Faculty of Science

Application of nonaqueous capillary electrophoresis in asphaltene analysis

Research project as part of the Master Chemistry, Analytical Sciences track

Supervisor : Dr. W. Th. Kok

Amsterdam, July 29 2011

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Preface This study is part of the Analytical Sciences track of the Master of Science Chemistry at the Universiteit van Amsterdam and the Vrije Universiteit. The study will continue the work of undermentioned investigations. The study will be focusing on the use of capillary electrophoresis to analyze asphaltenes and crude oils. Previous studies

- Characterization of Asphaltenes by Nonaqueous Capillary Electrophoresis, Kok, W. T.; Tudos, A. J.; Grutters, M.; Shepherd, A. G. Energy & Fuels 2011, 25, 208-214

- Asphaltenes characterization by non aqueous capillary electrophoresis, masterthesis of Maria Marioli, MSc.

Acknowledgments My gratitude goes out to: Wim Kok for not only supervising my masterproject, but also giving me the opportunity to do this study. Maria Marioli, my predecessor, for showing how the CE soft- and hardware works. Dominique Vanhoutte for helping me out with general CE operations and when the CE broke down. All the other PhD students, (associate) professors, technicians and students at the Analytical Chemistry group at the Universiteit van Amsterdam for their help.

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Contents Preface _____________________________________________________________ 3 Acknowledgments ___________________________________________________ 3 Contents ___________________________________________________________ 4 Abstract ____________________________________________________________ 5 Introduction ________________________________________________________ 6 Experimental ______________________________________________________ 14 Results and discussion _______________________________________________ 16 Conclusion _________________________________________________________ 27 Future Challenges __________________________________________________ 28 Bibliography _______________________________________________________ 29

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Abstract Crude oil and asphaltene samples are investigated by nonaqueous capillary electrophoresis (NACE). Taylor dispersion analysis (TDA) is used to determine the molecular weight (MW) of an asphaltene deposit and how the background electrolyte (BGE) affects the MW of the deposit sample. Changing the amount of acetonitrile (ACN) in tetrahydrofuran (THF), from 20% to 0%, increases the MW of the deposit sample from 10000 to 15000 Da. The increase of concentration of electrolyte, LiClO4, from 0 to 12 mM, increases the MW non-linear from 5000 to 15000 Da.

NACE separates the crude oil sample in a positive charged fraction and a neutral fraction. The size of the asphaltene charged fraction in crude oil seems to increase with total asphaltene content, although this is not always the case. NACE of asphaltene samples shows a valley instead of a peak at the place of the neutral fraction. This is probably a combination of the capillary refractive index and Rayleigh scattering.

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Introduction The global demand for fossil fuel has risen significantly over the years, which is predominantly caused by the upcoming economies of China and India. Furthermore, predictions are that the demand will increase with 1.2% per year.1 One of the consequences of this increased demand is a historically high oil price, which makes it profitable to win oil out of oil sands and the like. These resources contain crude oil which is very viscous and difficult to extract. More and more of these resources are being tapped into, and as a result the composition of the produced crude oil is containing more of the heavy petroleum distillates.

These heavy distillates include asphaltenes, which are defined as a polar polyaromatic hydrocarbon fraction of crude oil with high molecular weight that is insoluble in simple alkanes like n-heptane but is soluble in aromatic solvents like toluene and benzene.2 This is one of the most common ways of defining asphaltenes, namely on solubility. One method used is the SARA-fractionation (Saturates, Aromatics, Resins and Asphaltenes). In this method the maltene from the bitumen is separated on a (silica) column into fractions of saturates, aromatics, resins and asphaltenes.3,4 Asphaltenes are present in crude oil as a dissolved component or as nanoaggregates/colloids which are stabilized by adsorbed resins when the crude oil is still in the oil well. A lot of physical conditions of the oil well play a role in maintaining the (fragile) asphaltene dissolution equilibrium, such as temperature, pressure, composition of the crude oil and composition of the wall of the oil well. During crude oil extraction, these conditions obviously change and can lead to precipitation of the asphaltenes in the pipe which ultimately leads to the clogging of the pipe and costly repairs of the affected pipe segment(s).2

