Laser desorption techniques of nonvolatile organic substances

305

International Journal of Mass Spectrometry and Ion Physics, 45 (1982) 305-313 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

LASER

DESORPTION

TECHNIQUES

F. Hillenkamp Institut fiir Biophysik, D - 6000 Frankfurt/M.

OF NONVOLATILE

ORGANIC

Universitat Frankfurt, 70 (W. Germany)

SUBSTANCES

Theodor-Stern-Kai

7,

ABSTRACT A survey of the state of development of Laser Desorption Mass Spectrometry of organic samples is given. The greatly different experimental and instrumental approaches, used by the different research groups are described and possible influences on analytical results are discussed. The most important features, common to all laser desorption mass spectra are presented in the results section. The final chapter represents an attempt to summarize the present state of knowledge about the ion formation processes involved. It points to different mechanisms believed to be operative in different techniques and to some of the still unresolved problems in understanding. INTRODUCTION Laser desorption

masspectrometry

(LDMS)

but one of a still increasing variety of techniques that have come into use during years for a masspectrometric analysis of

of

organic

samples

is

so called desorption the last about twenty organic samples commonly

considered too nonvolatile or thermaly labile to be analyzed by thermal evaporation and subsequent ionization in the gas phase. All these techniques (e.g. field desorption (FDMS), secondary ion mass spectrometry (SIMS) and fast atomic bombardment mass spectrometry (FABMS) , plasma desorption (FFIDMS), electrohydrodynamic desorption), though very different in the experimental approach show some striking similarities in the spectra generated, which in turn sugyest similarities in a basic step of ion formation. Strong correllations are also observed in several respects to spectra obtained by chemical ionization in the gas phase. The developments of new desorption techniques particularly for the analysis of "middle mass molecules" (ref. 1 and 2) has received strong impulses both from the growing demand for the analysis of such molecules e.g. in biochemistry and pharmacology and the techniques themselves as it became clear that a formerly inacessible group of organic samples could at least in principle 0020-7381/82/0000-0000/$02.75

0 1982

Elsevier

Scientific

Publishing

Company

306

be successfully meters have years, but tory scale future.

investigated. been made available most of the and routine

INSTRUMENTAL APPROACHES Almost any conceivable rometer and sample stage

Some laser desorption commercially during

development large scale

is still applications

going

masspectrothe last

combination of suitable laser, mass specthas been used, most of them with success a fact that prevents an exhaustive

at least in some respect, survey and limits the comparability of results. Lasers in the whole wavelength range from the far infrared the far ultraviolett have been used. C02-gas lasers emitting 10.6 urn have been used by various groups (refs. 3 through 3.06 pm wavelength of the Neodymium-YAG or Neodymium-Glass in the near infrared has also aswell as the frequency-doubled 353 nm and particularly the far ultraviolet (refs. 15-19). at 347 nm has been applied by laser pumped dye laser at 483 titute excimer

of

few

on on a laboraare still in the

to at 11).

The

laser

frequently been used (refs. 6,12-14) line at 532 nm, the - tripled at quadroupled line at 265 nm in the The frequency doubled ruby line one group (refs. 22-24) aswell as a nm (ref. 25). A group at the Ins-

Spectroscopy in Moscow has used a N2-laser and different lasers at wavelengths of 337 nm, 308 nm and 249 nm (ref.26). the duration of the laser pulses important parameter,

Another or the exposure time, used to irradiate the samples, also varies over many orders of magnitude. From pulses in the picosecond (- lo+ s) domain (refs. 27 and 28), through the nanosecond (- 10B8s) (refs. 6,7,10,11,13, through 26), and us-domain uo-4-10-5s) (refs. 6,12) to sample irradiation for several minutes with continuous wave C02-lasers (refs. 3 through 8), all modes have been applied. With pulsed lasers, single pulse (e.g. refs. 13 through 26) analysis aswell as multiple pulse exposure (e.g. ref. 32) for single spectrum acquision have been used. Time of flight mass spectrometers (TOF) with or without time focusing ion reflectors have been used most frequently and seem to gain increasing acceptance for desorption techniques, because they render the most complete spectral information particularly for single pulse exposure and give the most direct access to the investigation of solid state vs. gas phase reactions (refs. 11,15 Governed more by what mass spectrometers happened through 24,26,28). to be available than by specific choice static or scanning quadru-

