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Comment on a Paper by Mullins, Martinez-Haya, and Marshall “Contrasting Perspective on Asphaltene Molecular Weight. This Comment vs the Overview of A. A. Herod, K. D. Bartle, and R. Kandiyoti” Alan A. Herod,*,† Keith D. Bartle,‡ and R. Kandiyoti† Department of Chemical Engineering, Imperial College London, London SW7 2AZ, United Kingdom, and Chemistry Department, UniVersity of Leeds, Leeds LS2 9JT, United Kingdom ReceiVed July 28, 2008. ReVised Manuscript ReceiVed September 15, 2008 Introduction This comment refutes a paper by O. C. Mullins, B. MartinezHaya, and A. G. Marshall,1 alleging that our analytical methods detect aggregates of asphaltenes and not molecules larger in mass than their publication claims, a range of 500-1000 units with a peak of intensity around 800 units. We discuss the detail of our methods and show why our results are correct and in agreement with experimental results from laboratory and plant, whereas the methods used by the authors of ref 1 detect only the small molecules by avoiding fractionation to isolate the larger molecules. Analytical caution is needed when dealing with polydisperse materials. Discussion There appear to be significant differences between our results and our position expressed in a recent paper2 and what Mullins, Martinez-Haya, and Marshall1 report having understood from it. Some clarification of our position would be useful. We would like to consider differences between our work and perceptions in ref 1 in order. 1. On Size-Exclusion Chromatography (SEC). The authors of ref 1 have suggested that our work considers asphaltene molecular-weight distributions to be bimodal, with one component in the megadalton range and the second component in the approximately 5 kDa range. We have indeed systematically reported obserVing bimodal size-exclusion chromatograms for nearly all samples with wide polydispersities. This applies to both coal- and petroleumderived heavy liquids.2-4 We have also reported that samples with low and relatively narrow molecular-mass distributions do * To whom correspondence should be addressed. Fax: +44-207-5945638. E-mail:
[email protected]. † Imperial College London. ‡ University of Leeds. (1) Mullins, O. C.; Martinez-Haya, B.; Marshall, A. G. Contrasting perspective on asphaltene molecular weight. This comment vs the overview of A. A. Herod, K. D. Bartle, and R. Kandiyoti. Energy Fuels 2008, 22, 1765–1773. (2) Herod, A. A.; Bartle, K. D.; Kandiyoti, R. The characterization of heavy hydrocarbons by chromatographic and mass spectrometric methods: An overview. Energy Fuels 2007, 21, 2176–2203. (3) Kandiyoti, R.; Herod, A. A.; Bartle, K. D. Solid Fuels and HeaVy Hydrocarbon Liquids: Thermal Characterisation and Analysis; Elsevier: Amsterdam, The Netherlands, 2006; ISBN: 0-08-044486-5. (4) Karaca, F.; Islas, C. A.; Millan, M.; Behrouzi, M.; Morgan, T. J.; Herod, A. A.; Kandiyoti, R. The calibration of size exclusion chromatography columns: Molecular mass distributions of heavy hydrocarbon liquids. Energy Fuels 2004, 18, 778–788.