Another problem associated with asphaltenes is the deactivation of catalysts used in cracking/upgrading since crude oil doesn’t have a high economical value from itself. In general, the lighter hydrocarbons used for gasoline and diesel have a high hydrogen to carbon ratio while the heavier fractions have a much lower hydrogen-carbon ratio. Thus the amount of hydrogen in the end product has to increase. Catalysts used for this process usually contain rare earth metals like palladium, which makes it expensive to renew, but this still happens on a daily basis. When the heavy fractions, for example bitumen, are catalytically processed, asphaltenes and aggregates of asphaltenes can deposit on the catalyst surface and/or into the pores of the support (in case of heterogeneous catalysis) which in turn lowers the catalytic activity. The deposition of the first asphaltenes promotes the deposition of more asphaltenes and leads to formation of a non-volatile, carbon rich solid named coke.5,6 The cause of the problem in both above examples is the deposition of the asphaltenes and other more aromatic compounds onto surfaces, coke formation.

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Asphaltene structure To understand why and how asphaltene deposition is happening on a macro scale, a closer look to the micro scale is necessary. There is a consensus about the general asphaltene structure which has been studied by diverse methods like NMR, X-ray Raman spectroscopy, X-ray absorption near edge spectroscopy, IR and scanning tunnelling microscopy.7 In 1967, the late Professor Teh Fuh Yen and co-workers developed a hierarchical model to understand how asphaltenes behave on a molecular scale. Later, a modified Yen-model is presented by Oliver Mullins and co-workers which is up until now one of the most common used models to describe asphaltene behaviour on a molecular scale. The modified Yen-model makes a distinction between three different aggregation states of the asphaltene molecule. See Figure 1.

The first state is just a single asphaltene molecule. An asphaltene molecule consists of one fused aromatic ring with four to ten aromatic rings, a few heteroatoms like nitrogen, sulphur and oxygen integrated in the aromatic ring system. Aliphatic (cyclo)alkanes are fixed on the aromatic ring. On a side note, an asphaltene molecule can be seen as a large polycyclic aromatic hydrocarbon (PAH) and after oil spills, PAH’s are one of the most difficult waste chemicals to remove because they are resistant to biodegradation. So asphaltenes do not only cause operational/technical problems but also environmental. The large aromatic ring system on asphaltene molecules allows for stacking of the systems on each other. This brings us to the second aggregation state, the nanoaggregates. Herein the asphaltene molecules have reached a critical concentration to interact with each other. Intermolecular forces between the molecules facilitate the formation of nanoaggregates. As seen in figure 1, the molecules are stacked by the aromatic ring system represented with a thick straight line. The aliphatic side chains are represented by thinner crooked lines.

Because the molecules are very diverse in aromatic ring size and aliphatic side chains, the stacking of them onto each other is not straight. There is a limit to the number of molecules that can be aggregated into the nanoaggregates. This is caused by the aliphatic side chains of the asphaltene molecules since they form a thin layer around the nanoaggregates and sterically repulsing other asphaltene molecules. Consequently, the other asphaltene molecules form additional nanoaggregates. The number of molecules in such nanoaggregates range from 6 to 8. Finally, the nanoaggregates aggregate with each other, forming clusters of nanoaggregates. The formation of these clusters is a result of aliphatic interactions of the side chains. Additionally, the form of such clusters depends on the media surrounding them, i.e. the nanoaggregates can be ordered more rectangular. The flocking of asphaltene clusters results in the precipitation of asphaltenes.8

