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Molecular mass determinations in coal-derived liquids by MALDI mass spectrometry and size-exclusion chromatography
ELSEVIER
PIh S0016-2361(97)00032-X
Fuel Vol. 76, No. 13, pp. 1225-1233, 1997 © 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0016-2361/97 $17.00+0.00
Molecular mass determinations in coal-derived liquids by MALDI mass spectrometry and size-exclusion chromatography Maria-Jesus Ldzaro a, Alan A. Herod a, Mike Cocksedge b, Mark Domin b and Rafael Kandiyoti a aDepartment of Chemical Engineering and Chemical Technology, Imperial College, University of London, London SW7 2BY, UK bDepartment of Pharmaceutical and Biological Chemistry, School of Pharmacy, University of London, Brunswick Square, London WClN lAX, UK (Received 28 November 1996; revised 12 December 1996) This study examined the effect of changes in instrument-related parameters on mass spectra obtained by MALDIm.s.: ion-extraction voltage, laser power and sample loading. Results were compared with those of size-exclusion chromatography (s.e.c.) using 1-methyl-2-pyrrolidinoneas mobile phase. A coal tar pitch and its pyridine-soluble and pyridine-insoluble fractions were used as samples. MALDI-mass spectra were obtained with no added matrix, since the sample absorbed strongly at the laser wavelength, 337 nm. Higher extraction voltages (up to 30 kV) led to the observation of higher masses in the whole pitch and its pyridine-insoluble fraction, where the presence of higher-molecular-mass material was indicated by s.e.c. Increasing the ion extraction voltage served only to accelerate species already ionized, without otherwise disturbing the sample: the use of higher ion extraction voltages must be therefore be considered as providing a more complete inventory of already ionized species. Increasing the laser power level and sample loading on the target led to similar changes in the spectra, although there were indications that for both these parameters, the results might no longer improve (and might eventually deteriorate) beyond optimum values, which appear to be sample-dependent. The low-power spectra revealed more structural information at low masses, where series of homologous peaks could be observed, compared with the spectra at high power. © 1997 Elsevier Science Ltd. (Keywords: coal tar pitch; molecular mass; MALDl-mass spectrometry)
The authors have recently investigated the application of laser desorption mass spectrometry (LD-m.s.) 1-6 and matrix-assisted laser desorption ionization mass spectrometry (MALDI-m.s.)6-10 to molecular mass determinations in coal-derived liquids. The work was initially undertaken to evaluate previous observations by size-exclusion chromatography (s.e.c.), by using an independent analytical technique. The s.e.c, work had suggested the presence of highmolecular-mass (MM) materials (up to 4000-6000 u) in a number of coal pyrolysis tars and liquefaction extracts 1i- 15. Preliminary work with a LIMA (laser ionization mass analysis) instrument gave single-shot spectra showing MMs up to the available detection limit (m/z 1 2 0 0 0 )I 'Z"4.5 In these experiments, the instrument was operated with a reflectron stage at an ion extraction voltage of 20 kV and with a defocused beam to prevent breaking-up sample molecules under the power of the laser. Subsequent experiments using a Kratos Kompact MALDI III instrument of more recent conception, in linear mode (i.e. in the absence of the reflectron stage), allowed co-addition of spectra: results showed masses between m/z 20000 and
30 000 in pyrolysis tars and liquefaction extracts 10 and up to m/z 270 000 in solid coals 9. These findings went far beyond the MM range previously identified by s.e.c., in which tetrahydrofuran (THF) had been used as mobile phase. Lafleur and Nakagawa 16 have demonstrated the successful use of the more polar 1-methyl2-pyrrolidinone (NMP) as mobile phase in s.e.c.; this modification overcame solubility limitations encountered in the use of THF and allowed the detection of a new range of high-MM materials near the exclusion limits of s.e.c. 17 16 columns . Lafleur and Nakagawa attempted to characterize the material eluting at the exclusion limit of the column by the use of standards and heated-probe mass spectrometry, which showed little ion abundance between masses 250 and 550 u, the upper limit of the scan. In the absence of m.s. evidence for high-MM materials, the materials at the exclusion limit of the column were perceived as polar rather than of large molecular size. These findings were interpreted in terms of a multimode mechanism for elution, based on molecular size with an earlier-than-expected elution of polar molecules. However, the perceived absence
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MALDI-MS and size exclusion chromatography: M.-J. L~zaro et al. of high-MM materials in heated probe m.s. could be reasonably interpreted in terms of limitations of evaporative and ionization processes involved in heated-probe m.s. compared with laser desorption. More recently, the authors have presented MALDI-TOF m.s. spectra of coal tar pitch fractions separated both by solvent fractionation6 (~ridine-solubles/insolubles) and by planar chromatography ; a Fisons VG TOFSPEC instrument was used in this latter part of the work. The bulk of material insoluble in THF and in pyridine showed up at the exclusion limit of the s.e.c, columns; higher MMs were found by m.s. in samples identified by s.e.c. (in NMP) to give larger peaks at shorter elution times. Furthermore, higher-MM materials could be observed when pyridineinsolubles were examined by MALDI-TOF m.s. on their own, compared with the whole pitch sample containing 15% pyridine-insolubles. A high mass limit above m/z 50000 was observed. The work provided good qualitative
agreement between MALDI-m.s. and s.e.c, and indicated that fractionation of complex mixtures allowed the observation of characteristics of the less abundant fractions with greater clarity 19. There is considerable evidence from the MALDI-m.s. spectra of polymer systems6 that agreement between molecular-mass distributions from MALDI-m.s. and s.e.c, can be improved when samples of low polydispersity are analysed. Preferential desorption and ionisation of lower-MM components has been observed in the MALDI-m.s. of simple mixtures of polystyrene standards 2°'21 Significant improvement in agreement between the two techniques was reported when the polydispersity of polysaccharide samples was reduced by fractionation, using preparative s.e.c, prior to MALDI-TOF m.s.22. In experiments with mixtures of polystyrene standards, it was also recently observed that the sensitivity of the MALDI instrument for the highest-MM polystyrene standard diminished with increasing polydispersity of the
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Figure 1 MALDI-TOFspectra at three different extraction voltages: 10, 20 and 30 kV: (a) "soft" pitch; (b) pyridine-insoluble fraction of
"soft" pitch; (c) pyridine-soluble fraction of "soft" pitch
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MALDI-MS and size exclusion chromatography: M.-J. Lazaro et al.