not show a peak in the short elution time range.5 They show single, resolved peaks. It is erroneous, however, to suggest that we interpret the early eluting peak (apparently very large sizedmolecules) to correspond to sample molecules of megadalton masses. We have instead explained2,3 that the early eluting peak in these bimodal distributions is positioned at elution times corresponding to elution times of polystyrene molecular-mass standards of very large mass. We have explained that we consider these materials to be of very large “apparent molecular mass”. We have furthermore suggested that the material giving signal under the excluded peak may appear to have very large masses (i.e., elute at short times) because of changed molecular conformations, which result in larger than expected hydrodynamic Volumes.2-4,6,7 Such molecules are thought to elute at times corresponding to much larger masses, when evaluated using the polystyrene calibration. For example, fullerenes (C60 and C70 of molecular masses 720 and 840 units) appear at elution times corresponding to the exclusion limits of our SEC columns. Clearly, the molecular size-hydrodynamic volume relationship exemplified by the linear calibration between molecular mass (of polystyrenes and small standard molecules) and elution time breaks down when three-dimensional conformations are considered. We consider therefore that the early eluting molecules may possibly correspond to material that has adopted threedimensional conformations (or large hydrodynamic volumes) and appear to have far larger masses than their actual values. We are thus unable to accept the interpretation put on our work by the authors of ref 1. Furthermore, we have been careful in stating that we offer this explanation as a hypothesis, which remains to be proven. Our hypothesis is, in part, supported by the observation that known standard colloidal silica samples and other particulate material, such as soot (as well as the fullerene samples cited above), all elute at short elution times corresponding to exclusion from SEC column porosity.2-4 To the extent that their diameters (5) Morgan, T. J.; Morden, W. E.; Al-Muhareb, E.; Herod, A. A.; Kandiyoti, R. Essential oils investigated by size exclusion chromatography and gas chromatography-mass spectrometry. Energy Fuels 2006, 20, 734– 737. (6) Johnson, B. R.; Bartle, K. D.; Herod, A. A.; Kandiyoti, R. Improved size exclusion chromatography of coal derived materials using N-methyl 2-pyrrolidinone as mobile phase. Prepr. Pap.-Am. Chem. Soc., DiV. Fuel Chem. 1995, 40 (3), 457–460. (7) Herod, A. A.; Zhang, S.-F.; Kandiyoti, R.; Johnson, B. R.; Bartle, K. D. Solubility limitations in the determination of molecular mass distributions of coal liquefaction and hydrocracking products: N-Methyl 2-pyrrolidinone as mobile phase in size exclusion chromatography. Energy Fuels 1996, 10, 743–750.
10.1021/ef8006036 CCC: $40.75 2008 American Chemical Society Published on Web 10/21/2008
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are known, they also give a good linear log(diameter) versus elution time relationship. To date, we have not been able to identify the mass ranges of material appearing under the excluded peaks. Isolation of these materials is difficult because of high levels of dilution in 1-methyl-2-pyrrolidinone (NMP) or other eluents. The chemical reactivity of NMP has made removal of excess solvent difficult, although we are at present continuing work to isolate these materials. Meanwhile, no other technique is able to distinguish between two- and three-dimensional structures through size as might be possible by SEC. Sample Aggregation in SEC. In evaluating SEC as a technique, the authors of ref 1 have also taken up the often repeated but never proven claim that the excluded material is made of aggregates of small polar molecules, rather than molecules of large hydrodynamic volume. This idea originates from the petroleum industry and adopted by Lafleur and Nakagawa,8 who found that small polar standard molecules tended to elute early, in their column. Indeed, they observed that acetone and dihydroxyanthraquinone eluted far earlier (by 11 and 17 min, respectively) than their molecular masses would suggest, when related to the polystyrene calibration. However, times change and so do SEC column compositions. In our calibration work, no compounds other than fullerenes were observed to elute more than about 3 min earlier than expected. When using mixed solvents9 (mixtures of NMP and chloroform), some oxygenates eluted later than expected by about 3 min. In addition, Lafleur and Nakagawa8 examined the excluded material of a coal extracted using pyridine by heated probe mass spectrometry. The instrument was reported to have an upper mass limit of m/z 500, and no signal was observed above m/z 250. However, much of this particular sample would simply not have been volatile in the ion source of the instrument. The low mass material detected would most likely have been dimers and oxidation products of the NMP solvent. It may be noted that, despite much speculation, no evidence has ever been provided to indicate the occurrence of molecular aggregation, at the Very low concentrations used in SEC (sample concentrations are shown elsewhere4,9). When a wide range of successively diluted samples were injected into the SEC system, the relative proportions of the two peaks of the bimodal sizeexclusion chromatograms remained unchanged. This shows the absence of any type of disaggregation upon, at times extreme, dilution. Nor have proponents of the presumed “aggregation” explanation been capable of showing that samples diluted to levels used in SEC experiments can be effectively “disaggregated” in any way. In our own search for evidence of aggregation, we have also collected sample fractions eluting at short times in SEC, corresponding to the excluded region of the chromatograms, of a pitch sample. Upon re-injection, it was observed that these materials eluted at the originally observed elution times.10 There was no evidence of small molecules liberated by “disaggregation” after further dilution. We have also shown that the presumed disaggregation of the early eluting peak, reported after the addition of ionic salts to the NMP eluent, had an entirely different origin. In fact, the salts promote surface effects that (8) Lafleur, A. L.; Nakagawa, Y. Multimode SEC for characterizing coalderived mixtures. Fuel 1989, 68, 741–752. ´ lvarez, P.; Millan, M.; (9) Berrueco, C.; Venditti, S.; Morgan, T. J.; A Herod, A. A.; Kandiyoti, R. Calibration of size exclusion chromatography columns with NMP/chloroform mixtures as eluent: Applications to petroleum derived samples. Energy Fuels 2008, 22, 3265–3274. (10) Lazaro, M. J.; Islas, C. A.; Herod, A. A.; Kandiyoti, R. Calibration of size exclusion in 1-methyl-2-pyrrolidinone for coal derived materials using standards and mass spectrometry. Energy Fuels 1999, 13, 1212–1222.
