RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2003; 17: 2034–2038

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1149

Direct detection of particles formed by laser ablation

of matrices during matrix-assisted laser

desorption/ionization{

Sandra Alves, Markus Kalberer and Renato Zenobi*Laboratory for Organic Chemistry, Swiss Federal Institute of Technology (ETH), CH-8093 Zurich, Switzerland

Received 6 June 2003; Revised 10 July 2003; Accepted 11 July 2003

We report the detection of nanoparticles formed by irradiating matrix-assisted laser desorption/

ionization (MALDI) matrix samples. This is direct evidence for the ejection of large size aggregates

in the MALDI process. Nanometer-size particles were generated via a tunable solid-state UV laser,

irradiating a sample placed in a nitrogen atmosphere. Size distribution measurements were per-

formed using a differential mobility analyzer and a condensation particle counter. Particles in

the 10–1000 nm size range were detected. The dependence of the particle size distribution on the

laser fluence, wavelength and matrix was investigated. The observed effects are discussed and

related to the MALDI ablation dynamics and gas-phase processes. Copyright # 2003 John Wiley

& Sons, Ltd.

Desorption and ionization processes in matrix-assisted laser

desorption/ionization (MALDI) have been re-addressed, in

recent years, to overcome apparent contradictions of the

models proposed.1 New theories have proposed that clusters

play an important role as initial carriers of charge in the early

stage of the MALDI event. Also, in order to optimize deso-

rption and ionization yields, it is important to know the com-

position of the plume, but only indirect evidence about

cluster ejection following laser ablation exists.1–3

In this study, a new approach is pursued; the size

separation of particles generated from laser-ablated matrices.

These experiments were conducted at atmospheric pressure

with a conventional apparatus for particle sizing using a

differential mobility analyzer (DMA). In fact, aerosol genera-

tion by pulsed laser ablation from various solid surfaces like

refractory materials,4,5 silicon,6 or metallic7,8 targets, has

already been studied because of applications in engineering,

particularly in nanostructure synthesis. Control of the particle

size and composition is a critical issue for nanostructure

synthesis. Thus, a number of publications have investigated

the processes leading to particle formation through direct

droplet ejection or gas-phase reactions. It has been shown that

ablation parameters4,5,8,9 and also the gas pressure6–8,10

govern the particle formation by laser ablation.11

Although our aim is different, i.e., the investigation of the

MALDI mechanism, the process for particle production is

quite similar, with the exception that a solid MALDI matrix,

i.e., an organic molecular solid, is used. Nanoparticles were

formed using pulsed laser ablation at atmospheric pressure.

A basic assumption of this approach is that ablation at

atmospheric pressure allows quenching of the particle size

distribution immediately after the ablation event. In the

present study, it is shown how the ablation parameters affect

the nanoparticle formation. The observed effects are dis-

cussed and related to the ablation dynamics and the plume

evolution. Nucleation, condensation and coagulation pro-

cesses are discussed as important factors determining the

particle size distribution. One of the most important findings

is that an important part of the ablated mass is ejected in the

form of particles in the 10 nm to 1 mm size range.

EXPERIMENTAL

A schematic of the experimental set-up is depicted in Fig. 1.

The experiments are conducted in a windowed cell that holds

the target during laser irradiation. Nitrogen gas is continually

supplied to the cell with a flow of 0.3 L/min. The aerosol is

produced by laser irradiation using a Nd:YAG pumped opti-

cal parametric oscillator (OPO) laser (l¼ 266–337 nm, with a

repetition rate of 10 Hz). The laser beam is focused to an

approximately circular spot with a diameter of 0.4 mm.

The aerosol produced is flushed out of the cell by a N2 flow

in copper tubing and is run through a radioactive 85Kr

neutralizer in order to obtain a well-defined Boltzmann

equilibrium charge distribution of the particles. 85Kr is a b�

emitter, which ionizes the carrier gas. The carrier gas ions can

then react with neutral and charged particles leading to

mainly neutral plus a small fraction of singly charged aerosol

particles, positive and negative. The Boltzmann charge

Copyright # 2003 John Wiley & Sons, Ltd.