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Figure 1: Aggregation states of asphaltenes according to the modified Yen-model, adapted from [8] Asphaltenes are classified as a solubility class of crude oil and oil fractions. The point is that asphaltenes are usually not really dissolved in the classical way unless the concentration is very dilute (less than 50 mg/L), which is when one asphaltene molecule is surrounded by solvent molecules. They are usually present as colloids, (clusters of) nanoaggregates, suspended in the liquid, making it more an emulsion than a solution. A study on this aspect of asphaltene structure makes clear that the critical nanoaggregate concentration of asphaltenes in toluene is around the 100 mg/L. 9 Molecular weight of asphaltenes To analyze an unknown compound, molecular weight (MW) determination is one of the first actions that should be taken. Since asphaltenes are rarely present as a single molecule and they are defined as solubility class, a polydisperse mass is more often than not the case. The MW of asphaltenes is one of the most discussed subjects in asphaltene science.10,11 Mass spectrometry (MS) is used often to determine the MW of asphaltenes. The issue with this technique is that traditional impact based techniques like electron ionization fragment the molecules, making it difficult to give a conclusive number for asphaltene molecules. Studies with electron impact FT-ICR- MS coupled with LIAD evaporation report values of 350 – 1050 Da, while the maximum lies around 750 Da.12 Other MS methods employ less destructive techniques like laser desorption ionization (LDI) which in combination with a ToF-MS gave a peak mass of 680 Da with an 2-sided uncertainty of 150 Da. 13

Non-destructive techniques like vapour pressure osmometry (VPO) and size-exclusion chromatography (SEC) are also available. SEC is often used to determine the MW of polymeric materials/compounds since it separates polymers on base of there physical size. Two SEC studies report different molecular weights, where one reports a MW of 1700 Da14 while the other reports a MW of 3000 to 6000 Da.15 The respective asphaltene concentrations were 1% and 3 % weight, which shows that aggregates of asphaltenes are measured and not single molecules.14,15 With VPO a molar mass around 1800 g/mol is found at asphaltene concentrations below 0.5 kg/m3. The molar mass increases with an increase of concentration up to masses of 10000 g/mol.16 Another study reports MW of 1900 ± 200 Da.17

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Charge of asphaltenes Due to oxidation of heteroatoms and/or transitions metals asphaltenes become charged. These heteroatoms include O, N and to a lesser extend S. A lot of these transition metals are incorporated into porphyrin structures.18 It is thought that these charged particles are more likely to precipitate. By gaining an understanding in what amount of asphaltenes is charged and how solvent conditions can affect the charge on asphaltenes can soften the challenge of asphaltene deposition. The asphaltene charge depends in aqueous solvents on the pH. In aqueous solvents, asphaltenes are negative around neutral pH values and higher and positive at pH values lower than 4. 19,20

Asphaltene charge in organic solvents depends on the composition of the solvent rather than the pH value. Asphaltenes in toluene/heptane mixtures with a toluene content of 12% (volume) or less are positively charged while those in mixtures with 16% toluene are neutral.21 Another study reports positive charged asphaltene deposition after they are precipitated from toluene by adding drops of heptane.22 This contradiction in results can be accounted to the different preparation methods used in both studies. Capillary electrophoresis Electrophoresis is the migration of charged particles in a solution under influence of an electric field. Particles move through the solution with a different velocity due to their size and charge and this is the principle of separation in electrophoresis. In capillary electrophoresis (CE) a glass tube with a very small diameter is used to contain the separation medium. To accommodate an electric field, an electrolyte is dissolved into the solvent that is used; these two together are called a background electrolyte (BGE). Since the capillary is made of glass, the usual detection method is by optical methods, such as UV-Vis-absorption, refractive index and fluorescence emission. Since the capillary used is small, diameters of 100 µm or less, it is also possible to couple the CE setup to ESI-MS detection in addition.The particle mobillty (µ) is independent on the electric field applied, but depends on its charge (q), size (r) and the viscosity of the BGE (η).