ruined the size separation mechanism.11-13 We have pointed out that simple blank tests with known standards would have shown clearly that the observed shifts had no relation to “aggregates” disaggregating. We thus have reason to think that the early eluting components are indeed molecules but of different size (hydrodynamic volumes) in relation to their masses, rather than aggregates that will not disaggregate. In consequence, our position regarding the molecular masses of the excluded peak, as discussed,2,3 is quite clear. However, the authors of ref 1 appear to have ignored the evidence that we have raised. By comparing to standard three-dimensional solid materials,2,3 we estimate the range of diameters of petroleum asphaltene molecules showing signal under the excluded peak to range from less than 1 to about 6 nm. This calibrated size range covers the range claimed by the authors of ref 1 for asphaltene molecules in their Figure 1. GC-MS of Alkanes. The authors of ref 1 have compared our bimodal distributions to evidence from the GC-MS of alkanes, etc., showing a continuous range of mass. Although a somewhat elementary point, we are sure most readers will not need reminding that the ranges of masses available to GC-MS instruments are far lower than the materials being considered in our work, outlined in refs 2 and 3. Had GC-MS methodology been applicable to samples under consideration, there would have been no need to use less exact methods, such as SEC or laser-desorption mass spectrometry, to attempt to gauge molecular-mass distributions. Clearly, even if alkanes did show bimodal distributions, GC-MS and SFC-MS would not have been able to detect the phenomenon. This is because the high-mass components would not be able to pass through the chromatographic columns. The authors of ref 1 appear to have ignored that the bulk of petroleum asphaltenes and most coal-derived liquids isolated as heptaneinsoluble, acetone-insoluble, or acetonitrile-insoluble material simply do not pass through GC columns. Although a relatively simple point, we have explained this difficulty repeatedly, notably in ref 2 in some detail. The comparison is not appropriate because the relevant components cannot be detected by GC. SEC Column Calibrations. The calibrations that we have used to date are based on determining the elution times of sets of molecular-mass standards. The standards used belong to many classes of compounds, including polystyrene standards and an array of other polymers with distinct structures (e.g., polymethylmethacrylates, polysaccharides, etc.), in addition to an extensive suite of known standard compounds (up to about 1000 units). The full list has been given.2,3,9,10 The work has shown that the small-mass end of the polystyrene (and other polymer) calibration range from 1000 down to 500 units overlaps with the upper end of the mass range of the known, relatively small mass, standard molecules.2-4,10,14 Because the structures of (11) Herod, A. A.; Shearman, J.; Lazaro, M.-J.; Johnson, B. R.; Bartle, K. D.; Kandiyoti, R. The effect of LiBr addition to 1-methyl-2-pyrrolidinone in the size exclusion chromatography of coal derived materials. Energy Fuels 1998, 12, 174–182. (12) Karaca, F.; Behrouzi, M.; Morgan, T. J.; Herod, A. A.; Kandiyoti, R. The effect of salts on the SEC profiles of heavy hydrocarbon liquids: New approach with salts dissolved in solvents used for planar chromatography. Energy Fuels 2005, 19, 187–199. (13) Karaca, F.; Morgan, T. J.; Behrouzi, M.; Herod, A. A.; Kandiyoti, R. Effect of several salts on the elution behaviour of heavy hydrocarbon liquids during size exclusion chromatography with 1-methyl-2-pyrrolidinone as eluent. Fuel 2005, 84, 1805–1811. (14) Paul-Dauphin, S.; Karaca, F.; Morgan, T. J.; Millan-Agorio, M.; Herod, A. A.; Kandiyoti, R. Probing size exclusion mechanisms of complex hydrocarbon mixtures: The effect of altering eluent compositions. Energy Fuels 2007, 21, 3484–3489.