EXPERIMENTAL
I00
(c)
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Samples The coal tar pitch sample has been studied previously 1"2'5-7. Its elemental composition (wt%) was C 91.4, H 4.1, N 1.3, S 0.76 and O 2.4 (by difference). In this work, solubility fractions were prepared using pyridine; the pyridine-insolubles were 15 wt% of the pitch. The pyridine-insoluble fraction and the whole pitch sample were dissolved in 1-methyl-2-pyrrolidinone for subsequent examination. The pyridine-soluble fraction was used in (pyridine) solution.
50
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MALDI-TOF spectra were obtained with a Fisons VG TOFSPEC mass spectrometer (VG Organic, Manchester, UK) fitted with a nitrogen u.v. laser (337 nm) and a VAX 400-base data system with OPUS software. The linear TOF mode was used with different accelerating voltages of 10, 20 and 30 kV and two levels of laser power. The full mass range of > 300000 u was examined. The sample was applied to the target in NMP (or pyridine) solution and dried in a vacuum oven for 2 h before insertion into the mass spectrometer ion source. To test the effect of the amount of sample deposition, solutions of the whole pitch and pyridine-solubles and -insolubles were added to target spots at increasing dilution. The deposited sample layer may not necessarily be considered to have spread evenly on the target after solvent evaporation. Approximately 50 spectra were summed for each spot and both the most intense range of the spectra and the highest-mass approach to instrument baseline were examined. In this work MALDI-mass spectra were obtained with no added matrix, since the sample absorbed strongly at the laser wavelength, 337 nm.
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m/z Figure 1 Continued polymer mixture, in favour of ion intensities of lower-MM polystyrene standards, although the high-mass peaks remained visible 23. Therefore major sample-related problems remain to be resolved before the findings outlined above can be translated into a coherent method to obtain quantitative molecular mass distributions of coal-derived liquid mixtures. The present study examines the effect of changes in a number of instrument-related experimental parameters such as laser power, ion extraction voltage and sample loading on the mass spectra of coal-derived liquids obtained by MALDI-m.s. The m.s.-derived results have been compared with data from size-exclusion chromatography performed using 1-methyl-2-pyrrolidinone as the mobile phase. In the current work, a coal tar pitch and its pyridine-soluble and pyridine-insoluble fractions were used to study the effect of changes in these experimental parameters. In a separate study 24, the effect of the use of different matrix materials on the spectra of the pyridineinsoluble fraction was examined.
S.e.c. using NMP as solvent was carried out using a 30 cm long, 7.5 mm o.d. polystyrene-divinylbenzene polymer-packed s.e.c, column (3 mm, Mixed E, Polymer Labs, UK) operated at 85°C with a flow rate of 0.45 mL min -I, using a variable-wavelength Perkin-Elmer LC290 u.v. detector set at 350 or 450nm. Calibrations against polystyrene standards were performed at 262 nm, since NMP is opaque at 254 nm but partly transparent at 262 nm. The elution volume corresponding to the total exclusion limit of the column was - 5 . 0 mL (polystyrene molar mass 1.84 million) but the upper range of polystyrenes corresponding to a linear relation between log(molar mass) and elution volume was - 3 0 000-40 000 u. RESULTS AND DISCUSSION
MALDI-m.s. Effect of ion extraction voltage level on spectra.
Spectra for the 'soft' pitch and the pyridine-soluble and pyridineinsoluble fractions were obtained at three extraction voltages (10, 20 and 30kV) using maximum laser power [Figure l(a,b,c)]. At the lowest ion extraction voltage (10 kV), the continuum of peaks in the spectra of the 'soft' pitch and its pyridine-insoluble fraction extended only to m/z 4000-5000. The continuum of masses extended to m/z 6000-7000 at 20 kV and up to m/z 100000 (for the 'soft' pitch) at the maximum available extraction voltage of 30 kV. The intense peaks at low mass were also observed to shift to higher masses with increasing ion-extraction
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MALDI-MS and size exclusion chromatography: M.-J. L~zaro et al.
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Figure 2 MALDI-TOF spectra at high (i) and low (ii) laser energy and at different sample Ioadings: (a) "soft" pitch, original solution (0) and third dilution (3); (b) pyridine-soluble fraction, original solution (0) and second dilution (2); (c) pyridine-insoluble fraction at high laser energy, all four dilutions; (d) pyridine-insoluble fraction at low laser energy, all four dilutions; (e) high-mass limits of the pyridine-insoluble fraction, original solution (0) and second (2) and third (3) dilutions.
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MALDI-MS and size exclusion chromatography: M.-J. L&zaro et al.
the pyridine-soluble fraction contained significantly less high-MM material.
