Laser desorption multiphoton ionization mass spectrometry

47

Journal of Analytical and Applied Pyrolysis, 20 (1991) 47-56

Elsevier Science Publishers B.V., Amsterdam

Laser desorption multiphoton mass spectrometry T.L. Weeding *, R.J.J.M. Steenvoorden,

ionization

P.G. Kistemaker and J.J. Boon

FOM Institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam (The Netherlands)

(Received June 15, 1990; accepted December 10, 1990)

ABSTRACT

The combination of laser desorption followed by multiphoton ionization (MPI) with time of flight mass spectrometry (TOF/MS) produces a versatile method of high sensitivity and selectivity. The first step is laser desorption in which molecules are desorbed from the sample intact or with little degradation. Laser ionization is employed as the second step to produce the ions and also induce fragmentation. Soft ionization, which yields primarily the parent ion, and hard ionization, which yields primarily the fragments, are both possible. Instrumental details are described and preliminary results from recent laser desorption MPI-TOF/MS experiments on a perbenzoylated sugar and on a porphyrin are presented. Examples from single photon ionization (SPI) experiments on two alkanes are also shown.

Laser desorption; multiphoton mass spectrometry.

ionization;

pyrolysis; single photon ionization;

time of flight

INTRODUCTION

Pyrolysis techniques, in combination with mass spectrometry, have proven to be very useful in the characterization of a wide variety of insoluble non-volatile materials including biopolymers, synthetic polymers, archeological samples, and fossil fuels. The result of this has been a rich literature in which different pyrolysis methods have been applied to many different compounds with the goal of understanding and optimizing analytical procedures. Amongst these procedures is laser desorption which can be coupled to various separation and ionization techniques. At the current time, laser desorption appears to be the most promising method for volatilizing large molecules intact. This was recently demonstrated when a DNA molecule with a molecular weight of 410 kDa was successfully ejected from a frozen aqueous solution and then analyzed by gel electrophoresis [l]. In this case 0165-2370/91/$03.50

0 1991 Elsevier Science Publishers B.V.

48

the molecule of interest was embedded in a matrix, the co-ablation of which could lead to unwanted background signals in a mass spectrum. However, an appropriately chosen matrix, the nicotinic acid first used by the group of Hillenkamp for example, can be very useful since it participates directly in, firstly, the desorption process by absorbing (near) resonant laser light and, secondly, in the ionization process by serving as a proton donor [2]. An unavoidable consequence of this method is again the appearance of matrix peaks in the low mass ( < 100 Da) region of the spectrum. High intensity infrared (IR) radiation from a Nd : YAG laser has been used in laser pyrolysis GC-MS experiments to analyze carbonaceous rocks [3]. In these experiments the production of a large number of small radical fragments and acetylene was demonstrated. In similar experiments, a CO, laser was used to pyrolyse organic plant materials such as cotton, sawdust and corn cobs [4]. Also in these experiments, a large number of small molecules such as acetylene, methane, carbon monoxide, carbon dioxide and water, were detected. In both of these laser pyrolysis experiments the intensities of the pyrolyzing lasers were very high (up to 10 J/pulse from the Nd : YAG) causing a great deal of molecular degradation. The intensity of the CO, pyrolysis laser strongly influences the extent of fragmentation as has been demonstrated in laser pyrolysis MS experiments on engineering polymers [5]. When the pyrolytic degradation was minimized by using lower CO, laser intensities, characterization of the composition of the polymer sample was simplified. As has been demonstrated by the extensive work of Schlag and co-workers [6], greater flexibility is possible when 2 lasers are used. The first laser, typically a CO, laser, is used for the desorption of the molecules from the probe. As virtually all molecules of interest, and also the probe itself, absorb infra-red radiation, no matrix is necessary. The second laser provides high energy photons for ionization. There are several advantages to this two laser approach: there is no interference in the spectrum from the matrix, the number of ions produced by post ionization is increased compared with single laser methods since the large number of desorbed neutrals can be ionized, the time delay between the desorption and ionization events can be controlled, the intensities of the desorption and ionizing lasers can be independently varied, and the wavelength of an ionizing dye laser used for MPI can be adjusted to selectively ionize a single component in a mixture. The sensitivity of the technique is high, in analytical applications Li and Lubman have shown that 5 to 10 ng of small aromatic molecules can be detected by laser desorbing from TLC plates and then ionizing with ultraviolet (UV) radiation [7]. In a spectrometer which employs no cooling and, therefore, no skimmer, subfemtomole levels of detection have been reported [8]. In our experiments, we follow this two laser approach. The first laser is a CO, laser of adjustable intensity and the second laser is either an ultraviolet laser (frequency-doubled output of a dye laser) for multiphoton ionization