*Correspondence to: R. Zenobi, Laboratory for Organic Chemis-try, ETH Honggerberg, CH-8093 Zurich, Switzerland.E-mail: zenobi@org.chem.ethz.ch{Dedicated to Professor Jean-Claude Tabet on the occasion ofhis 60th birthday.Contract/grant sponsor: Swiss National Science Foundation;contract/grant number: 2000-066752.

distribution is size-dependent. For each particle size, the

fraction of singly positively charged particles, which are

measured in the DMA, is well known and can be used to

calculate the total number of particles at this size. Copper

tubing transports the aerosol to the analysis and detector

instrument. The size distribution of the positively charged

particles is characterized by measuring their electrical

mobility (a function of their size) using the differential

mobility analyzer (DMA) connected to a condensation

particle counter (CPC; Grimm, Ainring, Germany). The

DMA consists of two ca. 60 cm long concentric cylindrical

electrodes, separated by a gap of 10 mm into which the

particles are introduced. The inner electrode is connected to a

positive high-voltage supply. Large particles are selectively

deposited on a plate due to their high inertia and thereby

separated from the smaller particles and the gas flow. Such a

cut-off impactor is positioned in front of the DMA in order to

eliminate particles larger than 1 mm. Under the influence of

the electric field, the positively charged particles are

separated according to their electrical mobility diameter

and are introduced into the particle counter. The CPC detects

particles in the sub-micrometer size range with an efficiency

approaching unity. In this instrument, the sample flow is

passed through a chamber saturated with butanol vapor.12

Particles larger than a threshold size (i.e., approx. 5 nm) grow

by butanol condensation in a cooled tube following the

saturator. These larger droplets are then measured optically

via light scattering.

Common MALDI matrices, such as sinapinic acid (SA), a-

cyano-4-hydroxycinnamic acid (HCCA), 2,5-dihydroxybe-

zoic acid (DHB), 3-hydroxypicolinic acid (3-HPA), 6-

azathiothymine (ATT), 2-(4-hdroxyphenylazo)benzoic acid

(HABA) and nicotinic acid (NA), were investigated. Pressed

matrix samples (produced in a hydraulic press) are used in

these experiments to ensure a stable supply of material

during several minutes, the time required for generating a

size distribution by the DMA. Some particle concentration

measurements (using the CPC instrument only) were

performed from dried-droplet depositions, which showed

similar particle concentrations compared to irradiation of the

matrix pellet. We assume a similar optical penetration depth

for both solid samples, the thin layer of matrix crystals and

the pellet.

RESULTS

The DMA instrument allows the recording of particle size

distributions in the 10–900 nm size range. Particles produced

by MALDI from different matrices were found to fall into this

diameter range. Size distributions with and without the 85Kr

neutralizer were compared in order to quantify the yield of

positively charged particles during the laser ablation process

into the cell and that of charged particles produced in the

radioactive Kr source under equilibrium conditions (data

not shown). Almost the same size distributions were

obtained with a slightly higher particle concentration in the

absence of the 85Kr source, which is probably due to some

additional particle loss in the neutralizer unit. This observa-

tion implies that the MALDI process itself generates a charge

distribution of the particles close to equilibrium. Within the

observation window, mobility measurements revealed a

bimodal distribution. A typical example is shown in Fig. 2,

where small nanoparticles with diameters less or equal to

10 nm coexist with a majority of particles in the 100–300 nm

size range. We also note the almost complete lack of particles

in the 10–50 nm size range. Particles larger than 1mm are also

produced, as suggested by the tail in the large size range. The

number of micrometer size particles, however, is small,

because they can only be observed visually as deposits in

the cell and on the cut-off impactor after hours of experimen-

tation. This general picture is in good agreement with litera-

ture data reporting typical size distributions measured in situ

with the DMA for various targets. Particles with diameters

smaller than 10 nm have generally been detected to-

gether with larger size particles (�100 nm).4,5,9,10 In general,

these two distinct peaks in the size distribution were attribu-

ted to different (but simultaneous) processes of material

ejection.

