Energy Fuels 2009, 23, 6003–6014 Published on Web 09/30/2009
: DOI:10.1021/ef9006966
Isolation of Size Exclusion Chromatography Elution-Fractions of Coal and Petroleum-Derived Samples and Analysis by Laser Desorption Mass Spectrometry Trevor J. Morgan,* Anthe George, Patricia Alvarez, Alan A. Herod, Marcos Millan, and Rafael Kandiyoti Department of Chemical Engineering and Chemical Technology Imperial College London, London SW7 2AZ, United Kingdom Received July 7, 2009. Revised Manuscript Received September 13, 2009
High-mass species in a coal- and a petroleum-derived sample were investigated by LD-MS of eluent fractions collected from SEC. Results were compared with polymer-based calibrations of SEC elution times. It was observed that polymer- and PAH-based calibrations provide good mass estimates for material eluting in the resolved (later eluting) part of SEC chromatograms. At elution times shorter that 15 min (i.e., in the “excluded” region) there was significant deviation from the PS calibration for both the coal tar pitch and the petroleum-derived samples. In all cases, however, material eluting early during SEC was observed by LD-MS to have higher average masses compared to later-eluting material. The data appears conclusive. An upper limit to the detection of high-mass material by LD-MS appears to have been reached. When comparing mass estimates by LD-MS of SEC elution-fractions of material from the coal tar pitch and petroleum asphaltene, similar molecular mass ranges were found for fractions collected at similar elution times. This provides significant confirmation for the suitability of SEC as a technique for estimating molecular masses of complex hydrocarbon mixtures. The analytical approach presented in this paper provides a valid and useful basis for exploring relationships between molecular size, molecular mass, and structure for complex coal and petroleum-derived materials.
heavy coal-derived liquids has been summarized elsewhere.3,4 More recently, these methods have been applied to petroleum-derived heavy fractions in an attempt to develop the characterization of these materials in terms of their structures and mass distributions.8,9 The presence of highmass materials in commercial distillate fractions (creosote and anthracene oil) of coal tars has been observed by these methods.10 Creek11 has given a useful overview of problems associated with the discussion and the study of petroleumderived materials; many of the methods apply equally well to the characterization of coal-derived liquids. A notable comment refers to the futility of considering average structures for these complex materials.11 The SEC work in the present study uses 1-methyl-2-pyrrolidinone (NMP) as solvent and eluent for coal-derived liquids, biomass tars, humic substances, and soots.12-16 SEC chromatograms obtained using NMP as eluent consistently give bimodal distributions, with an early eluting peak, showing signal for material excluded from column porosity and a later eluting retained peak giving the signal for material resolved by
Introduction Reliable estimates of molecular mass distributions of coaland petroleum-derived heavy hydrocarbon fractions are essential in developing a better understanding of their properties and behavior. To date, no single technique has been found capable of providing a detailed picture and there is little agreement in the literature regarding molecular mass distributions and structural features of these complex materials.1,2 Two promising methods for obtaining data about mass distributions are size exclusion chromatography (SEC), a version of HPLC, and laser desorption mass spectrometry (LD-MS).3-7 Although widely used for characterizing molecular mass distributions of complex hydrocarbon mixtures, both methods have their own peculiar limitations. One procedure that has been uniformly observed to improve the resolution of characterization methods is the fractionation of complex samples, to separate smaller molecular mass, and usually less polar, fractions from the larger mass (and more polar) samples. Previous work in this laboratory, on sample preparation and the selection of analytical methods for characterizing
(8) Herod, A. A.; Paul-Dauphin, S.; Karaca, F.; Morgan, T. J.; Millan, M.; Kandiyoti, R. Energy Fuel. 2007, 21 (6), 3484. (9) Berrueco, C.; Venditti, S.; Morgan, T. J.; Alvarez, P.; Millan, M.; Herod, A. A.; Kandiyoti, R. Energy Fuels 2008, 22, 3265. (10) Morgan, T. J.; George, A.; Alvarez, P.; Millan, M.; Herod, A. A.; Kandiyoti, R. Energy Fuels 2008, 22, 3275. (11) Creek, J. L. Energy Fuels 2005, 19, 1212. (12) Herod, A. A.; Zhuo, Y.; Kandiyoti, R. J. Biochem. Biophys. Methods 2003, 56, 335. (13) Morgan, T. J.; Herod, A. A.; Brain, S. A.; Chambers, F. M.; Kandiyoti, R. J Chromatogr., A 2005, 81-88, 1095. (14) Apicella, B.; Millan, M.; Alfe, M.; Herod, A. A.; Pucci, P.; Ciajolo, A. Rapid Commun. Mass Spectrom. 2006, 20, 1104. (15) Purevsuren, B.; Herod, A. A.; Kandiyoti, R.; Morgan, T. J.; Avid, B.; Gerelmaa, T.; Davaajav, Ya. Fuel. 2004, 83, 799. (16) Purevsuren, B.; Herod, A. A.; Kandiyoti, R.; Morgan, T. J.; Avid, B.; Davaajav, Ya. Eur. J. Mass Spec. 2004, 10, 101.
