Anal. Chem. 2007, 79, 7259-7266
Accelerated Articles
Properties of C84 and C24H12 Molecular Ion Sources for Routine TOF-SIMS Analysis Gregory X. Biddulph, Alan M. Piwowar, John S. Fletcher, Nicholas P. Lockyer, and John C. Vickerman*
Manchester Interdisciplinary Biocentre, University of Manchester, Manchester, M1 7DN, UK C84+ and coronene (C24H12+) have been studied as primary ions for use in secondary ion mass spectrometry. A representative range of samples has been used to compare the effectiveness of each primary ion with the existing C60+, Au+, and Au3+ primary ions. It was found that C84 is the most effective primary ion providing higher secondary ion yields and a high molecular to fragment ion ratio. Coronene had a performance similar to C60. Coronene and C60 primary ions were also used to extend a previous study of matrix suppression/enhancement effects. The C60 was found to ameliorate this effect, possibly due to the increase in protonation in polyatomic sputtering, and coronene was found to further reduce suppression showing evidence of a chemical effect.
Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has seen much development in recent years in the field of primary ion sources. Using atomic primary ion sources, TOF-SIMS has traditionally had a number of problems when analyzing organic samples. If primary ion beam currents of more than 1013 ions/ cm2 (the static SIMS limit) are used, then damage accumulates in the sample resulting in a loss of signal from intact molecular species.1 Another challenge is the small amount of sputtered material that is ionized and therefore detectable. The recent development of polyatomic primary ion sources has made progress toward solving these problems. Cluster ion sources, such as Aun+ and Bin+, and polyatomic beams, particularly C60+, have been shown to produce a 30-100× increase in secondary ion yield (counts/primary ion) over monatomic gallium sources with performance particularly enhanced at * To whom correspondence should be addressed. E-mail: John.Vickerman@ manchester.ac.uk. (1) Vickerman, J. C., Briggs, D., Eds. ToF-SIMS Surface Analysis By Mass Spectrometry; Surface Spectra, I M Publications: Manchester, UK,, 2001. 10.1021/ac071442x CCC: $37.00 Published on Web 09/07/2007
© 2007 American Chemical Society
higher mass where more chemically and biologically specific information is found.2,3 Molecular dynamics simulations suggest that this improvement is a result of a change in the sputtering mechanism between monatomic and polyatomic primary ions. The energy of the projectile is partitioned between the constituent atoms as the primary ion breaks up on impact causing multiple sputtering events with less penetration of the primary beam atoms.4,5 A coarse-grained molecular dynamics study by Smiley et al. suggests that at 5-keV impact energy there is an optimum size of fullerene projectile to deliver the highest sputter yields of benzene. C20+ and C60+ primary ions were found to give the highest sputter yields. It is suggested that the energy of these ions is deposited in a region 15-20 Å below the surface, which is particularly favorable for the emission of high yields of intact benzene molecules.6 This implies that there is no benefit to be gained from using a large fullerene molecule as the penetration depth may not be sufficient for the efficient sputtering of molecules and suggests that perhaps C60 is the optimum projectile at 5 keV. Experimental studies using massive gold clusters7 have shown a general increase in sputter yield with primary ion cluster size, when the gold cluster has more than five atoms. Gillen and Fahey have studied the effects of using a number of small carbon clusters for SIMS at 5.5 keV.8 They reported a large nonlinear enhancement in secondary ion yield as the size of a carbon cluster was (2) Davies, N.; Weibel, D. E.; Blenkinsopp, P.; Lockyer, N.; Hill, R.; Vickerman, J. C. Appl. Surf. Sci. 2003, 203-204, 223. (3) Weibel, D.; Wong, S.; Lockyer, N.; Blenkinsopp, P.; Hill, R; Vickerman, J. C. Anal. Chem. 2003, 75, 1754. (4) Nguyen, T. C.; Ward, D. W.; Townes, J. A.; White, A. K.; Krantzman, K. D.; Garrison, B. J. J. Phys. Chem. B 2000, 104, 8221. (5) Postawa, Z.; Czerwinski, B.; Szewczyk, M.; Smiley, E. J.; Winograd, N.; Garrison, B. J. J. Phys. Chem. B 2004, 108, 7831. (6) Smiley, E. J.; Winograd, N.; Garrison, B. J. Anal. Chem. 2007, 79, 494. (7) Guillermier, C.; Della Negra, S.; Rickman, R. D.; Pinnick, V.; Schweikert, E. A. Appl. Surf. Sci. 2007, 252, 6529. (8) Gillen, G.; Fahey, A. Appl. Surf. Sci. 2003, 203/204, 209.