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voltage: for the 'soft' pitch sample [Figure l(a)] the peak shifted from m/z 300 at 10 kV to m/z 2000 at 30 kV, whereas for the pyridine-insoluble fraction [Figure l(b)], the peak was observed to shift from m/z 300 at 10 kV to m/z ~ 1400 at 30 kV. However, for the pyridine-soluble fraction [Figure l(c)] no significant shift to higher mass could be observed with increasing extraction voltage. Since the increase in ion extraction voltage serves only to accelerate species already ionized, without otherwise disturbing the sample, use of higher ion extraction voltages must be considered as merely providing a more complete inventory of ionized species, apparently by enhancing the kinetic energy imparted to higher-MM materials. It is not yet known what the eventual optimum extraction voltage may turn out to be. These data show furthermore that higher extraction voltages lead to the observation of higher masses only for samples where higher-mass materials are actually present (e.g. whole pitch and pyridine-insolubles): s.e.c. chromatograms presented below strongly suggest that
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Effect of changes in sample loading on spectra. The whole 'soft' pitch sample, its pyridine-solubles and its pyridine-insolubles were examined at an ion extraction voltage of 20 W. Figure 2(a) presents spectra showing the effect of successive dilution of the (whole) 'soft' pitch sample at maximum laser power (arbitrary setting 4.2) and the equivalent spectra at a lower laser energy setting (arbitrary setting 3.2); only the spectra for the original solutions and the third dilutions are shown, since the intermediate ones were very similar to the spectra of the original solutions. Figure 2(b) presents analogous spectra for the pyridine-solubles at 'high' and 'low' laser power; once again, not all levels of dilution are shown, since the spectra of the intermediate dilutions resembled those of the original solutions. Figure 2(c,d) shows 'high' and 'low' laser power spectra of the pyridine-insoluble fraction; here, spectra are shown for all dilutions since there was in fact a change at high power. Considering Figure 2(a) for the 'soft' pitch sample, the spectra were generated from loadings of 27.5, 6.9, 1.7 and 0.43 mg on the target; higher sample loadings led to overloading of the intensity scale of the summed spectra in all cases except for the most dilute spot. However, spectra for the pyridine-soluble and pyridine-insoluble fractions did not show evidence of overloading with increased sample loading, probably because the sample masses deposited were lower than for the whole pitch. The problem arises from the difficulty in removing NMP and pyridine from the sample, thus preventing accurate sample loadings from being determined. Except for the most dilute samples, the data for the whole pitch and for the pyridine-soluble fraction did not appear very sensitive to sample loading on the target. At low loadings, changes in the shape of the overall profiles are apparent, probably reflecting the lower overall intensity of the summed spectrum and the greater difficulty in finding regions of the target from which a sufficient number of spectra could be acquired before the sample was completely removed. The spectrum of the pyridine-insoluble fraction did change with dilution at high laser power, Figure 2(c), with loss of the relatively intense signal between 1000 and 2000 u in favour of the intense peak between 200 and 300 u; the changes were less marked at low laser power, Figure 2(d). Figure 2(e) shows the high-mass limits in the spectra of the pyridine-insoluble fraction with dilution, indicating an approach to instrument baseline at mlz between 100 000 and 200000, although the precise upper limits are not easily discernible. Clearly, more nearly quantitative methods for estimating upper mass limits need to be developed. Similar spectra were obtained for the pitch and the pyridine-soluble fraction (not shown). Changes in MALDI-m.s. spectra with variation in laser energy level. Lower-power-level spectra usually reveal more structural information at low masses than spectra obtained at high power levels. In the authors' previous work, a common feature of low-power-level experiments was the appearance of low-MM homologous series of components in several spectra, noted particularly in the MALDI spectra of kerogens 25, pyrolysis tars and liquefaction extractsz4 and in the MALDI-m.s. characterization of the Argonne Premium Coal Sample set9. However, higher laser power levels were required to detect higher-MM species, although there are indications that results may no
MALDI-MS and size exclusion chromatography: M.-J. Lazaro et al. longer improve (and may even deteriorate) above optimum power levels which appear to be sample-dependent. Comparing spectra obtained at 'high' and 'low' laser energy levels in Figure 2, the peaks at low masses were more prominent in the low-power spectra than in the highpower spectra. The low-power spectra showed less variation in profile than the high-power spectra, particularly for the pyridine-insoluble fraction. The high-mass ends of the spectra were not found to vary significantly with laser power in any of the spectra; the spectra of Figure 2(e) were typical. However, the mass distribution for the pyridine-soluble fraction at the lower laser energy [Figure 2(b)] showed proportionally more signal in the m/z 500-3000 mass range than was observed at the higher laser energy [Figure 2(b)]. The influence of laser energy is thus seen to be sampledependent: where samples may be expected to contain highMM materials, the use of higher power levels appears necessary: however, where high masses are not expected to predominate, high power levels do not necessarily improve the spectrum and may even lead to its deterioration.
When the pyridine-insoluble fraction was examined in the presence of different matrix materials 24, mass spectra similar to those of the original solution in Figure 2(c) were obtained; the different matrices gave rise to variations in the position of the peak of maximum intensity. Clearly, the greatest loading of the fraction in this work acted as an effective matrix for the sample, but the nature of the chemical features of the material most significant for the process is not obvious.
Size-exclusion chromatography Figure 3(a) and (b) present size-exclusion chromatograms of the whole 'soft' pitch sample and its two solubility fractions, obtained by u.v. detection at 350 and 450 nm respectively. All three samples showed evidence of excluded material, which would presumably have approached the instrument baseline asymptotically in an s.e.c, column able to accommodate the range of molecular sizes, as do the mass spectra. Lafleur and Nakagawa 16 have indicated that polar molecules elute unexpectedly early;
SEC-NMP profiles (350 nm) 0.007 (a) 0.006 B
"4 0.005 0.004
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Time (minutes) SEC-NMP profiles (450 nm)
0.007 (b) 0.006 B
.~ 0.005 "4 0.004 •~ 0.003 = 0.002 0.001
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Time, minutes Figure 3 Size-exclusion chromatograms for the pyridine-soluble (curve A) and pyridine-insoluble (curve B) fractions and, for comparison, the whole 'soft' pitch (curve C). Area-normalized intensity of absorbance versus time; detection by u.v. at (a) 350 nm, (b) 450 nm.