*To whom correspondence should be addressed. E-mail: t.j.morgan@ic. ac.uk. (1) Mullins, O. C.; Martı´ nez-Haya, B.; Marshall, A. G. Energy Fuels 2008, 22 (3), 1765. (2) Herod, A. A.; Bartle, K. D.; Kandiyoti, R. Energy Fuels 2008, 22, 4312. (3) Herod, A. A.; Bartle, K. D.; Kandiyoti, R. Energy Fuels 2007, 21, 2176. (4) Kandiyoti, R.; Herod, A. A.; Bartle, K. D. Solid Fuels and Heavy Hydrocarbon Liquids: Thermal Characterisation and Analysis; Elsevier: 2006. ISBN: 0-08-044486-5. (5) Huang, J. Pet. Sci. Technol. 2007, 25, 1313. (6) Ali, F. A.; Ghaloum, N.; Hauser, A. Energy Fuels 2006, 20, 231. (7) Acevedo, S.; Gutierrez, L. B.; Negrin, G.; Pereira, C. J. Energy Fuels 2005, 19, 1548. r 2009 American Chemical Society
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the column. The two peaks usually appear as separated by a near-zero intensity valley. According to the polystyrene (PS) and polyaromatic hydrocarbon (PAH) calibrations used to calculate molecular masses from elution times, the excluded peak normally corresponds to apparent masses in excess of 200 000 u.17,18 Up to ∼3000 u, near-quantitative agreement has been found between the molecular masses of coal tar fractions, determined by LD-MS and MALDI-MS, and their elution times in SEC, as converted to molecular mass using PS, polymethylmethacrylate (PMMA), and polysaccharide (PSAC) molecular mass standards.17 Calibration curves obtained using the PS and PMMA molecular mass standards are statistically indistinguishable up to very high masses. Up to ∼15 000 u, furthermore, mass estimates using PS-PMMA standards on the one hand and the polysaccharide calibration on the other differ by about a factor of 2.5. Elution times corresponding to about 15 000 u by PS-PMMA give masses of about 6000 by the polysaccharide calibration. To date, polysaccharide standards have provided the largest observed deviation from the PS-PMMA curve. Thus, it is considered that molecular masses of complex hydrocarbon samples may be determined to within a factor of 2.5 at masses of up to 15 000 u (by PS-calibration). However, above masses of ∼15 000 u, the cited calibrations are not thought to correspond, in any degree of reliability, to actual molecular mass distributions of these complex materials.17 In more recent work, agreement between the PS-PMMA calibration and the PSAC calibration has been improved significantly by the use of a mixed eluent (a 6:1 volume ratio mixture of NMP/CHCl3).9 Until recently, however, it was not possible to recover samples of eluted material from the excluded region of the SEC chromatogram for separate analysis. We have previously suggested that the material showing signal under the early eluting peak may occupy larger hydrodynamic volumes than expected from their molecular weights, probably due to these materials adopting threedimensional (3D) conformations. In the absence of appropriate standard materials, however, it appears difficult to test this hypothesis. However, materials such as fullerenes are observed to elute far earlier than would be expected from their molecular masses.18 Another complication arises from the poor solubility of aliphatic samples in NMP. Alkanes are almost entirely insoluble in pure NMP, while petroleum asphaltenes only partially dissolve in this solvent.19,20 In recent work, however, we have shown that 6:1 (volume ratio) mixtures of NMP and CHCl3 are able to dissolve the NMP-insoluble fraction of petroleum asphaltenes. The mixed eluent-SEC method has been applied to coal tar distillate fractions as well as pitch fractions and was shown to give results similar to those obtained when pure NMP is used as eluent.9,21,22
Many researchers working on the characterization of petroleum asphaltenes prefer to operate with THF,5,23,24 chloroform,6 or toluene25 as SEC eluents. These are known to be good solvents for petroleum-derived samples. Unfortunately, none of these three solvents (THF, toluene, and chloroform) is suitable as eluent for the SEC of coal or petroleum-derived samples. Let us briefly explain. We have previously reported that when THF is used as eluent in the SEC of coal tar pitch derived fractions, or fractions from an Athabasca bitumen, interactions between the solute and the SEC column packing profoundly influence the shape of the chromatogram.3,26-28 Clearly, this makes THF entirely unsuitable as eluent in this type of chromatography. When chloroform or toluene are used as eluent, SEC chromatograms show an increase of elution time with increasing molecular mass for PAH standards.8,9 This is the opposite of the trend expected from a size exclusion based mechanism. In all three cases, furthermore, the solvent power does not appear adequate to prevent surface interactions between particles of column packing and sample molecules, further contributing to the distortion of the SEC chromatogram. In a study carried out elsewhere, CHCl3 was used as eluent during the SEC-based fractionation of petroleum fractions; THF was used as eluent to characterize these fractions in terms of molecular mass.6 Conclusions from this approach would need to be treated with some caution as both chloroform and THF tend to create problems of their own as outlined above. When working with pitch- and petroleum-derived asphaltenes, both pure NMP as well as the mixed NMP-chloroform solvent do appear to minimize interactions between solutes and column packing.8,9,29 Thus recent experience suggests the mixed solvent method to be the best, to date, for characterizing petroleum-derived materials by SEC. In discussing molecular mass distributions of petroleum asphaltenes, one major point of contention concerns the upper mass limits of these samples. Acevedo et al.7 compared results from VPO and LD-MS for petroleum asphaltenes and found mass distributions ranging from 100-10 000 u. These findings are consistent with results from other research groups using a variety of analytical techniques30,31 and with previous work in this laboratory.9,20,22 The lower mass range up to m/z 1000 selected by some authors1 appears to ignore much experimental evidence from our2-4 and other laboratories,7,30-36 (23) Garcia, M. C.; Urbina, A. Pet. Sci. Technol. 2003, 21 (5 & 6), 863. (24) Barth, T.; Hoeiland, S.; Fotland, P.; Askvik, M. K.; Myklebust, R.; Erstad, K. Energy Fuels 2005, 19, 1624. (25) Juyal, P.; Merino-Garcia, D.; Andersen, S. I. Energy Fuels 2005, 19, 1272. (26) Herod, A. A.; Kandiyoti, R. J. Planar Chromatogr. 1996, 9, 16. (27) Li, W.; Morgan, T. J.; Herod, A. A.; Kandiyoti, R. J. Chromatogr., A 2004, 1024, 227. (28) Domin, M.; Herod, A. A.; Kandiyoti, R.; Larsen, J. W.; Lazaro, M.-J.; Li, S.; Rahimi, P. Energy Fuels 1999, 13, 552. (29) Deelchand, J.-P.; Naqvi, Z.; Dubau, C.; Shearman, J.; Lazaro, M.-J.; Herod, A. A. J Chromatogr., A 1999, 830, 397. (30) Gonzalez, D. L.; Hirasaki, G. J.; Creek, J.; Chapman, W. G. Energy Fuels 2007, 21, 1231. (31) Akbarzadeh, K.; Bressler, D. C.; Wang, J.; Gawrys, K. L.; Gray, M. R.; Kilpatrick, P. K.; Yarranton, H. W. Energy Fuels 2005, 19, 1268. (32) Tanaka, R.; Sato, S.; Takanohashi, T.; Hunt, J.; Winans, R. Energy Fuels 2004, 18, 1405. (33) Ancheyta, J.; Centeno, G.; Trejo, F.; Marroquin, G.; Garcia, J. A.; Tenorio, E.; Torres, A. Energy Fuels 2002, 16 (5), 1121. (34) Strausz, O. P.; Safarik, I.; Lown, E. M.; Morales-Izquierdo, A. Energy Fuels 2008, 22 (2), 1156. (35) Zhang, L.; Yang, G.; Que, G.; Zhang, Q.; Yang, P. Energy Fuels 2006, 20 (5), 2008. (36) Seki, H.; Kumata, F. Energy Fuels 2000, 14 (5), 980.