Analytical Chemistry, Vol. 79, No. 19, October 1, 2007 7259
increased from one to eight with the C8- primary ion producing much more than 8 times the secondary ion yield of the C1primary. However, they reported that this nonlinear effect appears to saturate when there are between 8 and 10 constituents in these carbon clusters. In order to investigate whether C60+ is indeed the most effective of the fullerene primary ions, a larger fullerene, C84+, has been chosen to compare with C60+, Au+, and Au3+ primary ions. The related molecule coronene C24H12 will also be studied to investigate the effectiveness of a smaller carbon containing cluster primary ion. The chemical environment in which the molecule of interest is analyzed, known as the matrix, can have a profound effect on the detection of that molecule in TOF-SIMS.9,10 It has been shown that some molecules that are seen quite clearly when analyzed on their own can be almost completely suppressed in the presence of other molecules.12 In the case of the ionization of biomolecules by proton transfer (i.e., generation of [M ( H]( type ions), a strong correlation between suppression/enhancement and gasphase basicity has been observed.11,12 This observation is in agreement with those from MALDI experiments where the matrix effect is exploited to assist in the ejection and ionization of molecules.13 Although less widespread in SIMS, the application of a suitable matrix can lead to an increase in the detection of molecular species, particularly at higher mass (matrix-enhanced SIMS).14 A factor in the decision to investigate the use of coronene (C24H12) as a primary ion in SIMS was the hydrogen content of the molecule. It has been suggested in previous studies that increased proton availability during molecule emission may enhance the formation of [M + H]+ and related ions.15,16 It is hypothesized that hydrogen atoms will become available for reaction with the analyte in the selvedge when the coronene breaks up upon impact with the sample. There is also evidence that with clusters, C60 in particular, more protons are liberated during sputtering. A possible explanation for the nonlinear increase in reported secondary ion yields.16 Hence, in this investigation, we also assess the use of C24H12+ alongside C60+ with respect to the ability of these ions to ameliorate the suppression/enhancement observed in mixed chemical systems, using the same chemical systems where the effect has been reported by Jones et al.12 EXPERIMENTAL SECTION The analysis was performed on a Bio-TOF SIMS instrument that has been described in detail elsewhere.17 Concisely, the instrument comprises a fast entry load lock, sample preparation chamber and a main analysis chamber. A reflectron TOF mass analyzer is used with 20 kV postacceleration to aid the detection (9) Wu, K. J.; Odom, R. W. Anal. Chem. 1996, 68, 873. (10) Delcorte, A.; Me´dard, N.; Bertrand, P. Anal. Chem. 2002, 74, 4955. (11) Jones, E. A.; Lockyer, N.P.; Vickerman, J. C. Appl. Surf. Sci. 2006, 252, 6727. (12) Jones, E. A.; Lockyer, N. P.; Kordys, J.; Vickerman, J. C. J. Am. Soc. Mass Spectrom. 2007, 18, 1559. (13) Zenobi, R.; Knochenmuss, R. Mass Spectrom. Rev. 1998, 17, 337. (14) Altelaar, A. F. M.; Klinkert, I.; Jalink, K.; de Lange, R. P. J.; Adan, R. A. H.; Heeren, R. M. A.; Piersma, S. R. Anal. Chem. 2006, 734. (15) Wucher, A.; Sun, S.; Szakal, C.; Winograd. N. Anal. Chem. 2004, 76, 7234. (16) Conlan, X. A.; Lockyer, N. P.; Vickerman, J. C. Rapid Commun. Mass Spectrom. 2006, 20, 1327. (17) Braun, R. M.; Blenkinsopp, P.; Mullock, S. J.; Corlett, C.; Willey, K. F.; Vickerman, J. C.; Winograd, N. Rapid Commun. Mass Spectrom. 1998, 18, 1246.