49

or laser generated vacuum ultraviolet radiation (frequency-tripled output of the third harmonic of the Nd : YAG) for single photon ionization. Various pyrolysis-mass spectrometric and hybrid methods have been applied to the analysis and identification of polysaccharides including Py-MS, Py-GC/MS, Py-FIMS, DCIMS/(NH,) [9] and DC1 Py-MS/MS [lo]. However, if one wishes to use HPLC for the separation of large molecules such as polysaccharides and the pyrolysates of polysaccharides, detection can be a problem because many of the molecules do not absorb at the standard wavelength of detectors (254 nm). This problem can be solved by derivatization with substituents which do absorb in the UV, for example benzoyl groups. Perbenzoylation of cellulose and amylose pyrolysates followed by HPLC and MS analysis has shown promising results for the characterization of these materials [ll]. To be ionized with UV laser radiation, absorption of UV light is also required and so we selected a (per)benzoylated compound, cY-D-glucose pentabenzoate, for our initial study. A second class of molecules which we studied are the porphyrins which are often found at the active site of important biological systems such as chlorophyll and hemoglobin. In some fossil fuels, porphyrin molecules serve as biological markers indicating the origin of the materials [12]. To model natural porphyrin containing materials, we began with a commercial sample. A nonselective radiation source for molecules which do not absorb in the UV region of the spectrum is laser generated vacuum ultraviolet (VUV) radiation. Many molecules have very large absorption cross-sections of similar magnitudes in the VUV which suggests that VUV ionization would be very useful in quantitative applications. As examples of single photon ionization of compounds which have no UV absorption, SPI TOF mass spectra were measured for n-hexane and cyclohexane.

EXPERIMENTAL

The LD-MPI TOF mass spectrometer is composed of a CO, desorption laser (Pulse Systems LP-30), a Nd : YAG (Quanta Ray DCR-3) pumped dye laser (Spectra Physics PDL-3) with frequency doubling crystal ( P-BahO,), a reflectron time-of-flight [13] mass spectrometer (Bruker Franzen) with pulsed nozzle (Bosch), a delay generator (Stanford Research Systems DG535), a high speed digital oscilloscope (LeCroy 9450), digital plotter (HP 7475A) and a computer (Olivetti M280). Fig. 1 shows a block diagram of the configuration of the instrumentation. The spectrometer functions as follows: the surface of the sample on a stainless steel probe is irradiated with a pulse of intense IR light (10 pm, 10 msec pulse) from the desorption laser. A ZnSe polarizer is used to regulate the intensity of the CO, laser which has a maximum output of about 60 mJ/pulse. As the particles desorb from the surface, the nozzle between the

50

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gSp8ntor).

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(high pressure) gas reservoir and the vacuum chamber opens and sweeps the molecules towards the TOF/MS. Due to the adiabatic expansion of the Ar gas into the ionization chamber, the molecules are cooled. After the molecules have passed through a skimmer, an ionizing laser pulse of UV light (7 ns, 0.1 to 5 mJ/pulse) creates positive ions which are accelerated into the mass spectrometer. The ions pass through the drift region and are reflected by an electrostatic mirror toward the detector. The reflector serves to energy focus the ions: the ions of a given mass which have a higher kinetic energy penetrate deeper into the reflector before they are reflected and thereby travel a longer path than their slower neighbours with the same mass. All ions with the same mass to charge ratio arrive at the detector within a few nanoseconds of one another. The ion current is the time-dependent signal which is digitized and transferred to the computer for further manipulation; the minimum width of the signal is determined primarily by the laser pulse width, and is in our case about 8 ns. The Nd : YAG pump laser runs at 10 Hz so a maximum of 10 spectra per second can be collected; single shot spectra are also possible. The reflectron TOF/MS has much better resolution than typical linear TOF instruments while preserving the, in principle, unlimited mass range which typifies TOF spectrometers. We have measured