(17) Islas, C.; Suelves, I.; Millan, M.; Apicella, B.; Herod, A. A.; Kandiyoti, R. J. Sep. Sci. 2003, 26, 1422. (18) Karaca, F.; Islas, C. A.; Millan, M; Behrouzi, M.; Morgan, T. J.; Herod, A. A.; Kandiyoti, R. Energy Fuels 2004, 18 (3), 778. (19) Ascanius, B. E.; Merino-Garcia, D.; Andersen, S. I. Energy Fuels 2004, 18, 1827. (20) Al-Muhareb, E.; Morgan, T. J.; Herod, A. A.; Kandiyoti, R. Pet. Sci. Technol. 2007, 25, 81. (21) Karaca, F.; Morgan, T. J.; George, A.; Bull, I. D.; Herod, A. A.; Millan, M.; Kandiyoti, R. Rapid Commun. Mass Spectrom. 2009, 23, 2087. (22) Morgan, T. J. PhD Thesis, Imperial College London: 2008.
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Figure 1. SEC chromatogram of the whole PPI-N used to produce the SEC elution-fractions A and B. NMP was used as eluent with a heated (80 °C) Mixed-D column and detection by UV-A at 300 nm.
showing the presence of larger molecular mass materials. Although it is reasonably clear that sizable proportions of asphaltene molecules are of masses below 1000 u, the relevance of the discussion revolves around the effect of highmass molecules during extraction, production and refinery processing. The present study aims to examine the presence of high mass species in coal- and petroleum-derived samples by collecting eluent fractions from an SEC column for analysis by LD-MS. The recovery of the fractions from extremely dilute NMP (or NMP/CHCl3) solutions is complex, because NMP can oxidize and polymerize under the usual conditions used for evaporating NMP from solution.22,37 The problem was found to be severe even when mere traces of oxygen were present in the ambient atmosphere during the evaporation procedure. As a result, NMP distillation residues we found to contain high mass materials, which appear to have mass ranges similar to those of coal and petroleum derived samples. Blank tests have confirmed that there is some risk of interference from byproduct formed during the distillation of NMP. As described below, the solution to this problem was relatively simple. In the present work, fractions successively eluting from an SEC column were collected and examined by LD-MS. The pitch-derived sample was fractionated by eluting in pure NMP whereas a 6:1 (v/v) solution of NMP/CHCl3 was used for fractionating a sample of Maya asphaltene. This provides a method for corroborating polymer-based calibrations commonly used to convert elution times from SEC into estimates of molecular mass. The determination of molecular mass ranges for materials eluting in the excluded region of SEC were of particular interest. The work presented below provides a demonstration of how the novel mixed eluent system (NMP/CHCl3) can be used for characterizing petroleum derived materials by SEC.
Experimental Section Samples. The pitch pyridine-insoluble fraction of a coal tar pitch3,4,38 and an asphaltene fraction of Maya crude22,39,40 were examined. A 1% wt/vol (1 mg mL-1) slurry in NMP was made using the pyridine-insoluble fraction of the pitch. The slurry was mixed for 48 h at room temperature; the undissolved material was isolated using centrifugation and filtration at 2.5 μm; that insoluble sample is denoted as PPI-N. This sample can be dissolved in NMP at the low concentration needed for SEC and was studied, as it has a particularly large proportion of material that elutes in the excluded region of the SEC chromatogram (Figure 1). The second sample was the NMP-soluble fraction of Maya asphaltene (MNS), the procedure used to isolate this sample is described in Supporting Information S1. Size Exclusion Chromatography (SEC) Systems. A mixture of NMP and chloroform (volume ratio 6:1) was used as eluent in collecting SEC elution-fractions of the MNS sample. The operating conditions and methodology have been reported elsewhere.10,18 Briefly, a Mixed-D column (5 μm particle size, 300 mm 7.5 mm I.D.) packed with polystyrene/polydivinylbenzene beads, was operated at 80 °C with a Knauer M100 isocratic HPLC pump. The eluent was pumped at 0.5 mL min-1. Material eluting from the column was detected by UVabsorbance at 300 nm. A Perkin-Elmer LC290 single wavelength UV-A detector (supplied by Perkin-Elmer Beaconsfield, UK) was linked to the data acquisition system. The SEC system has been calibrated using standard polystyrene (PS), polymethylmethacrylate (PMMA), and polysaccharide (PSAC) samples, as well as numerous small standard PAH, O-PAH, and N-PAH compounds. For the PPI-N fractionation, an identical SEC system was used (Mixed-D column operating at 80 °C) with pure NMP as eluent. A description of the SEC system and its calibration have been presented elsewhere.18 SEC Fractionation. Samples were collected from the outlet of the SEC system over 2 min intervals (Figure 1). The (38) Morgan, T. J.; George, A.; Davis, D. B.; Herod, A. A.; Kandiyoti, R. Energy Fuels 2008, 22 (3), 1824. (39) Maity, S. K.; Ancheyta, J.; Rana, M. S. Energy Fuels 2005, 19 (2), 343. (40) Douda, J.; Alvarez, R.; Bolaos, J. N. Energy Fuels 2008, 22 (4), 2619.
(37) Berrueco, C.; Alvarez, P.; Venditti, S.; Morgan, T. J.; Herod, A. A.; Kandiyoti, R. Energy Fuels 2009, 23 (6), 3008.