7260
Analytical Chemistry, Vol. 79, No. 19, October 1, 2007
of higher mass species. The instrument is equipped with a massfiltered 40-kV C60 ion gun and a mass-filtered 25-kV Au/Ge liquid metal ion gun (both Ionoptika Ltd.), although in this study, Au+ and Au3+ data were acquired at 20 keV. Where necessary, charge compensation can be performed by irradiation of the sample with low-energy (25 eV) electrons between successive primary ion pulses. The C60 ion source has been described previously3 and utilizes electron impact to ionize C60 vapor following sublimation in a heated oven. As such, the gun can be used to produce C84 and C24H12 primary ions by replacing the C60 powder charge and reoptimizing the running temperature and electron impact energy for the desired primary species. C84. The C84 was purchased from Nano-C, Inc.. It is known from previous studies of fullerenes18,19 that C84 requires greater temperatures then C60 to achieve the same vapor pressure; for example, at 475 °C, the vapor pressure of C60 would be 1 × 10-3mbar compared to 2 × 10-6 mbar for C84. When running with C60, a source temperature in the 400-430 °C range is usual depending on the current requirements. Primary ion current of C84+ was generated in useful quantities over 440 °C, with the source temperature of 450 °C being maintained during the SIMS analysis. This operational temperature produced a continuous beam current in the order of 100 pA. Coronene. The coronene was purchased from Sigma-Aldrich (Poole, UK). Locklear et al. have reported sublimation temperatures and ionization efficiencies of coronene, and these were used as a starting point in the optimization of our ion source.20 Sublimation of the coronene was observed at temperatures above 200 °C. All analyses in this report were performed using an accelerating voltage of 40 kV for the C60+, C84+, and C24H12+ and 20 kV for Au3+ and Au+unless otherwise indicated. Sample Preparation. For the study of secondary ion yield as a function of primary projectile, a range of chemically dissimilar samples was selected. These were a metal (silver), a peptide (cyclosporin A), a drug (haloperidol), a biomolecule (cholesterol), and a phospholipid (dipalmitoyl phosphatidycholine, DPPC). All of the samples were supplied by Sigma-Aldrich with the exception of the silver foil, which was sourced from Goodfellow Cambridge Limited. The silver foil was washed in methanol and water prior to entry into the vacuum chamber, and the other chemicals were prepared as dried droplets onto 5 × 5 mm cleaned silicon wafers. The films analyzed in this way were many layers thick, and substrate SIMS signals were not observed. For studies of the matrix effect, 2,4,6 trihydroxyacetophenone (THAP), cytosine, and barbituric acid (BA) were purchased from Sigma-Aldrich and were analyzed without further purification. THAP was dissolved in a 1:1 v/v acetonitrile/water mixture at a concentration of 0.05 M while cytosine and BA were both dissolved in a 1:1 v/v methanol/water mixture at a concentration of 0.05 M. For a mixed sample, aliquots of the pure solutions were mixed as equal volumes. To prepare the film, 5 µL of the required (18) Piacente, V.; Palchetti, C.; Gigli, G.; Scardala, P. J. Phys. Chem. A Letters, 1997, 24, 101. (19) Piacente, V.; Gigli, G.; Scardala, P.; Giustini, A.; Ferro, D. J. Phys. Chem. 1995, 99, 14052. (20) Locklear, J. E.; Verkhoturov, S. V.; Schweikert, E. A. Int. J. Mass Spectrom. 2004, 238, 59.
Figure 1. Spectra of cyclosporin A with 40-keV C60+, C84+, and C24H12+ and 20-keV Au3+ primary ions. The [M + H]+ peak at m/z 1202 has been expanded in the y-axis by a factor of 5 for clarity.