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MALDI-MS and size exclusion chromatography: M.-J. Lazaro et al. however, the molecules of pitch are not particularly polar and the atomic composition indicates that at a molecular mass of 1000 u, the average molecule will contain 1.5 oxygen atoms (in non-polar furans), 1.5 nitrogen atoms (in pyrrole or pyridine) and 0.2 sulfur atoms (in non-polar thiophene). The shift to shorter elution times observed between 350 and 450 nm indicates the structural complexity of the mixture, in this case the presence of a high concentration of large polycyclic aromatic ring systems, able to absorb u.v. radiation at longer wavelengths. The chromatograms clearly showed the pyridine-insoluble fraction to contain a greater proportion of material eluting at or near the exclusion limit of the column than the whole 'soft pitch' sample or the pyridine-soluble fraction. The relative abundance of material in this region of the chromatograms reflects the magnitude of the MM distributions found by MALDI-m.s. (Figures 1 and 2). As explained above, the exclusion limit of the present s.e.c, column corresponds to - 3 0 0 0 0 4 0 0 0 0 u at an elution time of 12.5rain in terms of polystyrene standards, in the mass range where a linear relation was observed between log(molar mass) and elution volume. However, a polystyrene standard of molar mass 1.84 × 106 was observed to elute at 11.0 min, indicating that the column did not have a sharp cut-off at the exclusion limit; a similar problem in calibrating the interstitial volume of a size-exclusion column has been reported for aqueous protein systems 26. Although differences in structural characteristics do not allow s.e.c, elution times of polystyrenes to be equated with those of pitch molecules, the evidence that pitch constituents elute at times equivalent to such large polystyrene standards is consistent with the very high-MM materials observed by MALDI-m.s. Such large molecules are not normally detectable by standard methods, due to their limited solubilities in common solvents and their low volatilities when characterized by probe mass spectrometry and gas chromatography.
higher-molecular-mass species for the 'soft' pitch and the pyridine-insoluble fraction. Increased laser power levels did not increase the high-mass limit in spectra for the pyridinesoluble fraction, where size-exclusion chromatography indicated the presence of less material at the exclusion limit of the column. Self-matrix effect. The work has shown that the coal tar pitch-derived materials can give apparently satisfactory MALDI mass spectra with the sample acting as its own matrix. In MALDI-m.s., higher-mass materials can be observed with greater clarity when the polydispersity of the sample is reduced. The separation of the 'soft' pitch sample into a pyridine-soluble and a pyridine-insoluble fraction served to concentrate higher-mass materials in the pyridine-insoluble fraction. However, the polydispersity of all the samples examined in this study is likely to be far from unity; the MALDI-m.s.-derived molecular mass distributions are therefore thought to underestimate the actual distributions within the samples. General agreement was found between MALDI-m.s. profiles and size-exclusion chromatograms, showing greater concentrations of higher-molecular-mass materials in the pyridine-insoluble fraction than in the original 'soft' pitch sample; the pyridine-soluble fraction showed the lowest molecular mass region. Taken together, the results indicate that the influence of experimental parameters on MALDI mass spectra of coalderived materials depends partly on sample characteristics. ACKNOWLEDGEMENTS The authors would like to thank the Spanish Government, Ministerio de Educaci6n y Ciencia, for the award of a Postdoctoral Grant to M. J. L. Support for this work by the British Coal Utilisation Research Association under Grant No. b32(a) is gratefully acknowledged.
CONCLUSIONS This study of the effect of a number of instrument- and technique-related parameters on the MALDI-mass spectra of coal-derived materials has suggested the following conclusions. Effect of ion-extraction voltage. The intense peaks at low mass as well as the upper limit of the continuum of masses was found to extend to higher values with increasing ionextraction voltage. This effect was not observed for the pyridine-soluble fraction, which was shown by s.e.c, to contain less high-mass material. Since the increase in ion extraction voltage serves only to accelerate species already ionized, without otherwise disturbing the sample, use of higher ion extraction voltages is thought to provide a more complete inventory of already ionized species. Effect of sample loading. The technique was found to accommodate a wide range of sample loadings on the target, the main effect being an increase in summed intensity with increasing loading. However, sample loading does not appear to have a determining effect on the quality or reproducibility of the spectra. The upper mass limit of all the samples was found to lie between 100000 and 300000 u, irrespective of sample loading. Effect of laser power level. Spectra obtained at low laser power levels were found to reveal more structural information at low masses than spectra acquired at high laser power. However, higher laser power levels were required to detect
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PIh S0016-2361(97)00032-X
Fuel Vol. 76, No. 13, pp. 1225-1233, 1997 © 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0016-2361/97 $17.00+0.00
Molecular mass determinations in coal-derived liquids by MALDI mass spectrometry and size-exclusion chromatography Maria-Jesus Ldzaro a, Alan A. Herod a, Mike Cocksedge b, Mark Domin b and Rafael Kandiyoti a aDepartment of Chemical Engineering and Chemical Technology, Imperial College, University of London, London SW7 2BY, UK bDepartment of Pharmaceutical and Biological Chemistry, School of Pharmacy, University of London, Brunswick Square, London WClN lAX, UK (Received 28 November 1996; revised 12 December 1996) This study examined the effect of changes in instrument-related parameters on mass spectra obtained by MALDIm.s.: ion-extraction voltage, laser power and sample loading. Results were compared with those of size-exclusion chromatography (s.e.c.) using 1-methyl-2-pyrrolidinoneas mobile phase. A coal tar pitch and its pyridine-soluble and pyridine-insoluble fractions were used as samples. MALDI-mass spectra were obtained with no added matrix, since the sample absorbed strongly at the laser wavelength, 337 nm. Higher extraction voltages (up to 30 kV) led to the observation of higher masses in the whole pitch and its pyridine-insoluble fraction, where the presence of higher-molecular-mass material was indicated by s.e.c. Increasing the ion extraction voltage served only to accelerate species already ionized, without otherwise disturbing the sample: the use of higher ion extraction voltages must be therefore be considered as providing a more complete inventory of already ionized species. Increasing the laser power level and sample loading on the target led to similar changes in the spectra, although there were indications that for both these parameters, the results might no longer improve (and might eventually deteriorate) beyond optimum values, which appear to be sample-dependent. The low-power spectra revealed more structural information at low masses, where series of homologous peaks could be observed, compared with the spectra at high power. © 1997 Elsevier Science Ltd. (Keywords: coal tar pitch; molecular mass; MALDl-mass spectrometry)