307 poles (refs. 3 through 5,8,9,14), magnetic sector (refs. 6, 7) and double focusing instrumentscrefs. 6,7,10,11,72) have also been Interesting results with respect to ion formation processes used. have been obtained with double focusing instruments in the reverse order (MS/MS, ref. 13). Photoplates and, more commonly, open secondary electron multipliers aswell as channelplate have been used for ion detection. The sample-, irradiationand ion extraction-geometry also influence the analytical results. In all but

detectors

the

appears Laser-Micro-

to

Mass-Analyzer LAMMA areas of about 0.1 to several millimeters in In the LAMMA instruments (refs. 15 diameter have been irradiated. through 24,27) the sampled area is only about 1 urn in size. In all but most of the LAMMA experiments, samples were supported on bulk substrates and irradiated at angles between O" (refs. 6,7) and 80° to the surface normal, with the ions extracted at either 9o" (refs. 3 through 7) or O" to the normal. No special attention seems to have been given to the bulk substrates used, yet the degree of substrate absorption at the laser wavelength used, aswell as the thermal conductivity of the substrate may be of considerable influence on the results, as may be the thickness of the sample R layer. In the LAMMA 500 instrument thin sample layers are supported by organic foils of several ten nanometer in thickness, with the laser beam incident normally onto the film from one side and the Sample not only moreover relative

ions extracted normally to the other. preparation is also a very important factor. It influences the reproducibility of spectra to a varying degree it seems to influence fragmentation patterns aswell as intensities of most of the sample-specific ion signals.

This is e.g. obvious for the effect of alkali-metals often present as contaminants at unknown concentration or intentionally added as halide salts. It appears however that critical concentrations are not the same for the various techniques used. Hardin and Vestal (ref. 14) e.g. observed the (MLH + 2Na)+ -signal as base peak in many of their spectra of organics, even though no alkali halides had been added, whereas the (M + Na)+ or (M + H)+ ions are usually the more abundant ones in the spectra of the same substances obtained by other investigators (e.g. ref. inten23), even with tionally added alkali salts at considerable concentration. All results, so far published have been obtained from solid -6 - 10m3 mol/l samples, most prepared from solutions of about 10

308 concentration. the substrate Aerosols have

Varying amounts of solution have been dried onto in air or in vacuum, or electrosprayed onto it. also been prepared and impacted onto the substrate

or the spectra have been taken from powders or even organic solids (ref. powder granules e.g. of amino acids from those obtained after the powder solution dried onto a sample support

commercially available sample 29). Spectra obtained from are reproducibly different had been dissolved and the (ref. 30). The pH of the sol-

vent may also be of influence in certain cases. Among the parameters and different techniques that influence the spectra, sample preparation and sample-irradiation/ion-extraction geometry are the most difficult ones to asses and severly limit any intercomparison of results, obtained in different laboratories, this even more so, as reproducibility of results seems to be a problem with most if not all desorption techniques with or without lasers results

and is

very little accessible

information on the in the literature.

reproducibility

of

RESULTS Laser spectra complexes including cularly

desorption techniques have been successfully used to obtain of a large variety of organic or bioorganic molecules and such as oligosaccharides, glucosides, organic acids amino acids and oligopeptides, organic salts, partiquarternary ammonium salts, enzymes, polyand mononucleopharmaceutical agents such as tides and their building blocks, antibiotics and organic polymers. For detailed results the reader is referred to the original publications given in the references. In the many cases in which a given substance has been analyzed by more than one or even all the different experimental techniques, described above (this holds particularly for some saccharides, amino acids and nucleosides) the spectra show general qualitative agreement and moreover strongly resemble those, obtained with other non-laser desorption techniques. The most important common features are: - polarity is supportive, if not a prerequisite of desorption, in contrast to thermal evaporation where polar groups must, as a rule, be derivatized, to render a molecule volatile - ions are predominantly of the even electron type, protonation and deprotonation rather than electron abstraction or attachment Radical ions are only rarely generated from are common processes.

309

strongly aromatic compounds. - Cationization by alkali-metal nization observed

by other metal under suitable

ions

ions (e.g. conditions

is very Ay,Cu,Mg (refs.

frequent. etc.)

Catiohas also as has

13,19,20)

been

been anionization e.g. by chlorine (ref. 19). - Ions of both polarities are generated, usually at comparable abundances. They carry complementary information. For acidic (M - H)-) are found mostly in the compounds specific ions (e.g. negative ion spectra, for basic compounds specific ions (e.g. (M + Alkali)+) are more easily identified in the (M + H)+, positive ion spectra. frequent, even - Cluster ions such as (2M + Na)+ are relatively for parent molecules dalton. - For the techniques

with using

rel. very

molecular short,

a more or less smooth transition to observed with increasing irradiance. for the LAMMA technique this transition at identical irradiances for positive observation that deserves attention - Spectra of the

are qualitativly molecules analyzed,

influenced e.g. by

mass high

of

several

irradiance

hundred

laser

pulses

pyrolysis of the sample is It appears that at least does not always occur and negative ions, an for further clarification. by the immediate an organic matrix

So far, the vast majority of molecules investigated served had relative molecular masses below about 1000 occasionally ions up to M,/z = 2500 have been detected

surrounding (ref. 31). or ions dalton, (ref.

obbut 17,

18).