rq

particle πηµ

6=

Equation 1

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The walls of the capillaries are etched with sodium hydroxide which makes them negative. To compensate this charge, the cations of the BGE go to the walls leaving the anions in the middle of the capillary. When a positive voltage is applied, the resulting electric field exerts a force to the cations on the capillary wall. The resulting electro osmotic flow (EOF) will be in the direction of the outlet, provided the inlet is the cathode and the outlet the anode. This principle applies also to the analyte present in the BGE. All analyte particles migrate with either the same velocity as the EOF (neutral species), faster (cations) or slower (anions). The opposite is true when a negative voltage is applied. In Equation 2 the mathematical approach to this is displayed where E is the applied electric field. In most cases the EOF is larger than the velocities of the particles thus all particles will eventually migrate towards the outlet.23

Ev

Ev

Ev

anosman

osmneut

catosmcat

*)(

*

*)(

µµ

µ

µµ

+=

=

+=

Equation 2 There are different types of CE, like isotachophoresis (ITP), capillary isoelectric focusing (CIEF) and micellar electrokinetic chromatography (MEKC), but the most common type of CE is capillary zone electrophoresis (CZE). In CZE separation takes place on base of electrokinetic migration. This creates distinct zones of compounds and the resulting electropherogram shows these zones as peaks which are very similar to liquid and gas chromatograms.23

Water is commonly used in CE as the solvent because a wide range of electrolytes dissolve in water. However, asphaltenes will only precipitate when introduced to water. For this reason nonaqueous capillary electrophoresis (NACE) is used for the experiments in this report. NACE, as it name implies, uses organic solvents in the BGE. The first NACE experiments were done in 1984, but since then this technique has not seen a lot of use until 1990. The major problem was the evaporation of the solvents from sample and buffer vials and around 1990 manufacturers of analytical instruments developed better methods to seal the vials.

The advantages of NACE over regular CE are plentiful. A wide range of organic solvents are available and they can be also mixed to obtain optimal conditions. Additionally, organic solvents do not conduct very well compared to water. This results in very low currents while high voltages (>10 kV) can be applied to decrease run time. The low current prevents excessive heating due to Joule’s first law, so heat degradation and solvent evaporation will be less likely. There is also a major disadvantage in using NACE. The range of electrolytes to choose from is much smaller than CE because most of them will not dissolve in organic solvents. Another minor disadvantage is that the UV cut-off value can be high, but since there is plenty of choice of solvents (not to mention the number of mixtures) most of the time a workaround is possible.24

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Figure 2: Principle of CZE.

Figure 3: A typical CE setup.

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Taylor Dispersion Analysis (TDA) When a solute is injected hydrodynamically in a cylindrical tube, it has the form of a rectangular band when viewed from the longitudinal side. A pressure applied on the tube causes a laminar flow in which the middle parts of the flow have a higher velocity than the parts closer to the tube wall, creating a parabolic flow profile. This flow profile ensures that the longitudinal distribution of the solute in the tube is not even. Molecular diffusion takes place which causes the solute to disperse radially. Eventually, radial dispersion and longitudinal diffusion lead to a Gaussian distribution. See also Figure 4.

Figure 4: Through diffusion of analyte particles the assymmetric flow profile is counteracted resulting in a Gaussian distribution.

Taylor coupled the radial dispersion to the longitudinal diffusion, from which the molecular diffusion coefficient can be calculated by Equation 3. Herein σt

2 is the variance of the Gaussian distribution, d the diameter of the tube, D the diffusion coefficient of the analyte and t the time of the peak.

tDd

t *96

22 =σ

Equation 3 From the resulting peak, like in Figure 4, the width at half height can be measured. For standard Gaussian peaks this is 2.35σt which then can be used to calculate σt

2. By measuring σt

2 for the same sample at different flow velocities, a regression plot can be made with σt

2 as function of t. The slope of the regression line is then equal to d2/96D from which the diffusion coefficient can be calculated.