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Figure 2. Area-normalized SEC chromatogram of the PPI-N SEC elution-fractions A and B. NMP was used as eluent with a heated (80 °C) Mixed-D column and detection by UV-A at 300 nm.
elution-fractions were reinjected into the SEC system to check for any changes in elution time compared to when the fractions were initially collected (Figure 2). The SEC mass estimates presented in this work are based on the method outlined in Supporting Information S5. A number of different procedures were investigated to recover the samples from dilute NMP solution, while avoiding the formation of reaction products of NMP. It was found that the reactions of NMP were aided by the presence of oxygen dissolved in the NMP solution.37 The experimental methods to avoid polymerization and oxidation of NMP sample solutions has been outlined elsewhere.22,37 The method described below is primarily intended for the recovery of SEC elutionfractions, to be subsequently analyzed by LD-MS. Briefly, it involves spotting a small volume of solution onto a planar chromatography (PC) plate followed by evaporation at low temperature, aided by blowing nitrogen over the plate. Polyester or aluminum-backed 20 20 cm PC-plates with silica gel thickness of 250 μm (Whatman, UK) were cut into 1.5 cm 4 cm strips. More details on this method are given in the Supporting Information associated with this paper (S8). Introducing the sample into the LD-MS instrument using a PC plate thus reduces the likelihood of sample contamination with NMP reaction products, compared to the procedure where the solution is concentrated using a more conventional method (cf. Supporting Information S1 and refs 10,22, and 37). The focused nitrogen stream (high flow rate) sweeps the evaporated NMP away from the sample. Using a hot plate operating below 100 °C allows the NMP to quickly cool after evaporation and decreases the likelihood of reaction products contaminating the sample. As the PC-plate is inserted into the LD-MS high vacuum system, any residual NMP will be removed at the evacuation stage. This method was favored when the sample was needed solely for LD-MS analyses. It is not possible to recover the sample from the plate by solvent extraction, as some of the heavier components in the sample adhere to the silica particles of the PC plate.41 Blank NMP runs were performed simultaneously alongside the sample (i.e., on another area of the same PC-plate) to check for the formation of NMP reaction products during the recovery process; none were found.
Laser Desorption Mass Spectrometry (LD-MS). A Bruker Daltonics Reflex IV MALDI-TOF mass spectrometer equipped with a 337 nm laser was used for LD-MS. No matrices were used in this work. This is because all the samples investigated were colored; they absorb the laser light directly and act as selfmatrix. A HIMAS detector operating in linear mode was used to investigate the higher molecular mass region. The extraction voltage and detector voltage values were manipulated to attempt to detect the highest mass materials within the samples, without losing the smaller mass ions or overloading the detector. The effect of delays in applying the extraction voltage was also studied. The optimal LD-MS operating parameters and sample preparation procedures are not well-defined for coal- and petroleum-derived samples. A thorough study combining LD-MS with planar chromatography has recently been reported.10,22 The method developed in that study has formed the basis of the method used in this work. Briefly, LD-MS spectra were recorded over a wide range of laser powers for each sample to check the repeatability of the data. Also, spectra were acquired with and without the use of delayed ion extraction (DIE); this made it possible to more clearly observe the higher or lower m/z species, respectively. In all cases, the maximum value of the high-mass accelerator (HMA) voltage was used, 10 kV, to aid the detection of high-mass species. The extraction voltage was 20 kV. UV-Fluorescence (UV-F) Spectroscopy. The procedure used for this analysis has been described elsewhere,42 and a brief summary is given here. A quartz cell with 1 cm path length was used in a Perkin-Elmer LS50 luminescence spectrometer set to scan at 240 nm min-1 with a slit width of 5 nm. The spectrometer has automatic correction for changes in source intensity as a function of wavelength. Emission, excitation, and synchronous spectra of the samples have been acquired using NMP as solvent. Synchronous spectra were obtained at a constant wavelength difference of 20 nm. Normally, the solutions would be diluted in order to avoid self-absorption effects, that is, dilution was increased until the fluorescence signal intensity began to decrease. However, as the samples being investigated here are elution fractions recovered from the outlet of the SEC system, they were already very dilute. In addition, only limited
(41) Herod, A. A.; George, A.; Islas, C. A.; Suelves, I.; Kandiyoti, R. Energy Fuels 2003, 17, 862.
(42) Li, C.-Z.; Wu, F.; Cai, H.-Y.; Kandiyoti, R. Energy Fuels 1994, 8, 1039.
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Figure 3. LD-MS of the PPI-N SEC elution-fractions A (lp 40%, black) and B (lp 50%, red), where no DIE was used and HMA was set to 10 kV. The spectra were acquired from the summation of 10 laser pulses on the same spot. Table 1. Mass Estimates from SEC and LD-MS for SEC Elution-Fractions A and B from the PPI-N Sample SEC mass (u)a sample fraction A fraction B
peak max int. 305 000 630
c
lower limit c
32 600 160
LD-MS (m/z)b upper limit c
484 000 1660
peak max int.
lower limit
upper limit
1750 650
500 450
7100 2100
tail >40 000 ∼6600
a
For the SEC mass estimate the method used is described in Supporting Information (S5). b For the LD-MS mass estimate the method is described in ref 10, and the positions for the tail, upper, and lower limits were taken from data shown in Figure 3. c The mass estimates for samples eluting earlier than 15 min are thought to be overestimated and would not normally be accounted for,10,18 in this case they are shown to highlight the problem.
However, the observed SEC elution behavior of Fraction A is consistent with the “excluded” fraction remaining intact upon further dilution. The two elution-fractions were next analyzed using LDMS. Figure 3 presents LD-mass spectra of SEC elutionfractions A and B from the PPI-N sample, with labels indicating the areas of the spectrum used for the mass estimates displayed in Table 1. The two spectra were recorded under identical conditions, apart from the use of greater laser power (50%) for Fraction B, compared to 40% for fraction A. The greater laser power was required to generate data with a satisfactory signal-to-noise ratio from fraction B, as the sample was present in lower abundance on the target, or was more difficult to ionize, compared to fraction A. The spectrum for fraction B in Figure 3 showed a clear shift in the peak maximum intensity to lower mass compared, to fraction A. The spectra were obtained without the use of the delayed ion extraction (DIE) voltage, available on the instrument. This means that the full ranges of ions generated from the sample can be examined. When a DIE is used, the smaller ions are not observed. It should be noted that molecules with low mass (less than approximately 250 u) are generally lost in the high-vacuum system of the LD-MS instrument before ionization and are not normally observed as part of a spectrum. To compare the SEC-based mass estimates with those obtained by LD-MS, the PS/PMMA calibration was extrapolated to cover the region where fraction A had eluted.
volumes of sample could be recovered, and the fluorescence quantum yields from the heavier fractions are rather low. Therefore, it was not always possible to obtain fluorescence signal significantly greater than the background fluorescence.