The authors have recently investigated the application of laser desorption mass spectrometry (LD-m.s.) 1-6 and matrix-assisted laser desorption ionization mass spectrometry (MALDI-m.s.)6-10 to molecular mass determinations in coal-derived liquids. The work was initially undertaken to evaluate previous observations by size-exclusion chromatography (s.e.c.), by using an independent analytical technique. The s.e.c, work had suggested the presence of highmolecular-mass (MM) materials (up to 4000-6000 u) in a number of coal pyrolysis tars and liquefaction extracts 1i- 15. Preliminary work with a LIMA (laser ionization mass analysis) instrument gave single-shot spectra showing MMs up to the available detection limit (m/z 1 2 0 0 0 )I 'Z"4.5 In these experiments, the instrument was operated with a reflectron stage at an ion extraction voltage of 20 kV and with a defocused beam to prevent breaking-up sample molecules under the power of the laser. Subsequent experiments using a Kratos Kompact MALDI III instrument of more recent conception, in linear mode (i.e. in the absence of the reflectron stage), allowed co-addition of spectra: results showed masses between m/z 20000 and
30 000 in pyrolysis tars and liquefaction extracts 10 and up to m/z 270 000 in solid coals 9. These findings went far beyond the MM range previously identified by s.e.c., in which tetrahydrofuran (THF) had been used as mobile phase. Lafleur and Nakagawa 16 have demonstrated the successful use of the more polar 1-methyl2-pyrrolidinone (NMP) as mobile phase in s.e.c.; this modification overcame solubility limitations encountered in the use of THF and allowed the detection of a new range of high-MM materials near the exclusion limits of s.e.c. 17 16 columns . Lafleur and Nakagawa attempted to characterize the material eluting at the exclusion limit of the column by the use of standards and heated-probe mass spectrometry, which showed little ion abundance between masses 250 and 550 u, the upper limit of the scan. In the absence of m.s. evidence for high-MM materials, the materials at the exclusion limit of the column were perceived as polar rather than of large molecular size. These findings were interpreted in terms of a multimode mechanism for elution, based on molecular size with an earlier-than-expected elution of polar molecules. However, the perceived absence
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MALDI-MS and size exclusion chromatography: M.-J. L~zaro et al. of high-MM materials in heated probe m.s. could be reasonably interpreted in terms of limitations of evaporative and ionization processes involved in heated-probe m.s. compared with laser desorption. More recently, the authors have presented MALDI-TOF m.s. spectra of coal tar pitch fractions separated both by solvent fractionation6 (~ridine-solubles/insolubles) and by planar chromatography ; a Fisons VG TOFSPEC instrument was used in this latter part of the work. The bulk of material insoluble in THF and in pyridine showed up at the exclusion limit of the s.e.c, columns; higher MMs were found by m.s. in samples identified by s.e.c. (in NMP) to give larger peaks at shorter elution times. Furthermore, higher-MM materials could be observed when pyridineinsolubles were examined by MALDI-TOF m.s. on their own, compared with the whole pitch sample containing 15% pyridine-insolubles. A high mass limit above m/z 50000 was observed. The work provided good qualitative
agreement between MALDI-m.s. and s.e.c, and indicated that fractionation of complex mixtures allowed the observation of characteristics of the less abundant fractions with greater clarity 19. There is considerable evidence from the MALDI-m.s. spectra of polymer systems6 that agreement between molecular-mass distributions from MALDI-m.s. and s.e.c, can be improved when samples of low polydispersity are analysed. Preferential desorption and ionisation of lower-MM components has been observed in the MALDI-m.s. of simple mixtures of polystyrene standards 2°'21 Significant improvement in agreement between the two techniques was reported when the polydispersity of polysaccharide samples was reduced by fractionation, using preparative s.e.c, prior to MALDI-TOF m.s.22. In experiments with mixtures of polystyrene standards, it was also recently observed that the sensitivity of the MALDI instrument for the highest-MM polystyrene standard diminished with increasing polydispersity of the
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"soft" pitch; (c) pyridine-soluble fraction of "soft" pitch
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MALDI-MS and size exclusion chromatography: M.-J. Lazaro et al.
EXPERIMENTAL
I00
(c)
10
Samples The coal tar pitch sample has been studied previously 1"2'5-7. Its elemental composition (wt%) was C 91.4, H 4.1, N 1.3, S 0.76 and O 2.4 (by difference). In this work, solubility fractions were prepared using pyridine; the pyridine-insolubles were 15 wt% of the pitch. The pyridine-insoluble fraction and the whole pitch sample were dissolved in 1-methyl-2-pyrrolidinone for subsequent examination. The pyridine-soluble fraction was used in (pyridine) solution.
50
MALDI-m.s.
100
MALDI-TOF spectra were obtained with a Fisons VG TOFSPEC mass spectrometer (VG Organic, Manchester, UK) fitted with a nitrogen u.v. laser (337 nm) and a VAX 400-base data system with OPUS software. The linear TOF mode was used with different accelerating voltages of 10, 20 and 30 kV and two levels of laser power. The full mass range of > 300000 u was examined. The sample was applied to the target in NMP (or pyridine) solution and dried in a vacuum oven for 2 h before insertion into the mass spectrometer ion source. To test the effect of the amount of sample deposition, solutions of the whole pitch and pyridine-solubles and -insolubles were added to target spots at increasing dilution. The deposited sample layer may not necessarily be considered to have spread evenly on the target after solvent evaporation. Approximately 50 spectra were summed for each spot and both the most intense range of the spectra and the highest-mass approach to instrument baseline were examined. In this work MALDI-mass spectra were obtained with no added matrix, since the sample absorbed strongly at the laser wavelength, 337 nm.