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asphaltene molecules above the range amenable to analysis by GC and GC-MS are not known, it is only possible to aim to establish a calibration regime that is broadly based and attempts to establish elution times in a manner that is as little dependent upon molecular structure as possible. The mode of action selected was to compare elution times of asphaltenes with the SEC elution behavior of a diverse and broadly based range of model and standard materials. During the investigation of the molecular masses of these heavy hydrocarbon liquids, we also collected fractions of coal tar pitch, from preparative and analytical SEC columns, using NMP as an eluent. The molecular masses of these relatively low polydispersity fractions have been determined by LD-MS and MALDI-MS. The polystyrene calibration and the (smaller mass) standard compound calibration gave good agreement15 with the measured masses of the fractions up to a mass of about 3000 units, well beyond the range shown by the various ESI-MS measurements quoted by the authors of ref 1. It may be noted that the range of masses over which our two independent techniques (SEC and LD-MS) have been observed to agree is far above the range where the authors of ref 1 believe fossil-fuel-derived molecules exist. The fluorescence depolarization technique also showed far lower masses16 and could now be abandoned. Use of Mixed SolVents as Eluent. The foregoing and much of the accompanying experimental work has already been outlined in considerable detail.2,3 In more recent work, we have addressed problems arising from the fact that NMP does not completely dissolve petroleum asphaltenes. One way forward has been the use of mixtures of NMP with chloroform as solvent and eluent.9,14 Fractions of asphaltenes found to be insoluble in NMP alone were observed to dissolve completely in NMP/ chloroform mixtures. In SEC performed using mixed NMP/chloroform eluents, materials insoluble in pure NMP were observed to be of larger apparent size than the NMP solubles as shown by the following observations: (1) The later eluting (smaller molecule) peak of the fraction insoluble in pure NMP eluted earlier than the equivalent peak of the NMP-soluble fraction. (2) The excluded peak of the fraction insoluble in pure NMP was of increased proportion compared to the NMP-soluble fraction. We have observed this for several different crude oils as well as for several petroleum asphaltenes. The authors of ref 1 consider that THF is a better solVent for petroleum-derived asphaltenes than NMP, and we agree. However, THF is not a useful eluent for the SEC of petroleumderived asphaltenes, as explained elsewhere.2,3 Briefly, the continued use of THF as an eluent in SEC is known to lead to a gradual blocking of the column by deposited sample.17 In our experience, subsequent washing with NMP resulted in a black solution and an unblocked column. The problem is relatively straightforward: THF does not have enough solvent power to keep the whole sample in solution. Furthermore, standard polynuclear aromatic compounds do not elute in the expected order in THF. In other words, increasing elution times do not (15) Islas, C. A.; Suelves, I.; Millan, M.; Apicella, B.; Herod, A. A.; Kandiyoti, R. Matching average masses of pitch fractions of narrow polydispersity derived from matrix-assisted laser desorption ionisation time of flight mass spectrometry, with the polystyrene calibration of SEC. J. Sep. Sci. 2003, 26, 1422–1428. (16) Badre, S.; Goncalves, C. C.; Norinaga, K.; Gustavson, G.; Mullins, O. C. Molecular size and weight of asphaltene solubility fractions from coals, crude oils and bitumen. Fuel 2006, 85, 1–11. (17) Herod, A. A.; Johnson, B. R.; Bartle, K. D.; Carter, D. M.; Cocksedge, M. J.; Domin, M.; Kandiyoti, R. A reconciliation of mass ranges from MALDI-MS and size exclusion chromatography for coal-derived materials. Rapid Commun. Mass Spectrom. 1995, 9, 1446–1451.