51

a resolution of 3450 at m/z 690. Due to the multichannel nature of the TOF detection, a complete spectrum is obtained with every pulse and in many cases single shot spectra show a good signal to noise ratio. The VUV radiation, 118.2 nm (10.48 ev), with which these molecules were ionized was generated by frequency tripling the third harmonic (354.6 nm) of the Nd : YAG laser in a Xe cell. The experimental details of the laser generation of the VUV will be published elsewhere [14]. The hexanes were introduced directly into the ionization chamber via an effusive source which could be heated. Protoporphyrin IX dimethyl ester was obtained from Aldrich and (Y-Dglucose pentabenzoate (MW 700.7) was purchased from Sigma. Cyclohexane (99%) and n-hexane (99%) were obtained from Merck. The spectra shown are of the compounds as received without further purification.

RESULTS

AND DISCUSSION

The distance along the horizontal axis gives the time of flight of the ion; since the axis is linear in time, the distance along the axis is proportional to the square root of the mass to charge ratio of the ion. As has been observed in the past [15], the results of photofragmentation are quite different from those of electron impact (EI). Fig. 2 shows the mass spectrum of a-D-ghCOSe pentabenzoate obtained after summing the spectra obtained with UV multiphoton ionization after laser desorption. A dominant fragment peak is m/z = 105, the benzoyl group itself C,HSCO+, just as in the Py-MS spectrum. Loss of CO from the 105 fragment, or cleavage of the bond between the phenyl group and the rest of the molecule, gives C,Hf , m/z = 77. Other major fragments are those typical of a phenyl group at m/z = 51, C,H,+, and m/z = 27, C,Hl. The molecular ion (700 Da) is clearly visible in the laser desorption mass spectrum. Although not visible in the plot shown in Fig. 2, there are also resolved isotope peaks at 701 and at 702 Da. In the Py-MS mass spectrum (not shown), the molecular ion is extremely weak, but the large number of high mass fragments which do appear are very useful in structural analysis. In our MPI mass spectrum, no high mass fragments other than the peaks around 700 Da are visible; apparently the charge is carried by the benzoyl group and its smaller fragments. In Fig. 3 some results from laser desorption MPI mass spectra of protoporphyrin IX dimethyl ester ionized with 259.6 nm radiation are shown. This particular porphyrin molecule was selected for our initial experiments because this technique has been shown to he very sensitive for this molecule; it has been detected at subfemtomole levels by the group of Zare [7]. For the spectrum shown in Fig. 3(a), the ionizing laser intensity was quite low, 0.2 mJ/pulse, which when focused, corresponded to an intensity

52

z. 6. ?' 3 9g. : 2 0 2.

703 1

o~.:..~.~.,~....~~.~....~.........~~~~~~~~"'~""~ 100 120 140 TOF Cusl

160

180

200

Fig. 2. Laser desorption MPI-TOF mass spectrum of a-D-glucose pentabenzoate. In the drawing of the molecule, Phe indicates a phenyl group. The horizontal axis gives the time of flight, the peaks are labelled with their corresponding masses. This spectrum is obtained by summing the results of 33 laser pulses.