The mechanism of particle generation from MALDI

matrices is still largely unexplored. Small particles can be

Figure 1. Schematic of the experimental apparatus used for the production and analysis

of particles from irradiated matrices.

Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 2034–2038

Particle detection from irradiation of MALDI matrices 2035

produced by gas-phase nucleation followed by condensation

processes with vapor molecules. Generation of large particles

could also occur by direct ejection of chunks (or ‘droplets’),

stabilized by collisions with the ambient gas. Coagulation of

nanoparticles is possible if the particle number concentration

is sufficiently high. Furthermore, the reverse process,

evaporation from larger precursors, may occur. In the present

study, it was difficult to determine the contribution of

particles produced in the ablation process or by gas-phase

processes occurring later, because of the dynamic evolution

of the aerosol with time and particle concentration. The gas

flow should rapidly quench such gas-phase nucleation and

coagulation processes as the plume components are carried

away from the target and diluted rather fast. The main factors

responsible for determining the final size distribution are the

initial plume density and the lifetime of nascent particles,

whose size is expected to change between their creation and

detection in the DMA. Experiments performed under

different pressure conditions have shown that a multimodal

distribution reflects distinct nanoparticle formation mechan-

isms.4,5,9,10 The general interpretation is that 10 nm particles

originate from nucleation and condensation processes of

single molecules desorbed after laser irradiation, whereas

larger size particles stem from direct ablation from the target

surface.4,5,9

We studied the dependence of the particle size distribution

on the ablation parameters (matrix, laser fluence and laser

wavelength). Figure 2 displays the particle size distributions

measured by the DMA for different laser fluence values on

irradiating pressed matrix at both 337 and 266 nm. An

increase in the particle number concentration with increasing

laser power can be seen for ablation of HCCA and DHB at

different wavelengths. This has been observed before in

related work on graphite and tungsten targets.9 For ablation

of HCCA, the mean particle diameter increases slightly with

increasing laser fluence (Fig. 2(a)), which we interpret as a

consequence of faster coagulation of smaller nanoparticles

when more material is available in the gas phase. In other

words, coagulation of 10–100 nm size particles contributes to

the formation of particles in the 100–200 nm size range5

during plume expansion. This behavior is expected, as the

coagulation rate is known to strongly depend on particle

density. However, the resulting increase in particle size is

small taking into account the mean particle diameter and the

large change in laser fluence. The distribution for DHB did

not show a variation with increasing laser energy at 266 nm

(Fig. 2(b)). This behavior may be related to the laser

wavelength; DHB ablation at 337 nm does lead to a small

increase in the mean particle diameter on increasing the laser

energy (data not shown).

Figure 3 shows the comparison of size distributions for

different UV wavelengths and matrices at fixed laser energy.

The decrease in the mean size of HCCA particles on changing

from 337 to 266 nm could be explained by greater self-

absorption of the shorter laser wavelength by the parti-

cles,13,14 i.e., the particles produced absorb the laser light

leading to some particle dissociation. This behavior is also

observed from HABA samples, but other matrices (DHB,

3-HPA and SA) did not exhibit a change in size distribution

within the laser wavelength. The self-absorption process is

expected to be a function of the optical properties of the

matrix.

The mean particle diameter shows a clear dependence on

the matrix studied (Fig. 4). Several matrices were studied in

Figure 2. Particle size distributions measured by the differ-

ential mobility analyzer at different laser energy values for

irradiation of (a) HCCA at 337 nm and (b) DHB at 266 nm.