solution was pipetted onto a cleaned silicon shard and allowed to evaporate under ambient conditions. RESULTS AND DISCUSSION Cyclosporin A. Cyclosporin A is a cyclic polypeptide molecule mainly consisting of the amino acids leucine, valine, alanine, and glycine. It is used in medicine as an immunosuppressant drug following organ transplant surgery. Example spectra of cyclosporin A with 40-keV C60+, C84+, and C24H12+ and 20-keV Au3+ are shown in Figure 1: The most abundant form, used here, is cyclosporin A. The positive SIMS spectrum shows a strong [M + H]+ signal at m/z 1202 and a characteristic fragment at m/z 100. The immonium ions of the amino acids leucine (m/z 86), valine (m/z 72), alanine (m/z 44), and glycine (m/z 30) are also present and were used in yield calculations. Haloperidol. Haloperidol is a small drug molecule that is used in psychiatric medicine as an antipsychotic. The positive ion SIMS spectrum has three major peaks from the drug, [M + H]+ at m/z 376 and two characteristic fragments at m/z 165 and 123. DPPC. Dipalmitoyl phosphatidylcholine is a phospholipid with a phosphocholine hydrophilic head group and two equivalent palmitoyl fatty acid, hydrophobic tail groups and is highly abundant in many biological cell membranes. The positive ion SIMS spectrum of the molecule is dominated by an intense
fragment at m/z 184 that corresponds to the phosphatidylcholine head group. The [M + H]+ fragment is apparent at m/z 735 with a number of diagnostic fragments at lower m/z, [M - head group + H]+ at m/z 551 and single fatty acid chain with one fatty acid attached at m/z 313. Cholesterol. Cholesterol is a major constituent of cell membranes. The positive SIMS spectrum of cholesterol shows a large amount of low-mass organic signal. The most abundant of the molecular-type ions is the [M - H2O + H]+ fragment at m/z 369 overshadowing the [M - H]+ fragment at m/z 385, which in turn is more intense than the [M + H]+ fragment at m/z 387. Silver. Silver is a metal with two stable isotopes of 106.9 and 108.9 Da in the ratio of 52:48, respectively. The positive ion SIMS spectrum shows clusters of silver atoms, with odd-number clusters being more abundant in the spectra. The peak areas of the two isotopes were used for the n ) 1 silver data, with the sum of the isotope pattern used for odd-number clusters up to n ) 9 silver atoms. Secondary Ion Yields. Secondary ion yield data are shown in Table 1. The range of primary ion dose used for each of the ion beams is also shown in the table. To determine the yields shown in the table, at least three spectra were acquired from different locations on the samples and the yield spread in each case was less than 10-15%: The following plots are a graphical representation of the data in the table for clarity and ease of comparison. The secondary ion yields of the molecular ions for DPPC, haloperidol, cyclosporin A, and cholesterol, together with the m/z 184 fragment from DPPC are shown in Figure 2. The main trend is for C84+ primary ions to produce higher yields with C24H12+, Au3+, and C60+ delivering lower but similar yields whereas the yields from Au+ are much lower. Cholesterol is an exception in that the carbon-containing primary ions all generate similar secondary ion yields. Au3+ is very effective at sputtering haloperidol and DPPC but less so for the cyclosporin A molecular ion and curiously for cholesterol. In SIMS analysis, high mass molecular information is often the most useful. Figure 3 shows the ratio of the molecular ion signal to characteristic fragments. In the case of silver, the ratio of the n ) 9 peak to the sum of the n ) 1, 3, 5, 7 peaks was used. The fragments used were m/z 86, 184, 313, and 351 for the DPPC, the immonium ions of the amino acids for cyclosporin A, and m/z 165 and 123 for haloperidol. C84+ is the most effective primary ion for producing the more useful high-mass signal relative to fragment ions. C24H12+ and C60+ seem to behave similarly, which is perhaps surprising given the higher energy density of coronene C24H12+ primary ions. Au3+ is similar to C24H12+ and C60+ for the lower mass species and the silver clusters, but is less effective in delivering high-mass secondary ions from the larger peptide cyclosporin A. Au+ is generally much less effective, except in the case of the small drug molecule haloperidol. It should be noted that the use of the lower impact energy for Au+ and Au3+ would be expected to show a reduced amount of fragmentation than if they had 40-keV energy.21 A possible reason for the reduced fragmentation with C84+ primary ions is that the energy is distributed over a larger number of constituents compared to the other primary ions, which (21) Gilmore, I. S.; Seah, M. P. Appl. Surf. Sci. 2000, 161, 465.