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Goal of the study The CE setup can be used for both TDA and NACE experiments by simply turning off the voltage when performing TDA. With TDA the effect of the BGE on the MW and thus aggregation of asphaltenes is investigated. The composition of the solvent as well as the concentration of the electrolyte, LiClO4, will be investigated. NACE will be used to separate asphaltene and crude oils on base of their charge. Also the reliability of the NACE-method itself will be investigated

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Experimental Chemicals All commercially available solvents used are of analytical grade and obtained from Biosolve. No further purification of the solvents was performed. Millipore water was obtained from a Sartorius Arium 611 UV. Anhydrous lithium perchlorate (LiClO4) was obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). Sodium hydroxide was obtained from Fluka. Formic acid was obtained from Analar, while the trifluoroacetic acid was obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands).

Varying compositions of BGE were used ranging from 100% tetrahydrofuran (THF) to 80:20 mixtures of THF: acetonitrile (ACN). The concentration of lithium perchlorate was varied from 0 to 12 mM. Unstabilized THF is used. Samples All dilutions are with analytical grade toluene (Biosolve) unless mentioned. A diluted asphaltene deposit used in previous studies25,26 is used. The exact concentration is not known due to evaporation of toluene, but is estimated between 1% and 2% weight.

19 crude oil and 8 asphaltene samples, including an ultra-pure asphaltene sample, were obtained from Shell. In addition an asphaltene deposit (also from Shell) dissolved in toluene 2% weight from earlier studies was used. All crude oil and asphaltene samples were diluted.

Ten crude oil samples labelled as ‘CRU’ were diluted to a 1% weight solution. Nine liquid SARA asphaltene samples were diluted to a 3% weight solution. The ultra-pure asphaltene was diluted to a 0.5% weight solution. The solid SARA asphaltenes, with the exception of SH019307, were diluted to a 0.4% weight solution. The solids off the SH019307 sample were stuck on the wall of the sample vial which made it very difficult to scrape it off. 3 mL toluene was added to dilute the sample. The sample concentration is therefore unknown. All of the diluted samples were placed on a shaker for at least one night to homogenize.

CE measurements were done with 50 µL of the diluted sample added tot 950 µL of BGE.

With time, the BGE evaporates out of the sample vials. Every day the sample vials were replenished with BGE to a volume around 1 mL.

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Instrumentation/Measurements CE was performed on an Agilent HP 3D CE equipped with a diode array detector. The cathode is at the inlet. Online detection on base of UV-absorption was at 260 nm and 350 nm. At 260 nm, although asphaltenes also absorb, toluene is measured since it much more abundant. The toluene peak is used as marker for the neutral peak. At 350 nm asphaltenes are measured. Cassette temperature was 25 ºC. Fused silica capillaries with an internal diameter of 75 µm from Polymicro Technologies were used. The length of the capillary ranged from 0.45 – 0.57 m and the detection window ranged from 0.36 – 0.46 m. The coating on the detection window and the inlet end was burned away. Injection of the sample occurred hydrodynamic for all measurements.

For TDA measurements an injection pressure of 20 mbar was applied for 5 seconds. Pressures used for TDA runs were raised by steps of 10 mbar from 10 – 40 mbar. Between TDA runs the capillary was flushed (900 – 1000 mbar) 4 minutes with the BGE used. All runs were done in triplicate. Peak width was estimated by analyzing peak width at half height using the numerical data points of the electropherogram.

For all electrophoretic measurements an injection pressure of 50 or 30 mbar was applied for 4 seconds. A voltage of +30 kV and a pressure of 10 mbar were applied simultaneously. In between runs the capillary was flushed with toluene for 2 minutes and then with the BGE used for 2 minutes.

For method reliability testing, 5 µL of 1.3 M trifluoroacetic acid or 0.27 M formic acid in ACN was added to 945 µL BGE and 50 µL of sample. Then 4 consecutive runs were done with the sample in the same BGE.