Table 1. Measured mean particle sizes and full width half maximum values (FWHM) of size distributions after laser ablation of

different MALDI matrices for a laser energy of about 150 microjoules. Gas-phase proton affinities (PA, in kJ mol�1) and optical

absorption coefficients (e in cm�1) are also given

Matrix HCCA HABA DHB SA ATT 3-HPA NA

PA (kJ mol�1) from Ref. 19from Ref. 20

765 765 853 853 — — 899841 943 &850 887 — 896 907

e337 (105 cm�1) from Ref. 15 2 — 0.7 1.1 — — 0.2l¼ 337 nm mean size (nm) 205� 10 180� 10 165� 10 180� 10 200� 10 80� 10 —FWHM (nm) 280� 30 240� 40 200� 30 230� 10 240� 40 100� 20 —e266 (105 cm�1) from Ref. 15 0.8 — 1.6 0.5 — — 0.8l¼ 266 nm mean size (nm) 140� 20 110� 20 160� 20 — — 80� 10 130� 20FWHM (nm) 200� 20 180� 20 240� 40 — — 110� 20 160� 20

2036 S. Alves, M. Kalberer and R. Zenobi

Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 2034–2038

order to investigate the contribution of their physicochemical

properties in the particle formation. Laser ablation of 3-

hydroxypicolinic acid (3-HPA, see Fig. 4) produces a

particularly low particle size (80� 10 nm), while most other

matrices form particles in the 150–200 nm size range.

Which properties of the matrix determine the particle

formation process and size distributions? Certainly several

physicochemical properties can be considered. First, the

particle size distribution can be related to the amount of

ablated material, which depends on the penetration depth of

laser beam into the material, on the optical absorption

coefficient15 or on the matrix cohesive energy. However, the

available optical absorption coefficients of matrices (see

Table 1) cannot explain the differences in the self-absorption.

Other physical properties (ionization energy, sublimation

energy, etc.) were also considered without showing a clear

correlation. Secondly, only charged clusters are transported

across the DMA. The stability of charged particles in the gas

phase may be related to the matrix gas-phase properties such

as the ionization energy or the gas-phase basicity. For

example, a relation between basicity and cluster stability

has been reported in the works of Mautner16,17 and Kebarle18

using low molecular weight compounds, and can be expected

for MALDI matrices as well. Gas-phase stability of proto-

nated particles is expected to increase for less basic matrices.

However, large discrepancies exist between published

values of physical properties,15,19,20 and it was difficult to

find a clear correlation between the particle formation

process and the physicochemical properties of the matrices.

The craters formed by laser irradiation were also examined

by means of an optical profilometer to determine the ablation

volume. A total volume of about 107 mm3 was calculated for

� 1800 laser shots needed to record one size distribution. This

volume can be related to the total particle volume reaching

the particle sizer system. The total volume of detected

particles can then be estimated from the corrected size

distribution. This estimation gave &107mm3, implying that a

large part of the ablated material is detected in the form of

nanoparticles. This further implies that the mass contribu-

tions due to molecular desorption and ejection of micron-

sized chunks are not dominant, and that particle loss to the

walls of the cell and the tubing is minor.

Implication for MALDI mechanismsNew theories for MALDI ion production start with clusters as

the precursors of the observed ions1 in the early stage of the

MALDI process. In the model of Karas,1 the singly charged

ions produced are derived from charged clusters, but only

indirect evidence about cluster ejection following laser abla-

tion exists. The desorption of heterogeneous clusters of small

analyte and matrix molecules in a supersonic jet of argon, fol-

lowed by ionization through a multiphoton process, has been

studied; it was shown that singly charged analyte ions are

produced through intracluster proton or cation transfer reac-

tions.21 The possible contribution of such intracluster reac-

tions in MALDI ion formation has been discussed.

Molecular dynamics simulations3 show that laser ablation

from organic solids is characterized by two mechanisms of

molecular ejection. These authors reported that, below a

threshold value, mainly single molecules are desorbed from

the surface, while, at fluences exceeding the threshold, abla-

tion processes become more important and large molecular

clusters were present in the laser-induced plume. In other

research, simulations of the time-of-flight2 have shown that

ions are not directly produced from the target, but originate

from high mass precursors that desolvate under the high elec-

tric field during the pulsed ion extraction. Another study22 on

plume composition analysis was conducted using a trapping

plate in vacuum (10�6 Torr) and subsequent atomic force

microscope imaging of trapped ablation products. In addi-

tion to a thin molecular film corresponding to molecules or

small clusters, particles with diameter of less than 200 nm

were detected.