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m/z Figure 1 Continued polymer mixture, in favour of ion intensities of lower-MM polystyrene standards, although the high-mass peaks remained visible 23. Therefore major sample-related problems remain to be resolved before the findings outlined above can be translated into a coherent method to obtain quantitative molecular mass distributions of coal-derived liquid mixtures. The present study examines the effect of changes in a number of instrument-related experimental parameters such as laser power, ion extraction voltage and sample loading on the mass spectra of coal-derived liquids obtained by MALDI-m.s. The m.s.-derived results have been compared with data from size-exclusion chromatography performed using 1-methyl-2-pyrrolidinone as the mobile phase. In the current work, a coal tar pitch and its pyridine-soluble and pyridine-insoluble fractions were used to study the effect of changes in these experimental parameters. In a separate study 24, the effect of the use of different matrix materials on the spectra of the pyridineinsoluble fraction was examined.
S.e.c. using NMP as solvent was carried out using a 30 cm long, 7.5 mm o.d. polystyrene-divinylbenzene polymer-packed s.e.c, column (3 mm, Mixed E, Polymer Labs, UK) operated at 85°C with a flow rate of 0.45 mL min -I, using a variable-wavelength Perkin-Elmer LC290 u.v. detector set at 350 or 450nm. Calibrations against polystyrene standards were performed at 262 nm, since NMP is opaque at 254 nm but partly transparent at 262 nm. The elution volume corresponding to the total exclusion limit of the column was - 5 . 0 mL (polystyrene molar mass 1.84 million) but the upper range of polystyrenes corresponding to a linear relation between log(molar mass) and elution volume was - 3 0 000-40 000 u. RESULTS AND DISCUSSION
MALDI-m.s. Effect of ion extraction voltage level on spectra.
Spectra for the 'soft' pitch and the pyridine-soluble and pyridineinsoluble fractions were obtained at three extraction voltages (10, 20 and 30kV) using maximum laser power [Figure l(a,b,c)]. At the lowest ion extraction voltage (10 kV), the continuum of peaks in the spectra of the 'soft' pitch and its pyridine-insoluble fraction extended only to m/z 4000-5000. The continuum of masses extended to m/z 6000-7000 at 20 kV and up to m/z 100000 (for the 'soft' pitch) at the maximum available extraction voltage of 30 kV. The intense peaks at low mass were also observed to shift to higher masses with increasing ion-extraction
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MALDI-MS and size exclusion chromatography: M.-J. L~zaro et al.
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Figure 2 MALDI-TOF spectra at high (i) and low (ii) laser energy and at different sample Ioadings: (a) "soft" pitch, original solution (0) and third dilution (3); (b) pyridine-soluble fraction, original solution (0) and second dilution (2); (c) pyridine-insoluble fraction at high laser energy, all four dilutions; (d) pyridine-insoluble fraction at low laser energy, all four dilutions; (e) high-mass limits of the pyridine-insoluble fraction, original solution (0) and second (2) and third (3) dilutions.
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MALDI-MS and size exclusion chromatography: M.-J. L&zaro et al.
the pyridine-soluble fraction contained significantly less high-MM material.
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voltage: for the 'soft' pitch sample [Figure l(a)] the peak shifted from m/z 300 at 10 kV to m/z 2000 at 30 kV, whereas for the pyridine-insoluble fraction [Figure l(b)], the peak was observed to shift from m/z 300 at 10 kV to m/z ~ 1400 at 30 kV. However, for the pyridine-soluble fraction [Figure l(c)] no significant shift to higher mass could be observed with increasing extraction voltage. Since the increase in ion extraction voltage serves only to accelerate species already ionized, without otherwise disturbing the sample, use of higher ion extraction voltages must be considered as merely providing a more complete inventory of ionized species, apparently by enhancing the kinetic energy imparted to higher-MM materials. It is not yet known what the eventual optimum extraction voltage may turn out to be. These data show furthermore that higher extraction voltages lead to the observation of higher masses only for samples where higher-mass materials are actually present (e.g. whole pitch and pyridine-insolubles): s.e.c. chromatograms presented below strongly suggest that
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Effect of changes in sample loading on spectra. The whole 'soft' pitch sample, its pyridine-solubles and its pyridine-insolubles were examined at an ion extraction voltage of 20 W. Figure 2(a) presents spectra showing the effect of successive dilution of the (whole) 'soft' pitch sample at maximum laser power (arbitrary setting 4.2) and the equivalent spectra at a lower laser energy setting (arbitrary setting 3.2); only the spectra for the original solutions and the third dilutions are shown, since the intermediate ones were very similar to the spectra of the original solutions. Figure 2(b) presents analogous spectra for the pyridine-solubles at 'high' and 'low' laser power; once again, not all levels of dilution are shown, since the spectra of the intermediate dilutions resembled those of the original solutions. Figure 2(c,d) shows 'high' and 'low' laser power spectra of the pyridine-insoluble fraction; here, spectra are shown for all dilutions since there was in fact a change at high power. Considering Figure 2(a) for the 'soft' pitch sample, the spectra were generated from loadings of 27.5, 6.9, 1.7 and 0.43 mg on the target; higher sample loadings led to overloading of the intensity scale of the summed spectra in all cases except for the most dilute spot. However, spectra for the pyridine-soluble and pyridine-insoluble fractions did not show evidence of overloading with increased sample loading, probably because the sample masses deposited were lower than for the whole pitch. The problem arises from the difficulty in removing NMP and pyridine from the sample, thus preventing accurate sample loadings from being determined. Except for the most dilute samples, the data for the whole pitch and for the pyridine-soluble fraction did not appear very sensitive to sample loading on the target. At low loadings, changes in the shape of the overall profiles are apparent, probably