Analytical Chemistry, Vol. 79, No. 19, October 1, 2007
7261
Table 1. Secondary Ion Yields Per Primary Particle as a Result of Bombardment with 40-keV C60+, C84+, and C24H12 and 20-keV Au+ and Au3+ for Silver, Haloperidol, DPPC, Cholesterol, and Cyclosporin A secondary ion yield (×10-5) +
sample
C84+
C60
primary ion dose (1010 ions cm-2)
34
5
coronene 0.1-0.4
Au3+
Au+
2.4-4.7 3.3-3.7
also an approximately linear increase in secondary ion yield with increasing C60 impact energy. The constituent atoms of coronene and C60 will have more energy than those from C84 as the total kinetic energy is the same but there are fewer constituent atoms. The equation below is an attempt to normalize the yields so that each constituent of the primary ion has the same kinetic energy:
YN ) NcYs
Silver +:
cluster Agn n)1 n)3 n)5 n)7 n)9 total
807.3 865.8 288.2 158.2 124.9 13526
[M + H]+ m/z 376 m/z 165 m/z 123 total
1621 1193 2873 55319
[M + H]+ m/z 735 m/z 551 m/z 313 m/z 184 m/z 86 total
6.1 2.84 3.27 1844 1427 21184
[M + H]+ m/z 387 [M - H]+ m/z 385 [M - OH]+ m/z 369 m/z 15 total
29.7 140.6 874.9 12 41547
[M + H]+ m/z 1202 m/z 100 (leucine) m/z 86 (valine) m/z 72 (alanine) m/z 44 (glycine) m/z 30 m/z 15 total
2505 1416 338 162 128 15013
1186 447.1 88.2 66.1 68.7 12125
345.4 155.3 40.6 20.4 19.6 2288
489.2 63.4 26.0 4.3 3.8 2174
Haloperidol 4690 952.2 2265 1061 4512 1956 66993 26670
1840 1576 2926 31394
423.9 421.4 613.3 10180
6.75 6.26 2.07 1963 1868 32804
17.1 11.8 3.8 1555 1287 15041
0.14 0.18 0.47 61.0 43.9 2231
Cholesterol 30.3 41.7 109.1 203 794.9 838 68.8 44.8 47629 48745
4.89 15.2 52.4 17.6 14340
0.50 1.81 6.46 26.6 6199
Cyclosporin 702 2247 1233 3364 5982 4726 951.4 1493 1535 542.6 921.4 820.5 151.7 498.9 124.5 287.5 696.9 473 151.7 574.6 244.4 53816 105354 74012
394.5 3207 751.9 340.5 1205 81.4 37.5 32375
43.6 1087 207.8 97.4 335.3 17.9 32.3 10076
DPPC 63.4 17 14.2 4037 2893 54174
Table 2. Damage Cross Sections and Efficiencies of Cholesterol [M - H]+, m/z 385, and the [M + H]+, m/z 376 Ion of Haloperidol with 40-keV C84+and C60+primary Ions cholesterol [M - H]+ primary ion C84+ C60+
σ
(10-14
cm2)
3.18 2.56
haloperidol [M + H]+
efficiency (cm-2)
σ (10-14 cm2)
efficiency (cm-2)
3.4 × 1010 5.5 × 1010
0.56 4.58
8.4 × 1012 3.5 × 1011
produces a softer impact per carbon atom resulting in less fragmentation. The effect of the increased number of simultaneous impacts may also produce a better cooperative liftoff effect for larger molecules in the selvedge. Under primary ion impact, the cluster molecule is considered to fragment entirely, partitioning the kinetic energy between the constituent atoms. It is long established that a linear enhancement in secondary ion yield is observed with increasing primary ion energy.22,23 Fletcher et al.24 have demonstrated that for many secondary ion species there is 7262
Analytical Chemistry, Vol. 79, No. 19, October 1, 2007
where YN is the normalized yield, Ys is the secondary ion yield, and Nc is the normalization factor of the mass of the primary ion relative to the mass of C84, being 0.71 for C60, 1 for C84, and 0.30 for C24H12. This analysis suggests that C84 is most effective in delivering high yield. Since the approach should factor out the energy effect to some degree, it suggests that the larger number of atoms and maybe the structure of C84 is beneficial in delivering higher yield. The increased yields observed for C60 over atomic primary ions has largely been attributed to increased sputter yields with these polyatomic ions.3 It is likely that this is true in the case of C84 although there may be a degree of enhanced ion formation; see below. Disappearance Cross sections. The disappearance cross section is a measure of the average surface area depleted of a surface species as a result of a single ion impact25 and when combined with the secondary ion yield of a particular mass fragment may be used to calculate the efficiency of a primary particle for the generation of secondary ions from a specific sample. The cross section, measured as the rate of decay of the signal corresponding to the peak of interest with respect to increasing ion dose, can be a result of two parameters; damage (destruction of chemical structure by ion impact) and removal. On a thin sample with a coverage of e1 monolayer, the loss of signal intensity will