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Results and discussion NACE method There are snags on the road of the method. In the previous study26 it was mentioned that the composition of the BGE changes when an electric field is applied. This change in composition in BGE, changes also the ratio charged:neutral of the asphaltene peaks. At the inlet, where the anode is, oxidation takes place and H+-ions are formed. Since asphaltenes contain basic groups and are positively charged in organic media, the previous study26 suggests that H+-ions are the cause of the increased charge:neutral ratio.

Therefore, measurements with relatively strong acids have been done to investigate this. In Table 1 the results of these measurements are plotted. Samples with formic acid have been injected with a pressure of 50 mbar for 4 seconds, samples with TFA with 30 mbar for 4 seconds. Formic  Acid  

  350  nm  Charged   350  nm  Neutral   %  charged  vs  total  Run  1   949   2467   28%  Run  2     1249   2561   33%  Run  3     1232   2389   34%  Run  4   1205   2385   34%  Trifluoroacetic  Acid  

  350  nm  Charged   350  nm  Neutral   %  charged  vs  total  Run  1   596   1515   28%  Run  2     755   1615   32%  Run  3     712   1582   31%  Run  4   734   1474   33%  Table 1: Peak areas of electropherograms with formic acid and trifluoroacetic acid.

The measurements show an increase in ratio with consecutive runs in the same BGE. The concentration of acid was 0.2 M, which is an overdose with respect to the concentration of asphaltene in the sample. Considering this, if H+-ions are the cause of the increased charge:neutral ratio, then the ratio would not change in consecutive runs compared to the first run. This contradicts with the previous suggestion26. But the H+-ions do have an effect on the charge:neutral ratio. The ratios of the runs with acid are larger in comparison with the runs without acid (see also Table 2). The runs with acid also reach their maximum ratio at the run 2 while the ones without acid reach their maximum at run 3. It seems that a positive species is formed during electrophoresis and H+-ions are promoting this formation. This would mean that H+-ions are most likely involved in an indirect manner. These positive species then interact with the neutral asphaltenes to make them positively charged. It is still unknown what the direct cause is of the increase. The oxidation of (one of) the BGE(-components) can be the cause of this increase but it is still unknown which part of the BGE.

.

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Not only which species are interesting to research, but also how the charged asphaltene fraction itself will behave in different BGE’s. In Table 2 the results of the runs can be found. All runs are done in triplicate with fresh BGE after each complete run. CRU042 is used since this crude oil gave the highest percentage of charged asphaltene. The sample is injected with a pressure of 50 mbar for 4 seconds. 20%  ACN  

  350  nm  Charged   350  nm  Neutral   %  charged  vs  total  Run  1   528   2332   18%  Run  2     678   2329   23%  Run  3     792   2288   26%  Run  4   798   2083   28%  

10%  ACN  

  350  nm  Charged   350  nm  Neutral   %  charged  vs  total  Run  1   725   2734   21%  Run  2     730   2569   22%  Run  3     864   2809   24%  Run  4   961   2671   26%  No  ACN  

  350  nm  Charged   350  nm  Neutral   %  charged  vs  total  Run  1   526   4372   11%  Run  2     542   4545   11%  Run  3     551   4604   11%  Run  4   544   4551   11%  Table 2: Average values of triplicate runs with different volumes of ACN. The concentration of LiClO4 is 12 mM in all.

In runs with ACN the percentage of charged fraction is increasing while in pure THF the percentage remains the same. One possible way to account this is the low conductivity of THF, which only gives a current of 0.45 µA while those with ACN are at least ten times higher (Table 3). This lack of conductivity perturbs the degree of oxidation of the BGE since the low current is apparently not strong enough to oxidate more BGE over time. Another point of view could be that not oxidation products of the BGE are responsible for the increase in charged fraction, but the electrons that are produced simultaneously. Since the asphaltenes are injected as one band, the charged and neutral species are mixed. More electrons are produced in the second, third and fourth run, so the force caused by these surplus electrons can take away more charged asphaltene.   Current  in  μA  100%  THF   ±  0.45  10  %  ACN   ±  5  20%  ACN   8.5  -­‐  11  Table 3: Current in three different BGE’s with 12 mM LiClO4.