Our experiments are quite different from the latter study as

they are conducted at atmospheric pressure, where processes

such as nucleation or coagulation can contribute to particle

formation and growth. Experimentally, neither the laser

Figure 3. Particle size distributions measured by the differ-

ential mobility analyzer after irradiation of (a) HCCA and (b)

DHB at l¼ 337 nm and l¼ 266 nm, respectively, for a laser

energy of about 150 microjoules.

Figure 4. Particle size distribution measured by the differ-

ential mobility analyzer after irradiation of 3-hydroxypicolinic

acid (3-HPA) at 337 nm for a laser energy of about 150

microjoules.

Particle detection from irradiation of MALDI matrices 2037

Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 2034–2038

fluence nor the wavelength (both have an influence on the

particle density) strongly affects the mean particle size, at

least in the observed size range. The gas flow also did not

greatly influence the mean particle size (data not shown).

Nucleation and condensation processes are thought to only

weakly increase the size of particles in the 100–200 nm size

range. Our interpretation is that the observed peak in the

particle size distributions is mainly a result of coagulation of

smaller particles following the laser ablation event; given the

rapid dilution of the particles as they leave the ablation

plume, coagulation beyond a size of a few 100 nm simply

becomes too inefficient. We cannot make any statements

about the nascent particle size distribution in the very early

stages of the plume development. However, our results

provide direct evidence for production of nanoparticles by

the laser pulse, as has been already suggested for other

materials.4,5,9,10

These experimental observations can be interpreted as

direct evidence for the production of large size particles

by laser irradiation of MALDI matrices, certainly for the case

of atmospheric pressure MALDI. It is less straightforward to

relate these observations to a conventional vacuum MALDI

ion source. In both MALDI sources the initial processes of

laser light absorption and primary ion formation are the same,

and similar ejection of clusters is expected. Krutchinsky and

Chait23 have demonstrated that cluster ions composed mainly

of matrix molecules contribute to the chemical noise in

MALDI mass spectra. However, processes such as thermali-

zation8 of vibrationally excited ions, ion–ion and ion–

molecule reactions, and particle coagulation processes,

should be enhanced at atmospheric pressure. Indeed, a

charge distribution of the ejected particles close to equilibrium

is observed in our apparatus, probably due to multiple

collisions. Such a thermodynamic equilibrium is less probable

in vacuum. The existence of such cooling processes could

explain the softness of the atmospheric pressure MALDI

compared to a vacuum source. Finally, a somewhat smaller

particle diameter is expected at pressures typical for conven-

tional MALDI experiments, ca. 10�6 mbar. Nucleation

reactions with gas-phase molecules are less important under

vacuum conditions, and coagulation should be less promi-

nent also because of smaller plume confinement, whereas

such processes cannot be excluded at atmospheric pressure,

implying that smaller nanoclusters exist initially in the plume.

CONCLUSIONS

In conclusion, we have used a differential mobility analyzer

to study particle formation during laser ablation of organic

solids. We report the first direct detection of nanoparticles

generated by laser irradiation of MALDI matrices. The

experiments were conducted at atmospheric pressure and

were performed with a selection of several matrices. Two dif-

ferent particle sizes were detected: small particles with dia-

meters smaller than 10 nm, and larger particles in the 100–

200 nm size range. Nanoparticle production by laser ablation

was investigated using different laser energies and wave-

lengths. The effects of the laser parameters on the mean par-

ticle size and on the total particle concentration were

investigated for several matrices. We interpret our findings

to be due to nucleation and condensation processes for the

formation of the small particles, and coagulation processes

for the formation of the 100–200 nm particles, as previously

suggested for other materials.4,5,9,10 In addition, we have evi-

dence for direct ejection of particles by laser irradiation, at

least for atmospheric pressure MALDI conditions. An impor-

tant finding is that the particles account for the majority of the

ablated mass in MALDI.

AcknowledgementsFinancial support for this work from the Swiss National

Science Foundation (Grant no. 2000-066752) is gratefully

acknowledged.

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2038 S. Alves, M. Kalberer and R. Zenobi

Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 2034–2038