MECHANISMS OF ION FORMATION Any discussion of ion formation ly start from the striking similarity wavelengths used from the far UV,

in

laser desorption of spectra for

must certainall laser-

where many of the molecules analyzed exhibit strong electronic absorption,throughout the visible with little or no absorption to the far IR,where most organics again show strong, vibrational (e.g. C-O streching mode) absorption, aswell as for long CW-irradiation with thermal equilibrium in the sample, through intermediate time regimes down to pulses of 30 ps in time and irradiances of up to 10 12 Wcm-* in small spots, under which conditions thermodynamic equilibrium will most probably not be reached. techniques

This which

even also

holds in generate

comparison to the other similar spectra. There

desorption now seems

to

310

be a general agreement for these similarities of spectra to reflect that - no matter what the primary mode of sample excitation there is always a decisive step in the ion formation process that is governed by the chemical properties (i.e. molecular structure and structural stability, affinities, polarity etc.) of the molecules much as they are known to govern liquidor gas phase chemical reactions. One can most probably even reverse the argument to say that such a step is a necessary prerequisite for any ion formation from the majority of organic, particularly bioorganic molecules. There is good reason to believe that beyond this unifying, but very general principle important differences exist between the different desorption techniques that need to be investigated for a full exploitation and optimization of the various methods. In this respect, one must look for the more subtle differences in the results because the similarities in the results tend to obscure the differences in mechanisms rather than bringing them out. Two extreme examples may serve as a demonstration. In two recent publications (ref. 5,8) the groups of Rijllgen and Kistemaker were able to show conclusivly that ions in "desorption" spectra obtained with CW - CO2 lasers of only a few tens of watts power, are at least predominantly generated by ion-molecule gas phase reactions rather than being desorbed from the solid sample. For this technique to work, one needs areas of greatly different temperatures on the sample or substrate for a separate thermal evaporation of the alkali ions at high and organic molecules at lower temperature respectivly. Stall and Rollgen (ref. 32) aswell as Cotter and Yergev (ref. 33) have moreover independently shown that ions from certain organic compounds such as quarternary ammonium salts can indeed sample.

be generated by purely conductive thermal heating of the These results makes one wonder as to how involatile many of the organic molecules and ions really are, a question that certainly requires more thourough investigation. For several reasons (IO ns laser pulses, < 1 urn2 laser focus) the LAMMA technique is the other extreme. From an analysis of the line width of parent molecule-aswell as fragment ion peaks and the fact that spectra e.g. of amino acids or quarternary ammonium salts recorded with a straight TOF masspectrometer and one, equipped with an ion reflector, show no qualitative differences, it can be concluded that gas phase reactions, if present, are of only minor importance in

311

this

case.

The

observation out of pure

of cluster ions graphite samples

of very high (ref. 34) is

desorbed e.g. demonstration of this fact. For the same reasons decay of metastable ions in the gas phase can be samples analyzed with both spectrometers during 10 ns after an ion leaves the sample surface and

order, another

even unimolecular excluded for a time of about the some ten to

hundred microseconds it needs to reach the detector. believed that ion desorption in the LAMMA instrument predominantly a collective nonequilibrium process phase (ref. 21,34,35) with possibly an intermediate

It is now is at least in the condensed "liquid" state

to allow for the chemistry to take place. To what extent gas phaseor condensed phase -processes, or even other processes such as preformation of ions, contribute to the results of the other techniques described, is still a largely open question. One should be cautioned

however

to

conclude

equal

or

similar

formation

mecha-

nisms just from similarities in the spectra obtained, or to generalize principles without sufficient and detailed experimental proof. Quite in contrast to the results of all other investigators, one group has reported strong resonance effects for the ion yield of molecules that exhibit electronic absorption at the laser wavelength used (ref. 26). Unless it could be proved that these findings reflect gas phase reactions for which resonances would be expected or e.g. resonance enhanced field ionization (ref. 36), they would certainly cast some doubt on the concept of a truely collective excitation of the solid sample. The dependence of ion yield of various oligosaccharides on their absorption at the laser wavelength under CW - C02-Laser desorption, as reported by Stall et.al. (ref. 5), also poses the question, as to whether vibrational excitation enhances the ion-molecule gas phase reaction, or whether e.g. the temporal and spatial temperature gradients, induced by the laser irradiation evaporation of

in sample

the sample, molecules.

influence

the

rate

of

thermal

CONCLUSION Laser desorption has proved to be a simple, fast and very sensitive masspectrometric technique. Much work still needs to be done to explore its full capabilities, particularly with respect to the analysis of molecules with relative molecular mass above 1000. Future research will hopefully also result in a better understanding of the underlying physical and chemical processes and

312

thereby certainly

to

optimized still far

for the matrices principle.

detection is another

systems with is the future, of

organic promise,

constituents the

This survey paper could and very helpful discussions field group.

and particularly This help is

better performance. an analytical ion

not

with greatly

in

technique

complex,

holds

have been written with many of the the members acknowledged

Though microscope

at

even

organic

least

without colleagues

of our Frankfurt by the author.

in the in

many the

research

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