of approximately 9 X lo6 W/cm2. Figures 3(b) and 3(c) show the increased fragmentation which occurs when the ionizing laser intensity is increased. (The relatively intense peak at 65.5 msec, m/z = 90, in Fig. 3(b) is caused by an impurity (methyl benzoate) from an earlier experiment.) For all three spectra the desorption laser intensity was constant, 5 mJ/pulse, and was focused to a spot with a diameter of about 1 mm (6 x lo4 W/cm2). Concentrating our attention on Fig. 3(c) which shows the most fragmentation, we see that the largest peak in the high mass region of the spectrum is the molecular ion at 590 Da. Next to this are the isotope peaks at 591 Da and 592 Da. The spectrum of mesoporphyrin IX dimethyl ester measured by Grotemeyer et al. [6b] showed similar fragmentation behaviour. (The only difference between the two porphyrin molecules is that there are vinyl rather than ethyl groups at carbons 8 and 13 in the protoporphyrin IX dimethyl ester.) As is typical for a substituted porphyrin macrocycle [16], the high mass peaks are all from fragmentations of the side chains and not of the macrocycle itself. Other than the molecular ion peak at 590 Da (M)+, there are also some other lower intensity, high mass peaks most of which can be easily assigned to fragmentations of the ester side chains: 576 (M - CH2)+, 518,519 (M - CHCOOCH,)+, 444 (M - CH,COOCH,, - CH2COOCH3)+,

53

03..

0

.,.........,.........,.........,.........I

20

40

60

80

160

180

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80

100

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120

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~~~~....,.~....~..,.,,.,.,,, 0

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Fig. 3. Laser desorption TOF mass spectra of protoporphyrin IX dimethyl ester. Wavelength of ionization laser for MPI was 259.6 nm. COz laser intensity 5 ml/pulse. The m/z ratio of the fragments is labelled: (a) “Soft” MPI, spectrum is sum from 21 laser pulses with 0.2 mJ/pulse; (b) “Partially hard” MPI spectrum is sum from 36 laser pulses; (c) “Hard” MPI, spectrum is sum from just 6 laser pulses with 0.4 ml/pulse.

54

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- CH2CH2COOCH3)+, and 416 (M - CH,CH, 430 (M - CH,COOCH,, The apparent loss of CH, (576 Da) is COOCH,, -CH2CHzCOOCH3)+. due to incomplete esterification of the acid. In the low mass, short time, region of the spectrum is m/z = 12, Cf, followed by smaller peaks at m/z = 13, CH+, and 14, CH:, and then a larger peak at m/z = 15 which probably has contributions from both CHZ and NH+. In the following groups, this pattern of well resolved peaks increasing by single mass units is continued. The intensity of the laser beam in the centre of the ionization volume is sufficient to completely break up the molecule. In contrast, Figs. 4(a) and 4(b) demonstrate that VUV can be used to ionize n-hexane and cyclohexane with negligible fragmentation. The molecular ion and its isotope peak are the only peaks present in the time of flight spectrum. If one desires to do so, the fragmentation of these alkanes can be increased by increasing the temperature of the effusion source. If both the 118 nm and the (remaining) 335 nm radiation are focussed on the sample, the fragmentation is also increased because the ions formed by the VUV irradiation absorb the UV and further dissociate [14]. The application of the methods of laser desorption and laser ionization offers the possibility of exploring a variety of problems typically investigated with pyrolysis methods. The advantages of this technique include facile control of the degree of fragmentation especially when SPI is utilized, additional data for identification of unknowns from the wavelength depen-

55

0

40 TOF Cusl

60

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, ,

80

Fig. 4. SPI-TOF mass spectra of three hexanes at 20°C. Wavelength of ionization laser was 118.2 nm: (a) n-hexane, average of 256 pulses; (b) Cyclohexane, average of 51 pulses.

dence of the UV absorption in MPI, the very short duration of the desorption event, and the occurrence (photo)chemical fragmentations which can aid in structural assignments.

ACKNOWLEDGEMENTS

We would like to thank P.W. Arisz for the Py/MS spectrum of a-D-glucase pentabenzoate and useful discussion. We thank J. Puroveen for the Py/Ms spectrum of protoporphyrin IX dimethyl ester. This work is part of the research program of the Foundation for Fundamental Research on Matter, FOM (Fundamenteel Onderzoek der Materie) with financial support (in part) from the Netherlands Technology Foundation, STW (Stichting voor de Technische Wetenschappen).

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