reflecting the lower overall intensity of the summed spectrum and the greater difficulty in finding regions of the target from which a sufficient number of spectra could be acquired before the sample was completely removed. The spectrum of the pyridine-insoluble fraction did change with dilution at high laser power, Figure 2(c), with loss of the relatively intense signal between 1000 and 2000 u in favour of the intense peak between 200 and 300 u; the changes were less marked at low laser power, Figure 2(d). Figure 2(e) shows the high-mass limits in the spectra of the pyridine-insoluble fraction with dilution, indicating an approach to instrument baseline at mlz between 100 000 and 200000, although the precise upper limits are not easily discernible. Clearly, more nearly quantitative methods for estimating upper mass limits need to be developed. Similar spectra were obtained for the pitch and the pyridine-soluble fraction (not shown). Changes in MALDI-m.s. spectra with variation in laser energy level. Lower-power-level spectra usually reveal more structural information at low masses than spectra obtained at high power levels. In the authors' previous work, a common feature of low-power-level experiments was the appearance of low-MM homologous series of components in several spectra, noted particularly in the MALDI spectra of kerogens 25, pyrolysis tars and liquefaction extractsz4 and in the MALDI-m.s. characterization of the Argonne Premium Coal Sample set9. However, higher laser power levels were required to detect higher-MM species, although there are indications that results may no
MALDI-MS and size exclusion chromatography: M.-J. Lazaro et al. longer improve (and may even deteriorate) above optimum power levels which appear to be sample-dependent. Comparing spectra obtained at 'high' and 'low' laser energy levels in Figure 2, the peaks at low masses were more prominent in the low-power spectra than in the highpower spectra. The low-power spectra showed less variation in profile than the high-power spectra, particularly for the pyridine-insoluble fraction. The high-mass ends of the spectra were not found to vary significantly with laser power in any of the spectra; the spectra of Figure 2(e) were typical. However, the mass distribution for the pyridine-soluble fraction at the lower laser energy [Figure 2(b)] showed proportionally more signal in the m/z 500-3000 mass range than was observed at the higher laser energy [Figure 2(b)]. The influence of laser energy is thus seen to be sampledependent: where samples may be expected to contain highMM materials, the use of higher power levels appears necessary: however, where high masses are not expected to predominate, high power levels do not necessarily improve the spectrum and may even lead to its deterioration.
When the pyridine-insoluble fraction was examined in the presence of different matrix materials 24, mass spectra similar to those of the original solution in Figure 2(c) were obtained; the different matrices gave rise to variations in the position of the peak of maximum intensity. Clearly, the greatest loading of the fraction in this work acted as an effective matrix for the sample, but the nature of the chemical features of the material most significant for the process is not obvious.
Size-exclusion chromatography Figure 3(a) and (b) present size-exclusion chromatograms of the whole 'soft' pitch sample and its two solubility fractions, obtained by u.v. detection at 350 and 450 nm respectively. All three samples showed evidence of excluded material, which would presumably have approached the instrument baseline asymptotically in an s.e.c, column able to accommodate the range of molecular sizes, as do the mass spectra. Lafleur and Nakagawa 16 have indicated that polar molecules elute unexpectedly early;
SEC-NMP profiles (350 nm) 0.007 (a) 0.006 B
"4 0.005 0.004
- 0.003 0.002 0.001
10
15
20
25
30
Time (minutes) SEC-NMP profiles (450 nm)
0.007 (b) 0.006 B
.~ 0.005 "4 0.004 •~ 0.003 = 0.002 0.001
5
10
15
20
25
30
Time, minutes Figure 3 Size-exclusion chromatograms for the pyridine-soluble (curve A) and pyridine-insoluble (curve B) fractions and, for comparison, the whole 'soft' pitch (curve C). Area-normalized intensity of absorbance versus time; detection by u.v. at (a) 350 nm, (b) 450 nm.
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MALDI-MS and size exclusion chromatography: M.-J. Lazaro et al. however, the molecules of pitch are not particularly polar and the atomic composition indicates that at a molecular mass of 1000 u, the average molecule will contain 1.5 oxygen atoms (in non-polar furans), 1.5 nitrogen atoms (in pyrrole or pyridine) and 0.2 sulfur atoms (in non-polar thiophene). The shift to shorter elution times observed between 350 and 450 nm indicates the structural complexity of the mixture, in this case the presence of a high concentration of large polycyclic aromatic ring systems, able to absorb u.v. radiation at longer wavelengths. The chromatograms clearly showed the pyridine-insoluble fraction to contain a greater proportion of material eluting at or near the exclusion limit of the column than the whole 'soft pitch' sample or the pyridine-soluble fraction. The relative abundance of material in this region of the chromatograms reflects the magnitude of the MM distributions found by MALDI-m.s. (Figures 1 and 2). As explained above, the exclusion limit of the present s.e.c, column corresponds to - 3 0 0 0 0 4 0 0 0 0 u at an elution time of 12.5rain in terms of polystyrene standards, in the mass range where a linear relation was observed between log(molar mass) and elution volume. However, a polystyrene standard of molar mass 1.84 × 106 was observed to elute at 11.0 min, indicating that the column did not have a sharp cut-off at the exclusion limit; a similar problem in calibrating the interstitial volume of a size-exclusion column has been reported for aqueous protein systems 26. Although differences in structural characteristics do not allow s.e.c, elution times of polystyrenes to be equated with those of pitch molecules, the evidence that pitch constituents elute at times equivalent to such large polystyrene standards is consistent with the very high-MM materials observed by MALDI-m.s. Such large molecules are not normally detectable by standard methods, due to their limited solubilities in common solvents and their low volatilities when characterized by probe mass spectrometry and gas chromatography.