Discussion of Results ; I Coal Tar Pitch, Pyridine-Insoluble Sample (NMP Washed, PPI-N) - Fractionated by SEC. Figure 1 shows the SEC chromatogram of the PPI-N sample used in the fractionation. In this figure, the time bands corresponding to the successively recovered elution-fractions are indicated. Fraction A corresponds to an elution time between 9.5 and 11.5 min (excluded peak) and fraction B from 18.0 to 20.0 min (retained peak). Figure 2 shows the SEC chromatograms of elution-separated fractions, A and B. The solutions were not concentrated before reinjection. Due to the level of dilution, the chromatograms are somewhat noisier than usual. It is clear, however, that the two fractions have appeared at elutiontimes similar to the times observed upon their initial elution. It may be recalled that others have suggested these apparently large mass materials to be formed of aggregates of smaller molecules.1 Had material observed in the excluded region (Fraction A) indeed been composed of aggregates of smaller molecules, however, some dis-aggregation would have been expected during further dilution, when the fraction was reinjected and mixed with the eluent stream. No indication of such disaggregation has been observed. It is difficult to arrive at a method for “proving” a negative. 6007
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Figure 4. Area-normalized SEC chromatogram of the PPI-N SEC elution-fractions (F1-F4, excluded region) alongside that of the whole sample. NMP was used as eluent with a heated (80 °C) Mixed-D column and detection by UV-A at 300 nm.
Normally this would not be done, as it is known that in the excluded region (8-15 min) the PS calibration no longer correlates well with the mass of coal-derived materials.18 The mass estimates of fractions A and B, obtained by LD-MS and by SEC, are presented in Table 1. Details of the method used to generate the SEC-based mass estimates are presented in the Supporting Information (S5). The positions in the LD-mass spectra where the mass estimates were recorded were selected to obtain conservative estimates of the upper limit. This was achieved by taking the values from where there was a clear deviation from the baseline (cf. Figure 3). The method used does not, however, clearly distinguish between the ”trace” values relating to actual signal from sample material and material representing multimer ions or clusters. The mass estimates by SEC in Table 1 for material eluting earlier than about 15 min (Fraction A) are very high and probably overestimate actual values. However, peak maxima and upper limit values for fraction B, obtained from SEC and LD-MS were within ∼20% of each other. The lower limits showed larger differences as expected; as already signaled, lighter material are mostly lost under the high vacuum of the LD-MS instrument. Furthermore, smaller molecules show stronger UV-absorbance in the detector stage of SEC. This translates into a disproportionately large detector response for smaller mass material compared to larger mass material. The effect tends to skew results toward lower masses. The LD-MS data clearly showed larger peak maximum intensity mass and average mass values for fraction A (excluded region) compared to fraction B (retained region in Figure 1). There still remain, however, significant differences between masses predicted by SEC (using the PS-PMMA calibration) and those obtained by LD-MS. Narrower elution-time fractions were studied, in order to identify the molecular mass levels at which these differences become measurable. Examining Narrower Elution-Time Fractions. Ten successive SEC elution-fractions were produced from the PPI-N sample, each fraction covering ∼70 s of elution time. Figure S9.1 (Supporting Information) shows the SEC
chromatogram of the whole PPI-N sample used to produce the 10 fractions; the time bands have been indicated. Figures 4 and 5 present SEC chromatograms obtained from the reinjection of these elution-fractions. For clarity, elutionfractions 5 and 6 are not included; these two chromatograms gave low intensity responses and poor signal-to-noise ratios. The time ranges of the SEC elution-fractions collected from the PPI-N sample are presented in Table 2. In Figures 5 and 6, better segregation could be observed between successive elution-fractions recovered from the retained region (F6-F10), compared to those recovered from the excluded region (F1-F5). Fractions F5-F7 all showed peaks in both the excluded and retained regions, although F5 was collected from the tail edge of the excluded peak and F6 and F7 were recovered from the front edge of the retained peak. This suggests that the column was slightly overloaded and the separation less than totally complete. Evidence for this can be seen in Figure 1, where the intensity of the valley at 15 min is clearly not close to zero. Overloading the column tends to delay elution of excluded material into the valley and into the retained peak area (e. g., chromatogram of fraction 7). Meanwhile, fraction 4 could be observed to elute earlier than the time of collection on reinjection at a lower concentration. To see if a better separation could be achieved, the experiment was repeated using a lower sample concentration. When the elution-fractions from this sample were reinjected into the SEC system, the low concentrations made it difficult to discern signal above background noise (not shown). It was difficult to determine if the apparent reduction in smearing of the signal was due to better separation or to signal loss to baseline level. There is a compromise that needs to be made between working with sample concentrations in SEC that allow enough material for recovery and further analysis and working at a low enough concentration to avoid overloading the SEC column. A way around this problem may be to use two columns in series, to improve resolution. However, this has not been investigated as part of the present study. Table 3 summarizes SEC elution times and mass estimates for the PPI-N elutionfractions. 6008
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Figure 5. Area-normalized SEC chromatogram of the PPI-N SEC elution-fractions (F7-F10, retained region) alongside that of the whole sample. NMP was used as eluent with a heated (80 °C) Mixed-D column and detection by UV-A at 300 nm.
Figure 6. Plot of elution time (peak max) from SEC vs log10(m/z) of the peak max obtained from LD-MS analysis, for the PPI-N SEC elutionfractions. Mixed-D column was used with NMP as eluent.