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Taylor Dispersion Analysis Measurements were done on the asphaltene deposit. First, the effect of the solvent on the MW was measured. In Figure 5 plot is shown to illustrate the calculation of the diffusion coefficient. The same method is also applied to the diffusion coefficient of toluene.

20% ACN 12 mM

y = 0,2852xR2 = 0,9643

0

20

40

60

80

100

120

140

160

0 50 100 150 200 250 300 350 400 450 500

tmax in s

var

Figure 5: Plot of TDA runs of asphaltene deposit in 80:20 THF:ACN with 12 mM LiClO4. Plotted is the variance of the peak against the time the top of the peak. Peak times decrease with increasing pressure.

In Table 4 MW’s are estimated from the molecular diffusion. The ratio of the diffusion coefficients of 350nm/260 nm is proportional to the ratio MW0.5(toluene)/MW0.5(asphaltene). Using this will result in the average MW of the asphaltene deposit. BGE  (12  mM)   MW  (Da)   Diff.  (350  nm)   Diff.  (260  nm)   R2  

20%  ACN   9983   2.05x10-­‐10   2.14x10-­‐9   0.96  10%  ACN   13517   1.75x10-­‐10   2.12x10-­‐9   0.93  THF   14658   1.47x10-­‐10   1.86x10-­‐9   0.77  Table 4: TDA results after plotting.

As seen in Table 4, the asphaltenes in the deposit are present as aggregates since the MW is far higher than that of a single asphaltene molecule (~750 Da)8. The MW of asphaltenes in mixtures with ACN is lower than in pure THF which indicate there is less aggregation of asphaltenes in media with ACN. THF and ACN are both polar aprotic solvents and both are solvents in which a wide range of compounds will dissolve. One possible explanation why asphaltenes are ‘happier’ in ACN than THF is the fact that ACN has higher dipole moment than THF (3.92 against 1.63)27. This higher dipole moment makes ACN more polar than THF. Since asphaltenes are one of the most polar components of crude oil, they will be less likely to aggregate in ACN. The MW is not consistent with earlier studies on the same asphaltene deposit, which gave MW of 3000-4000 Da25 and around 6000 Da26. This indicates that the asphaltenes are present in the form of nanoaggregates. Because a part of the toluene evaporated over time, although the sample vessel was closed, the exact concentration is not known. The concentration of deposit used in this study is probably higher,

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which can account for the higher MW. Assuming that the asphaltenes are present in nanoaggregates, the number of molecules would range from 13 to 20 molecules per nanoaggregate. Since this number of molecules would be too high for nanoaggregates, which contain 6 to 8 molecules, they should be present as clusters which consist of 2 or 3 nanoaggregates.

Another point to be addressed is the precision of the THF only measurement, since it is very low compared to the other two. As can be seen in Figure 6, the plot suffers from outliers. This can be traced back to the original electropherograms of the runs. These plots suffer from a low signal-to-noise ratio which makes it difficult to exactly identify the peak from the baseline. Furthermore, the baseline itself is not straight and shows a drift upward during the runs. If the outliers are removed, as seen in Figure 7, then a more precise regression line (R2 = 0.89) is plotted. With this increase in precision, the slope of the line changes to 0.3168 which is lower than the 10% ACN run. This will decrease the MW of asphaltene to 10072 Da. This decrease in MW is surprising since it would suggest that the same number of clusters is present in pure THF as in THF mixed with 20% ACN.

THF 12 mM

y = 0,3973xR2 = 0,7652

0

50

100

150

200

250

300

350

0 100 200 300 400 500 600

tmax in s

var

Figure 6: Plot of TDA runs of asphaltene deposit in 80:20 THF:ACN with 12 mM LiClO4. Plotted is the variance of the peak against the time the top of the peak.