be predominantly a result of the removal by sputtering of the analyte by the primary beam. On thicker multilayer/bulk samples, the observed intensity changes are a result of the modification of the surface chemistry (or damage) by the primary particle. In organic samples, this modification is most often coupled to the accumulation of chemical damage and is manifest in the mass spectrum as a loss of intensity of higher mass chemically distinct fragments and an increase in low mass and atomic ions; this is known as the damage cross section, σ. Presented here are the damage cross sections calculated from the initial damage region. The damage cross sections for C84 and C60 are of the same order as a previous study.26 For cholesterol, the efficiencies of C84+ and C60+ are of the same order. This reflects the similar secondary ion yields observed. It maybe that the sputter yield (22) Anderson, H. H.; Bay, H. L. In Sputtering by Particle Bombardment; Behrisch, R., Ed.; Springer: Berlin, 1981; Vol. 1, pp 145-218. (23) Sigmund, P. In Sputtering by Particle Bombardment; Behrisch, R., Ed.; Springer: Berlin, 1981; Vol. 1, pp 9-71. (24) Fletcher, J. S.; Conlan, X. A.; Jones, E. A.; Biddulph, G. X.; Lockyer, N. P.; Vickerman, J. C. Anal. Chem. 2006, 78, 1827. (25) Ko ¨tter, F.; Benninghoven, A. Appl. Surf. Sci. 1998, 113, 47. (26) Jones, E. A.; Lockyer, N.P.; Vickerman, J. C. Int. J. Mass Spectrom. 2007, 260, 146.
Figure 2. Yield of m/z 184 phosphocholine head group fragment, [M + H]+ ions of DPPC, haloperidol, and cyclosporin A and [M - H2O + H]+ ion for cholesterol with 40-keV C60+, C84+, and C24H12+ and 20-keV Au3+ and Au+ primary ions.
Figure 3. Ratio of molecular ion to fragment ion yield for DPPC and haloperidol, ratio of molecular ion of cyclosporin A to the amino acid peaks and for silver the ratio of Ag9 to the sum of Ag1, Ag3, Ag5, and Ag7 with 40-keV C60+, C84+, and C24H12+ and 20-keV Au+ and Au3+ primary ions.
using the two primary ions is also very similar. The secondary ion yields observed for haloperidol [M + H]+ from C84 and C60 differ by a factor ∼3 (Table 1). The damage cross section is a factor of 10 less and this may reflect a higher sputter rate under C84+ bombardment. The efficiency using C84+ is 1 order of magnitude higher, reflecting the increase in the secondary ion yield and the lower damage cross section. Matrix Effects. The chemical environment or matrix in which an analyte of interest is found can influence the ionization of that species. Jones et al.12 have demonstrated that the gas-phase basicity plays a strong role in these matrix effects. Their results indicate that a species with higher gas-phase basicity has an increased chance for protonation over an analyte with lower gasphase basicity. As suggested in a previous study of amino acids and nucleic bases in ice,16 a possible means of ameliorating some of the unwanted matrix effects is to provide an excess source of hydrogen, which would supply an alternative means of forming the protonated molecular ion, [M + H]+. This section explores the idea that the primary ion beam may be able to supply those extra protons via the use of a hydrogenated polyatomic ion. In continuing with the study of Jones et al., we investigated both
pure and mixed systems containing cytosine, BA, and THAP with Au+, C60+, and C24H12+ primary ion beams. Cytosine. Cytosine is a nucleic base and is a major component of DNA and RNA. It has a gas-phase basicity of 918 kJ mol-1.27 When mixed with THAP (which has a lower gas-phase basicity) and analyzed using Au+, it has been reported that the [M + H]+ signal of cytosine is enhanced compared to its predicted yield. The predicted yield is calculated using spectra of a pure sample and considering the change in concentration in the mixture.12 Barbituric Acid. BA is an organic molecule that is the parent compound of many physiologically active barbiturates. The gasphase basicity of BA is 782 kJ mol-1.28 THAP has a higher gasphase basicity than BA and in mixed samples Jones et al. observed a suppression of the BA [M + H]+ signal, compared to the predicted signal, with a concomitant increase in the THAP [M + H]+ signal.12 (27) Russo, N.; Toscano, M.; Grand, A.; Jolibois, F. J. Comput. Chem. 1998, 19, 989. (28) Alvares, E. J.; Brodbelt, J. S. J. Mass Spectrom. 1996, 31, 901.
