom. Ion Phys. 1980,34, 197. (14)Schueler, B.; Krueger, R. F. Org. Mass Spectrom. 1980, 15, 295. (15)Karas, M.;Bachmann, D.; Hillenkamp, F. Anal. Chem. 1985,57, 2935. (16)Karas, M.;Bachmann, D.; Bahr, U.;Hillenkamp, F. Int. J . Mass Spectrom. Ion Phys., in press. (17)Parker, C. D.; Hercules, D. M.; Anal. Chem. 1986,58, 25.
0 1987 American Chemical Society
The Journal of Physical Chemistry, Vol. 91, No. 26, 1987 6503
LD-MS of Biomolecules a t 248 and 193 nm
variable laser optics
......
...........................
.........
"
....
.............
ion reflector
ion lens
Figure 1. Scheme of the mass spectrometer for UV laser ionization (MULI). not hold for nanosecond visible'* and UV laser desorption. This paper proposes a model of cationization by a nonequilibrium process. It may be in the end more misleading than helpful to differentiate strictly between condensed- and gas-phase processes because the observed reaction products result from the very fast, dynamic process of a transition between the two. Experimental Section All experiments were done with a mass spectrometer for UV laser ionization, MULI (Figure l ) , a laboratory-built system equipped with a pulsed laser for ion generation and a time-of-flight (TOF) system for mass separation, similar to the LAMMA lo00 instrument.lg The relevant experimental parameters are listed in Table I. The wavelengths of 248 and 193 nm were chosen because results obtained at 248 nm should be readily comparable to those obtained with LAMMA instruments at 266 nm; 193 nm is the shortest laser wavelength available for such experiments. It has a quantum energy of 6.4 eV, allegedly causing efficient photodissociation.20 The far field of the laser was sampled for a statistically significant number of shots with a reticon array detector. An approximately Gaussian beam profile with no hot spots was found. Focus diameters of 42 and 48 pm, respectively, at the two wavelengths were similar enough to exclude a sizable influence of the focus size, which was observed in other experiments?' The presented spectra are singleshot spectra, and all values of intensity in the graphics were averaged over ca.10 representative single-shot spectra. All samples were prepared by drying 2-pL aqueous solutions of ca. mol/L concentration onto aluminum substrates. Solutions were prepared either with water and only trace contaminants of NaCl or, for comparison, by admixing an equimolar amount of NaCl. Peptides (seven dipeptides and two polypeptides) were used as test substances because of their amphoteric character resulting in easy protonation and deprotonation and their well-investigated fragment pattern at 265 nm.15 In this paper results for only three dipeptides are reported. Their results are representative for the whole group. They show zero to strong absorption at 248 nm, depending on the content of aromatic residues. At 193 nm the delocalized a-electron of the peptide bond creates a strong absorption of 7 X lo3 L/(mol-cm) for all of them.z2J3 In addition salicin, with a moderate absorption at 248 nm and a strong absorption at 193 nm, was analyzed. Contrary to the peptides, the chromophore of the latter is not located at a bond that is easy to fragment. The saccharide gentiobiose, also tested for com(18) Hardin, E. D.; Vestal, M. L. Anal. Chem. 1981, 53, 1492. (19) Feigl, P.; Schueler, B.; Hillenkamp, F. In?. J. Mass Spectrom. Ion Phys. 1983, 47, 15. (20) Garrison, B. J.; Srinivasan, R. Appl. Phys. Lett. 1984, 44, 849. (21) Bahr, U.; Karas, M.; Hillenkamp, F. Proceedings ofthe Third In-
ternational Laser Microprobe Mass Spectrometry Workshop;University of Antwerp: Antwerp (Belgium), 1986; pp 17-19. (22) Wetlaufer, D. B. In Advances in Protein Chemistry;Anfinsen, C . B., Ed.; Academic: New York, 1962; p 326. (23) Hillenkamp, F., unpublished results.
70 IAI20f
20
LO
60
1M3
200
1 9
250
300 m/ z
4=2,5.106W/cmZ
Figure 2. Mass spectra of the dipeptide valylleucine at 248-nm desorption wavelength at threshold irradiance. [M-H*ZNal'
72 t
275 I
'
---
IM+NaI+
i
:iI
I
la,
E=?3.1O6Wlcm2 = 13.E,
1%
I
I
1
200
EO
300
m/z
Figure 3. Mass spectra of the dipeptide valylleucine at 248-nm desorption wavelength at high irradiance.
=> -
4-
G=0,5 106Wlcm2
6504 The Journal of Physical Chemistry, Vol. 91, No. 26, 1987 'Val-451'
?,
I M*HI'
, L y 5 1 '
50
20
'00
Spengler et al.
248 nm
2 31
'50
200
EO
193 nm
300 m/z
W/ c ' m
E = 8 6 106W/cm2 = 1 8 E b Figure 5. Mass spectra of the dipeptide valylleucine at 193-nm desorption
wavelength at 18 times threshold irradiance. ,Nqcll* 23
81
Figure 7. Intensities in relative units of the quasimolecular ions and 83 {M-H+ZNaI' 275
fragments of valylleucine versus irradiance E at 248 and 193 nm. The lower limit of irradiance for the onset of Na' and/or fragment ions is indicated in the figure by a left-terminated arrow.
50
2.48 nm
20
50
100
150
200
250
193 nm
300
m h
E: 2 6 5 106 W I c m ' = 5 2 EL Figure 6. Mass spectra of the dipeptide valylleucine at 193-nm desorption
wavelength at 52 times threshold irradiance. TABLE II: Molar Extinction Coefficients and Ratios of Observed Threshold Irradiances of the Used Test Substances C193r
substance
L/(mol.cm) 7.5 x 103 5.2 x 104
€248,
Eth 248/
L/(mol-cm) Eth193 6 X lo4