The spectra in Figures S10.1 and S10.2 (cf. Supporting Information) provide clear evidence that material eluting at times indicating exclusion from column porosity (15 min, fraction F6-F10, cf. Table 4). To examine the relationship between the SEC elution times of the fractions and their mass ranges from LD-MS, peak maximum intensity values from these two independent techniques were plotted against one another (Figure 6). For some of the SEC fractions, the m/z peak maximum intensity (from LD-MS) was unclear (cf. Table 4). This is probably due to the low abundance of some of the SEC fractions and was found to be independent of laser power. In these cases the lower estimate was used in the plots shown in Figures 6 and 7. A nearly linear relationship was observed between the elution times and the m/z peak maxima of elution-fractions collected during the retained (resolved, later eluting) part of
Table 2. Elution Time Pans of the SEC Elution-Fractions Collected from the PPI-N Sample excluded region fraction F1 F2 F3 F4 F5
time range (min) 8.50-9.65 9. 65-10.80 10.80-11.95 11.95-13.10 13.10-14.25
retained region fraction F6 F7 F8 F9 F10
time range (min) 16.30-18.00 18.00-19.50 19.50-21.00 21.00-22.50 22.50-24.00
Figures presenting the LD-MS spectra of the PPI-N SEC elution-fractions, with no DIE and 600 ns DIE are shown in the Supporting Information (Figures S10.1 and S10.2, respectively). The overlap between the fractions was clearly discernible. In both types of data, however, a clear trend from higher masses to lower masses is observed, going from F1 to F10. Table 4 compares the mass estimates from LD-MS and SEC, for the PPI-N SEC elution-fractions. 6009
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Table 3. Elution Time and Mass Estimates from SECa of the PPI-N SEC Elution-Fractions (F1-F10) peak maximum intensity sample fraction 1 fraction 2 fraction 3 fraction 4 fraction 5 fraction 6 fraction 7 fraction 8 fraction 9 fraction 10
time (min) 10.1 11.0 10.7 10.8 11.5 17.4 18.1 19.0 19.9 21.0
mass (u) b
300 000 150 000b 190 000b 180 000b 104 000b 1100 900 650 450 300
main peak
second peak
time range (min)
time range (min)
9.0-13.5 9.6-15.0 9.4-15.0 9.4-15.0 9.5-15.0 15.0-20.5 15.5-21.0 16.0-23.0 17.0-24.2 18.0 -25.0
main peak upper limit (u) b
n/a n/a 16.5-20 16.5-20 16.5-20 9.5-15 9.5-15 n/a n/a n/a
lower limit (u) 33 000b 10 000 10 000 10 000 10 000 450 350 150 100 100
490 000 300 000b 360 000b 360 000b 330 000b 4800 3200 2000 1000 770
a The SEC mass estimates are based on the method outlined in Supporting Information (S5). b The mass estimates for samples eluting earlier than 15 min are thought to be overestimated and would not normally be accounted for,10,18 in this case they are shown to highlight the problem.
Table 4. Mass Estimates from SEC and LD-MS for SEC Elution-Fractions (F1-F10) from the PPI-N Sample SEC mass (u)a
LD-MS (m/z) b
sample
peak max int.
lower limit
upper limit
fraction 1 fraction 2 fraction 3 fraction 4 fraction 5 fraction 6 fraction 7 fraction 8 fraction 9 fraction 10
300 000 150 000 190 000 180 000 100 000 1100 900 650 450 300
33 000 10 000 10 000 10 000 10 000 450 350 150 100 100
480 000 300 000 360 000 360 000 330 000 4800 3200 2200 1000 770
peak max int. 1800 2100 1800 1500-1600 1400 1300-1500 1000-1100 750-900 600 400-1000
lower limit
upper limit
tail
700 750 700 700 600 600 500 500 450 300
6500 6000 5000 4600 4000 3300 2500 2500 2000 2100
>10 000 >10 000 >8000 ∼7500 ∼7000 ∼6500 ∼5500 ∼6000 ∼5000 ∼4500
a The SEC mass estimates are based on the method outlined in Supporting Information (S5). b The LD-MS mass estimates are based on the method described above (cf. Figure 3). For some of the fractions the peak maximum intensity was unclear; in these cases a range is given.
Figure 7. Plot of elution time (peak max) from SEC vs log10 (m/z) of the peak max obtained from LD-MS analysis, for the PPI-N SEC elutionfractions, alongside PS and PAH standards. A Mixed-D column was used with NMP as eluent.
the PPI-N chromatogram. The fractions recovered from samples eluting in the excluded part of the chromatogram showed a less clear trend and a large discontinuity was observed between the two groups of data points. Furthermore, it was not possible to ascertain that the entire sample had been uniformly ablated from the PC surface by the laser, and it has been shown that these heavy fractions cannot be recovered completely by use of solvents from silica in column and in planar chromatography.41
The results in Figure 6 were compared with SEC calibrations prepared using standard samples.10,18 Figure 7 shows the PPI-N SEC elution-fractions plotted alongside PS and PAH standards. Fractions F6-F10 fall close the PS and PAH results. These data confirm earlier calibration work in this laboratory, showing good agreement between SEC and LD-MS results up to a little over 3000 u. The data show that using PS and PAH standards to calibrate this region of the SEC chromatogram provides a good approximation for coal-derived materials. 6010
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The results for fractions F1-F5, however, present further evidence of the unusual behavior of the material eluting in the excluded region. Similar results have been obtained previously.17,43,44 In earlier work, these observations were attributed to the failure of laser desorption to ablate the full range of larger mass species. The possible significance of the separation of excluded and retained peaks as likely pointing to a change of molecular conformation was not realized. Other materials observed in our previous work to show similarly early elution times compared to their masses were spherical samples: fullerenes, soots, and silica particles.18 This finding suggest that the material showing signal under the excluded peak might be adopting quasi-spherical or cagelike conformations, which show up at earlier elution times than their molecular masses would lead us to expect. Alternatively, it is possible that these results represent an upper limit of LD-MS, when used for these types of samples, but there is no obvious reason why this should be so. A suitable matrix may help increase the upper mass limit of material that can be observed in its entirety. A further investigation of the LD-MS results is presented in the Supporting Information (S12), where number average molecular mass (Mn) and the weight average molecular mass (Mw) were calculated and plotted against SEC elution times. The Mn and Mw estimates were also used to examine the polidispersity index (PDI = Mw/Mn) for the PPI-N and MNS elution fractions (cf. Table S12.2 and S19.2 respectively). The method outlined in Supporting Information (S14) was used to calculated Mn and Mw from the LD-MS data.