higher-molecular-mass species for the 'soft' pitch and the pyridine-insoluble fraction. Increased laser power levels did not increase the high-mass limit in spectra for the pyridinesoluble fraction, where size-exclusion chromatography indicated the presence of less material at the exclusion limit of the column. Self-matrix effect. The work has shown that the coal tar pitch-derived materials can give apparently satisfactory MALDI mass spectra with the sample acting as its own matrix. In MALDI-m.s., higher-mass materials can be observed with greater clarity when the polydispersity of the sample is reduced. The separation of the 'soft' pitch sample into a pyridine-soluble and a pyridine-insoluble fraction served to concentrate higher-mass materials in the pyridine-insoluble fraction. However, the polydispersity of all the samples examined in this study is likely to be far from unity; the MALDI-m.s.-derived molecular mass distributions are therefore thought to underestimate the actual distributions within the samples. General agreement was found between MALDI-m.s. profiles and size-exclusion chromatograms, showing greater concentrations of higher-molecular-mass materials in the pyridine-insoluble fraction than in the original 'soft' pitch sample; the pyridine-soluble fraction showed the lowest molecular mass region. Taken together, the results indicate that the influence of experimental parameters on MALDI mass spectra of coalderived materials depends partly on sample characteristics. ACKNOWLEDGEMENTS The authors would like to thank the Spanish Government, Ministerio de Educaci6n y Ciencia, for the award of a Postdoctoral Grant to M. J. L. Support for this work by the British Coal Utilisation Research Association under Grant No. b32(a) is gratefully acknowledged.
CONCLUSIONS This study of the effect of a number of instrument- and technique-related parameters on the MALDI-mass spectra of coal-derived materials has suggested the following conclusions. Effect of ion-extraction voltage. The intense peaks at low mass as well as the upper limit of the continuum of masses was found to extend to higher values with increasing ionextraction voltage. This effect was not observed for the pyridine-soluble fraction, which was shown by s.e.c, to contain less high-mass material. Since the increase in ion extraction voltage serves only to accelerate species already ionized, without otherwise disturbing the sample, use of higher ion extraction voltages is thought to provide a more complete inventory of already ionized species. Effect of sample loading. The technique was found to accommodate a wide range of sample loadings on the target, the main effect being an increase in summed intensity with increasing loading. However, sample loading does not appear to have a determining effect on the quality or reproducibility of the spectra. The upper mass limit of all the samples was found to lie between 100000 and 300000 u, irrespective of sample loading. Effect of laser power level. Spectra obtained at low laser power levels were found to reveal more structural information at low masses than spectra acquired at high laser power. However, higher laser power levels were required to detect
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4 5 6 7 8 9
Parker, J. E., Johnson, C. A. F., John, P., Smith, G. P., Herod, A. A., Stokes, B. J. and Kandiyoti, R., Fuel, 1993, 72, 1381. Herod, A. A., Kandiyoti, R., Parker, J. E., Johnson, C. A. F., John, P., Smith, G. P. and Li, C.-Z., Chemical Communications, 1993, 767. Herod, A. A., Stokes, B. J., Hancock, P., Kandiyoti, R., Parker, J. E., Johnson, C. A. F., John, P. and Smith, G. P., Journal of the Chemical Society, Perkin Transactions 2, 1994, 499. Herod, A. A., Kandiyoti, R., Parker, J. E., Johnson, C. A. F., John, P., Smith, G. P. and Li, C.-Z., Rapid Communications in Mass Spectrometry, 1993, 7, 360. John, P., Johnson, C. A. F., Parker, J. E., Smith, G. P., Herod, A. A., Gaines, A. F., Li, C.-Z. and Kandiyoti, R., Rapid Communications in Mass Spectrometry, 1991,5, 364. Herod, A. A., Johnson, B. R., Bartle, K. D., Carter, D. M., Cocksedge, M. J., Domin, M. and Kandiyoti, R., Rapid Communications in Mass Spectrometry, 1995, 9, 1446. John, P., Johnson, C. A. F., Parker, J. E., Smith, G. P., Herod, A. A., Li, C.-Z. and Kandiyoti, R., Rapid Communications in Mass Spectrometry, 1993, 7, 795. John, P., Johnson, C. A. F., Parker, J. E., Smith, G. P., Herod, A. A., Li, C.-Z., Humphrey, P., Chapman, J. R. and Kandiyoti, R., Fuel, 1994, 73, 1606. Herod, A. A., Li, C.-Z., Parker, J. E., John, P., Johnson, C. A. F., Smith, G. P., Humphrey, P., Chapman, J. R. and
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Herod, A. A. and Kandiyoti, R., Journal of Chromatography A, 1995, 708, 143. Mowat, I. A., Donovan, R. J., Bruce, M. and Monaghan, J. J., in Proceedings of the 43rd ASMS Conference on MS & Allied Topics. 1995, p. 968. Martin, K., Spickermann, J., R~der, H. J. and MiJllen, K., Rapid Communications in Mass Spectrometry, 1996, 10, 1471. Garozzo, D., Impallomeni, G., Spina, E., Sturiale, L. and Zanetti, F., Rapid Communications in Mass Spectrometry, 1995, 9, 963. Domin, M., Herod, A. A. and Kandiyoti, R., Paper to BMSS Conference, Swansea, 1996; Rapid Communications in Mass Spectrometry (submitted). Domin, M., Lazaro, M.-J., Herod, A. A. and Kandiyoti, R., Paper to BMSS Conference, Swansea, 1996; Rapid Communications in Mass Spectrometry (in press). Li, C.-Z., Herod, A. A., John, P., Johnson, C. A. F., Parker, J. E., Smith, G. P., Humphrey, P., Chapman, J. R., Rahman, M., Kinghorn, R. R. F. and Kandiyoti, R., Rapid Communications in Mass Spectrometry, 1994, 8, 823. Barnikol, W. K. R. and Potschke, H., Journal of Chromatography A, 1994, 685, 221.
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