Analytical Chemistry, Vol. 79, No. 19, October 1, 2007
7263
Figure 4. Normalized yield of phosphocholine head group (m/z 184) and [M + H]+ ions of DPPC. [M + H]+ ions of haloperidol and cyclosporin A and [M - H2O + H]+ of cholesterol, with 40-keV primary ions. Normalized to the kinetic energy/mass unit of the C84+ primary ion.
Figure 5. Comparison of the ratio of yields of the [M+H]+ ions of BA, THAP, and cytosine between pure samples (predicted, red) and in the mixtures (observed, black). For clarity, the data are normalized to predicted at 100%.
2,4,6-Trihydroxyacetophenone. THAP is widely used in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry as a matrix enhancement molecule. The gas-phase basicity of THAP is 852 kJ mol-1.29 When it is mixed with cytosine, THAP’s [M + H]+ intensity is interpreted as decreasing relative to its unmixed [M + H]+ intensity when a Au+ primary ion beam is used. Conversely, when THAP is mixed with BA, THAP’s [M + H]+ is interpreted as increasing relative to its unmixed [M + H]+ intensity when using a Au+ primary ion beam.12 Each of the three compounds was analyzed individually and compared to mixed 1:1 molar systems consisting of THAP/ cytosine and THAP/BA using each of the three primary ion (29) Breuker, K.; Knochenmuss, R.; Zhang, J.; Stortelder, A.; Zenobi, R. Int. J. Mass Spectrom. 2003, 226, 211.
7264 Analytical Chemistry, Vol. 79, No. 19, October 1, 2007
beams, Au+, C60+, and C24H12+. The purpose of the study was to discover whether the hydrogen atoms from the coronene primary ion particle were chemically active in the desorption and ionization processes. C60+ primary ions were also used to see if the different sputtering mechanism associated with polyatomic ions had any influence on the suppression/enhancement phenomenon previously observed with atomic projectiles. Figure 5 displays the normalized secondary ion yield ratios for the analytes under investigation when collected using 20-keV Au+, 40-keV C60+, and C24H24+ primary ions. For data interpretation, the secondary ion yield for the molecular ion of each analyte was recorded from both pure and mixed systems. A predicted value for a 1:1 molar mixture, assuming no suppression/enhancement, was calculated for each compound used and expressed as a percentage as in the equation below
YA,pure × 100 YA,pure + YB,pure where A is the analyte of interest, B is the other analyte in system YA pure is the secondary ion yield of the protonated molecular ion [M + H]+ of A and pure refers to a single-component system, similarly for analyte B. This method provides a means of comparing an ideal secondary ion yield ratio for the analytes to those observed in a mixture. An assessment of the extent of suppression/enhancement can be made by comparing these values to those calculated in the same manner from the mixed samples:
YA,mix × 100 YA,mix + YB,mix where mix refers to a two-component mixture. In Figure 5, the pure sample yields are shown as the predicted values alongside the observed from the mixed systems. The pure yields were obtained with each primary ion under the same conditions for each analyte, making the results directly comparable. For ease in comparing the data visually, Figure 5 shows the predicted values set to 100% and the observed values normalized to this. The difference between the ratio of the [M + H]+ secondary ion yields in the pure systems to the [M + H]+ in the mixed systems shows whether the other component in the mixture is acting to suppress the ion of interest. The cytosine/THAP data (A) shows that using a C60+ primary ion source produces the greatest enhancement of cytosine when compared to Au+ and C24H12+ primary ion beams, with coronene displaying the lowest deviation from the expected experimental value. The enhancement of cytosine is in agreement with the predicted outcome based on the gas-phase basicities. For THAP in the cytosine/THAP (B), it is shown that a Au+ primary ion beam produces the most dramatic suppression of the THAP [M + H]+, while C60+ and C24H12+ display an ability to attenuate the suppression effect, with the C24H12+ primary ion beam providing the most relief from the matrix effect. BA/THAP (C) shows a trend different from that observed in the cytosine/THAP system. On the basis of the gas-phase basicity, the presence of the THAP with BA is expected to suppress the BA [M + H]+ as observed in the data collected using the Au+ primary ion beam. However, from Figure 5A it can be seen that the use of polyatomic primary ion beams appears to produce an excess of hydrogen allowing for the observed molecular ion of BA in the mixed system to be greatly enhanced over the expected value calculated from the pure sample. C24H12+ produces nearly a 560% increase over the expected result for the BA [M + H]+, while C60+ produces nearly a 200% increase. The results for the polyatomic data are contrary to what is predicted from the gasphase basicities and observed with atomic projectiles. The analysis of the THAP in the mixed BA/THAP (D) system using Au+ primary ions again shows the trend expected based on gas-phase basicities, i.e., an enhancement of the THAP [M + H]+ over the expected result, albeit in a less dramatic way. The data collected with the polyatomic primary ion sources appear to almost completely ameliorate the enhancement/suppression ef-