Table 5. Time Ranges of the SEC Elution-Fractions Collected from the MNS Sample excluded region fraction F1 F2
time range 10.50-11.65 11.65-12.80
retained region fraction F3 F4 F5 F6 F7
time range 15.65-16.80 16.80-18.00 18.00-19.15 19.15-20.50 20.50-22.00
and identified in earlier work on coal tar pitch.43 Table 6 summarizes the SEC elution times and mass estimates of the MNS SEC elution-fractions. The LD-mass spectra of these elution-fractions are shown in Figure S17.1 (Supporting Information); no DIE was used in these runs. Figure S17.2 presents the spectra obtained using 600 ns DIE. As in the case of the PPI-N results, a nearly uniform trend can be seen from higher to lower average m/z values. This is most apparent for fractions F1-F5. However, fractions F6 and F7 appeared to shift to higher masses compared to F4 and F5. It is possible that the laser is unable to ablate these materials completely, as they contain significantly more aliphatic groups than the coal-derived PPI-N sample. For example, lower masses were observed for the PPI-N sample by LD-MS under similar conditions, even though the PPI-N is thought to contain higher average molecular masses.35 This is a clear indication that the lower mass components of the MNS sample were not observed. These results suggest that the MNS sample contains less detectable low-mass (m/z < 800) species by LD-MS than the PPI-N sample. It is likely that the lower mass components of the MNS sample (elution fractions F6 and F7) are not sufficiently aromatic or have structures that could not be ionized under the LD-MS conditions used. It is known that it is more difficult to ionize aliphatic species during LD-MS.17 These results are discussed further in the Supporting Information (S19). The relevant mass estimates from SEC and LD-MS have been summarized in Table 7. Figure 9 shows the elution time of the MNS fractions (peak max. int.) plotted against the m/z peak maximum intensity, obtained from LD-MS analysis. In general it was more difficult to obtain LD-MS data for the MNS elution fractions and these data were less repeatable. Each fraction seemed to contain a larger range of species compared to the PPI-N fractions. In Figures S17.1 and S17.1, where ranges of values (typically over 100-500 units) were obtained for the m/z peak maxima (Table 7), the average of these values was used. Figure 10 shows the MNS elution times of the fractions plotted alongside those of PS and N-PAH molecular mass standards. Figures S17.1 and S17.2 show that the MNS elutionfractions produced a more scattered correlation between mass (m/z from LD-MS) and elution time in SEC compared to the PPI-N fractions. In particular, the fractions recovered from the retained region do not fit the PS and N-PAH calibration as closely (i.e., F6 and F7 appear at higher than expected mass from LD-MS analysis). As before, there was a large discontinuity between the fractions recovered from the retained and excluded regions.9,17,18 However, despite the less well-defined nature of these data compared to the PPI-N results, similar general overall trends were found. Both sets of data showed increasing mass (by LD-MS) with decreasing SEC-elution time. In particular, the material eluting in the excluded region was of significantly higher average mass
Discussion of Results ; II Maya Crude Oil Asphaltenes - Fractionated by SEC. Similar methods were employed to examine the NMP soluble fraction of an asphaltene recovered from Maya crude oil (denoted as MNS). The SEC column used was a similar “Mixed-D” column. However, the eluent was a 6:1 mixture of NMP and CHCl3, instead of pure NMP, which is routinely used when examining coal-derived samples. The elution-time bands of the fractions collected are listed in Table 5 (cf. Figure S16.1 in the Supporting Information). Figure 8 presents chromatograms of the elution-fractions, following reinjection into the SEC system. Qualitatively, results from the SEC characterization of the MNS elution-fractions were broadly similar to those of the PPI-N sample. However, the MNS chromatogram showed a less intense excluded peak relative to the retained peak, spanning a shorter time range (10 min 30 s to 13 min 00 s) compared to that of the PPI-N sample (8 min 30 s to 14 min 00 s). Fewer fractions of excluded material could be collected and less segregation was observed between the two successive fractions collected in the excluded region, F1 and F2. When reinjected, Fraction 2 eluted slightly earlier than Fraction 1, although the difference between the two peaks was relatively small (∼20 s). This was probably a consequence of the column being overloaded, indicated by a nonzero intensity at around 14 min, where a zero intensity valley would normally be expected. The shift to an earlier elution time of F2 is similar to that observed in the PPI-N fractions above (43) Lazaro, M. J.; Islas, C. A.; Herod, A. A.; Kandiyoti, R. Energy Fuels 1999, 13, 1212. (44) Islas, C. A. PhD Thesis, University of London: 2001.
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Figure 8. Area-normalized SEC chromatogram of the MNS SEC elution-fractions (F1-F7). A mixture of NMP and CHCl3 (6:1) was used as eluent with a heated (80 °C) Mixed-D column and detection by UV-A at 300 nm. Table 6. Elution Time and Mass Estimates from SEC of the MNS Elution-Fractions (F1-F7) peak maximum intensity sample
time (min)
fraction 1 fraction 2 fraction 3 fraction 4 fraction 5 fraction 6 fraction 7
11.6 11.4 16.9 17.9 19.0 20.0 21.2
main peak
mass (u)
time range (min)
200 000b 230 000b 3600 1700 750 350 240
10.3-13.0 10.3-13.0 15.6-18.7 16.3-19.7 17.0-21.5 18.0-22.2 18.9-23.3
main peak upper limit (u)
lower limit (u)
360 000b 360 000b 6600 3900 2300 1100 550
100 000b 100 000b 1350 650 250 200 150
a
The SEC mass estimates are based on the method outlined in Supporting Information (S5) b The mass estimates for samples eluting earlier than 15 min are thought to be overestimated and would not normally be accounted for,9 in this case they are shown to highlight the problem.