correlate with decreasing molecular mass, as would be expected. For example, in THF, toluene (of mass 92 units) elutes18 well before perylene (of mass 252 units). We have found the same problem when using pure chloroform9,14 as an eluent in SEC. The unsuitable characteristics of THF for SEC have also been observed and reported in work involving Athabasca bitumen fractions separated by preparative SEC, using THF as an eluent.19 The fractionation was followed by analytical SEC of the fractions using NMP as an eluent. It was found that the materials observed under the excluded peak of the SEC chromatogram (in NMP) were in fact distributed throughout the chromatographic distribution, when THF had earlier been used as an eluent during the preparative process. This was identified by the presence of equivalent amounts of excluded material in the successive THF-eluted fractions. In fact, there should have been diminishing amounts of excluded material (or none) in the material eluting later, during the preparative process.19 Furthermore, the later-eluting peak in NMP was identical in the last three of the five fractions collected from the preparative column in THF, with no evidence of decreasing molecular size. The work indicated that fraction collection from SEC in THF eluent was little better than using a teaspoon to generate the fractions! We abandoned the use of THF as an eluent in SEC in the early 1990s and have repeatedly recommended others, still using the THF system for samples containing aromatic compounds, to do the same.2,3 2. On Mass Spectrometry. We next consider the mass spectrometric evidence for the upper mass of petroleum-derived asphaltenes. Most materials classed as petroleum-derived asphaltenes are relatively involatile and give little or no signal by GC-MS or by solids-probe mass spectrometry. It is consistently observed that sample introduction systems of mass spectrometers that rely on asphaltene volatility under the vacuum of the ion source of the instrument are likely to generate little volatile material (if at all) and those from the smallest molecules present in the sample. When higher temperatures (say 350-375 °C or above) are applied to volatilize the sample, sample decomposition occurs (possibly followed by devolatilization), rather than straightforward evaporation. It seems clear, therefore, that field-ionization mass spectrometry would not be capable of detecting very large molecules in asphaltenes. We have in the past tested samples of coal liquids by this method but did not publish the results because they were inadequate in our view. In a comparison of several ionization techniques,20 including FI and FD, the mass spectra by these two methods of a coal asphaltene showed differences in ion mass ranges, with the FD spectrum being higher, up to m/z 1100. In that work, the FD spectrum appeared to consist of ions not generated by the other ionization methods and the ion mass range matched those shown by the authors of ref 1, but comparing to other techniques, we did not consider that much of the high mass material had been detected. It may be noted that most mass spectrometric ionization methods have been demonstrated using single compounds or relatively simple mixtures. In some cases, the ionization method may be demonstrated using pure substances of large molecular mass (e.g., proteins). Mass discrimination effects in complex (18) Herod, A. A.; Kandiyoti, R. Fractionation by planar chromatography of a coal tar pitch for characterisation by size exclusion chromatography, UV-fluorescence and probe-mass spectrometry. J. Chromatogr., A 1995, 708, 143–160. (19) Domin, M.; Herod, A. A.; Kandiyoti, R.; Larsen, J. W.; Lazaro, M.-J.; Li, S.; Rahimi, P. A comparative study of bitumen molecular weight distributions. Energy Fuels 1999, 13, 552–557. (20) Herod, A. A.; Stokes, B. J.; Schulten, H.-R. Coal tar analysis by mass spectrometrysA comparison of methods. Fuel 1993, 72, 31–43.
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mixtures, however, are observed with greater difficulty. These effects become significant when ionizing a sample of wide but unknown polydispersity, such as asphaltenes. Laser desorption is one such method. We have shown such mass discrimination effects with deliberately prepared mixtures of polystyrene standards.21 Furthermore, it is easy to show that only the smaller molecules are desorbed at low laser power. The simple requirement proposed by the authors of ref 1 to use only the lowest laser power and very thin sample layers (giving low gas phase densities in the reactive plume) would indeed produce the result they observe, because such settings would cause desorption of only the smallest mass molecules within the samples. On the other hand, we have recently shown22 that using too high a laser power and too thick a layer of sample on the target may indeed give rise to ionized clusters. These appear above m/z 10 000 and may range up to 100 000. We have found that the clusters form from small molecules and obscure some of the genuine high mass ion signal, so that the high mass limit cannot be defined precisely. As reported nearly a decade ago,21 the problem can be minimized by reducing the polydispersity of the samples, before exposure to the laser. The careful fractionation essential to distinguish the large molecules from clusters of smaller mass materials has been outlined22 in some detail. The smallest molecules of the tar or asphaltene do require low laser power to ionize and evaporate, but the heavier molecules require significantly higher laser power to generate ions. The work reported22 shows that the laser power needed to ionize the larger molecules was sufficient to produce artifacts (cluster ions or multimer ions) from small molecular-mass material (approximately