fects by producing observed values that are very close to the expected values. From the analysis, it is clear that the suppression/enhancement effects based on the gas-phase basicities are most noticeable for the monatomic primary ion source, Au+. The results from the use of the polyatomic sources vary from the monatomic results, suggesting that the use of a polyatomic primary ion source may ameliorate some of the unwanted matrix effects. This is perhaps not unexpected in light of the previous studies that showed increased yields of protons when ice and indeed hydrocarbon systems are bombarded by C60.15,16 The increased density of hydrogen atoms or ions should provide for enhanced proton attachment perhaps reducing the tendency to preferential protonation by the analytes with the highest gas-phase basicity. Further, the use of a hydrogenated polyatomic primary ion source (C24H12+) is shown in some instances to provide additional benefits. More work with these systems is forthcoming. CONCLUSIONS For the yield study, in every case, excluding cholesterol C84+ primary ions produced higher yields than the other primary ions. The simulations that provided some of the impetus for this study suggested that the optimum cluster size would be between C20 and C60.6 However, these studies were carried out at 5 keV although the effect of increasing projectile energy on the yield and penetration depth for C60 bombardment was briefly investigated. The penetration depth did not change significantly up to 40-keV impact, but sputter yields increased linearly with energy. Thus, the increased energy would not be expected to change the optimum cluster size significantly. However, the simulations deal with the sputter yield rather than the secondary ion yield measured here. It is possible that there is an increased ion yield using C84 as a consequence of increased proton formation along the lines suggested for C60 and coronene. Equally significantly, C84+ primary ions produced an increase in the useful molecular signals when compared to the low-mass fragments. C24H12+ was similar in behavior to C60+ primary ions over the mass range of species studied, but may be expected to perform less well at producing secondary ions over m/z 1000, as is seen in the case of cyclosporin A. The gold data were included for comparison. While Au3+ delivers good yields in the lower mass range, its performance drops off markedly at higher m/z as has been reported widely elsewhere. Au+ is the least effective primary ion in all cases in terms of yield and relative fragmentation. For both the gold beams, yields drop dramatically with ion fluence due to bombardment-induced damage, whereas as is seen above the damage, cross sections for the carbon projectiles are low resulting in high efficiencies and the possibility of operating beyond the static limit. Practically, C84+ proved more difficult to use because the ion gun hardware had been optimized for C60+ and the source oven works most effectively at temperatures lower than C84 needs to produce high current; the result is that the C84+data were collected with primary ion currents typically 1 order of magnitude lower than C60+. Technically, however, this is a problem that should not be too difficult to overcome. On the other hand, C84 powder is also less available and very much more expensive than C60. There are distinct benefits from using C84+, but the cost-benefit analysis will be dependent on the application and the user. Analytical Chemistry, Vol. 79, No. 19, October 1, 2007
7265
Coronene was relatively easy to tune, but the source lifetime was very sensitive to the temperature of the oven, and paradoxically, it proved quite difficult to maintain a steady low temperature. Compared to C60, there are no real benefits from its use in terms of yields. The matrix suppression/enhancement study has shown that there are definite benefits from using polyatomic primary ions. There is a clear reduction in the suppression of the species with the lower gas-phase basicity when C60+ is used, and it becomes even more evident using C24H12+ as the primary ion. It appears that adding hydrogen to the sputter region reduces the matrix suppression effects even further. It is likely that C84 would have shown further beneficial results; unfortunately, our supply of C84
7266
Analytical Chemistry, Vol. 79, No. 19, October 1, 2007
was exhausted before these experiments could be started. It is clear, however. that further studies using C84 and other hydrogen containing polyatomic projectiles are merited. ACKNOWLEDGMENT We thank Dr. Emrys Jones for helpful discussions and Sadia Rabbani for access to unpublished data. The financial support of BBSRC, the Royal Society of Chemistry, and the EPSRC Life Sciences Interface is gratefully acknowledged. Received for review July 6, 2007. Accepted August 7, 2007. AC071442X