Table 7. Mass Estimates from SEC and LD-MS for SEC Elution-Fractions (F1-F7) from the MNS Sample SEC mass (u)a
LD-MS (m/z) b
sample
peak max. int.
lower limit
upper limit
fraction 1 fraction 2 fraction 3 fraction 4 fraction 5 fraction 6 fraction 7
200 000 230 000 3600 1700 750 350 250
100 000 100 000 1400 650 250 200 130
360 000 360 000 6600 3900 2300 1100 550
peak max. int.c 1700-2200 1300-1500 1200-1400 1000 900 900-1400 800-1200
lower limit
upper limit
tail
600 450 350 300 300 500 400
5000 3300 3000 3500 1800 2300 2500
>10 000 >6000 >5000 >5000 ∼3500 >6000 ∼5000
a
The SEC mass estimates are based on the method outlined in Supporting Information (S5). b The LD-MS mass estimates are based on the method described above (cf. Figure 3) and elsewhere.10 c For some of the fractions the peak maximum intensity was unclear; in these cases a range is given.
than the material recovered in the retained part of the chromatogram. To investigate the higher than expected m/z values for MNS SEC fractions F6 and F7 (expected to be the fractions that contain the smallest molecules) UV-F spectroscopy was used to examine the sizes of aromatic chromophores present. These results are discussed in Supporting Information (S21) for the PPI-N and MNS samples. The key findings are included in the summary below.
polymer-based calibrations, commonly used to convert SEC elution times into estimates of molecular mass. When comparing mass estimates by LD-MS of SEC elution-fractions, similar molecular mass ranges were found for fractions collected at similar elution times, for material from the coal tar pitch and petroleum asphaltene. This suggests that the two samples studied show a similar relationship between size and mass. Polydispersity indexes calculated from Mw and Mn estimates were fairly consistent for the PPI-N and MNS elution fractions (cf. Table S12.2 and S19.2 respectively). These findings provide a degree of confirmation for the suitability of SEC as a technique for estimating molecular masses of complex hydrocarbon mixtures. LD-MS results for the petroleum-derived materials were less coherent compared to those for the coal-derived sample.
Summary of the Results High-mass species in a coal and a petroleum derived sample were studied by LD-MS of eluent fractions collected from SEC. Results from this method were compared with 6012
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Figure 9. Plot of elution time (peak max) from SEC vs log10 (m/z) of the peak max obtained from LD-MS, for the MNS SEC elution-fractions.
Figure 10. Plot of elution time (peak max) from SEC vs log10 (m/z) of the peak max obtained from LD-MS analysis, for the MNS SEC elutionfractions, alongside PS and PAH standards. A Mixed-D column was used with a mixture of NMP and CHCl3 (6:1) as eluent.
A possible explanation of the unusual LD-MS results obtained for MNS elution fractions F6 and F7 compared to the SEC results comes from UV-F analysis and a follow-up NMR analysis (the NMR work is being prepared for publication).22 In that NMR-based study, the bulk NMP soluble fraction of the Maya asphaltene (MNS) and the NMPinsoluble material (MNI) were examined. The findings showed that the aromaticity of the MNS sample was 45% ((2%), and that of the MNI sample was 55% ((2%). Through the use of average structural parameter calculations it was concluded that the MNS samples contained molecules with structures made up of a number of relatively small aromatic units connected through long alkyl side chains and naphthenic rings. Moreover, the UV-F results presented in S21 confirm the small size of the aromatic chromophores in the MNS samples (elution fractions F6 and F7 in particular). For the coalderived elution fractions, a clear trend of increased fluorescence intensity and shifts to shorter wavelengths were observed with decreasing molecular size, as would be expected.
For the MNS ones; this trend was not observed. Within the context of the present work, the isolated nature of the aromatic chromophores and their relatively small sizes would probably account for the difficulty in obtain satisfactory LD-MS spectra for the MNS samples. The effect would be accentuated in the case of the lowest molecular mass components, notably elution fractions F6 and F7 as indicated by UV-F results. This explanation may be viewed in the context of difficulties encountered, for example, in trying to ionize PS molecules in LD-MS. Observing PS in LD-MS analysis is only possible when a particular combination of matrices is used.17 It appears necessary to identify a suitable matrix to help with the examination of samples that are more aliphatic/hydroaromatic in nature. Conclusions We are safely able to conclude that the PS and PAH calibrations provide a reasonable estimate of mass for 6013
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material eluting in the retained (resolved - later eluting) region of SEC chromatograms. At elution times shorter that 15 min (i.e., in the excluded region) there was significant deviation from the PS calibration for both the coal tar pitch and the petroleum-derived samples. In all cases, however, materials eluting early and showing signals in the excluded region were observed to be of significantly higher average masses compared to material eluting at longer times, in the retained region. The data appears conclusive. It should be noted, however, that the excluded region itself cannot be subdivided into a higher and lower mass region; it has to be taken as a whole. No evidence was found, furthermore, to show that the early eluting material could be composed simply of aggregates of smaller molecules. Indications were found that not all the higher molecular mass material could be observed by LD-MS. An upper limit to the detection of high-mass material by LD-MS appears to have been reached for the samples used in this study (i.e., coal tar pitch and petroleum asphaltene). It is possible that the range of masses observed by LD-MS could be increased by
identifying and using matrix materials appropriate to these samples. The analytical approach presented in this work provides a valid and useful basis for exploring relationships between molecular size, molecular mass, and structure for complex coal- and petroleum-derived materials. More work, however, is still needed on a wider range of samples to better understand the differences in behavior observed during this study. Supporting Information Available: S1 - Procedure for isolating the NMP soluble fraction of Maya asphaltene. S5 - Method for estimating mass numbers from the SEC data. S8 - Method for recovering the SEC elution fractions. S9 - SEC elution time bands for the PPI-N fractionation. S10 - Results of the LD-MS analysis of PPI-N samples. S12 - Estimating average mass numbers for the PPI-N samples. S14 - Estimating average mass numbers from the LD-MS data. S16 - SEC elution time bands for the MNS fractionation. S17 - Results of the LD-MS analysis of MNS samples. S19 - Estimating average mass numbers for the MNS samples. S21 - UV-F results for the MNS and PPI-N samples. This information is available free of charge via the Internet at http://pubs.acs.org/.
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