20

THF 12 mM, w/o outliers

y = 0,3168xR2 = 0,8932

0

20

40

60

80

100

120

140

160

180

200

0 100 200 300 400 500 600

tmax in s

var i

n s^

2

Figure 7: Plot of TDA runs of asphaltene deposit in 80:20 THF:ACN with 12 mM LiClO4. Plotted is the variance of the peak against the time the top of the peak. Herein two outliers who lie above the regression line from Figure 6 are removed.

Secondly, the effect of the electrolyte on the MW is investigated. During the sample runs, random valleys in the peaks are seen which makes it impossible to do TDA. It seemed this was not the case with the THF-only measurement, so solutions of 0, 1, 5 and 12 mM LiClO4 in THF were used for the sample runs. In Figure 8 the resulting MW are plotted. It is clear that the MW depends on the concentration of the electrolyte. Concentrations from 0 to 5 mM do not show a large difference in MW, ranging from 5000 to 5700 Da, while the difference from 5 to 12 mM is big (>8000 Da). This suggests that asphaltene nanoaggregates consisting of 6 or 7 molecules are present in the BGE of 5 mM and lower.

A closer look to the electrolyte is needed. It is known that transition metals like Ni, Fe and V are present in asphaltenes as ions trapped in porphyrin rings.18 Li is not a transition metal and its cation is much smaller than aforementioned transition metals. It is very unlikely that the cation is included in the porphyrin rings. Li+ is a high ionic strength species among the cations and thus will interact with electronegative species. Since asphaltenes contain heteroatoms, these species could interact with Li+. A study suggests that a Ca2+-ion can act as a bridge between separate negative asphaltene molecules. Herein a CaCl2-solution in water was prepared.28 The same concept could be applied here, although Li+ is monovalent and smaller than Ca2+, with the difference that Li+ would act as a bridge between heteroatoms since the asphaltenes are in organic media and thus positive. The Li+ would reside in the nanoaggregates. But this effect is only achieved at higher concentrations and/or marginal since the MW does not change a lot between concentrations of 0 and 5 mM. As asphaltenes are usually positively charged in organic media, the perchlorate-ion, ClO4

-, can play a role in stabilizing the electrostatic interactions in the nanoaggregates. The perchlorate-ion could also play a role as an oxidator. Although the Cl has an oxidation state of 7+, perchlorate has a lower oxidation potential than other less oxidated chlorate species. Oxidation of heteroatoms and/or carbons can result in more electropositive molecules which in turn would promote formation of nanoaggregates.

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Effect of conc LiClO4 THF

0

2000

4000

6000

8000

10000

12000

14000

16000

12 5 1 0

Conc LiClO4 in mM

MW

in D

a

Series1

Figure 8: MW of asphaltene deposit in THF with different concentrations of LiClO4. The MW of 12 mM is the one with the outliers.

22

Nonaqueous Capillary Electrophoresis Measurements were done on all samples available. First, the percentage of charged fraction with regards to the total fraction that absorb at 350 nm in all available samples is investigated. This is done by integrating the peak areas at 350 nm. All integrations are done manually because most of the electropherograms do not have baseline separation. Integration of peak areas is done manual as shown in Figure 9.

Since the charged asphaltene fraction increases when the same BGE is used, all data obtained are averages from two sample runs after an electric field of +30 kV has been applied for at least 40 minutes to the used BGE.

Figure 9: Manual integration of peak areas in electropherograms. Crude oil series The first batch of crude oils is measured with an injection pressure of 50 mbar for 4 seconds. BGE used was 12 mM LiClO4 in 20% ACN. Results are in Figure 10:

% Charged of total, CRU series

0

500

1000

1500

2000

2500

3000

3500

CRU024 CRU030 CRU041 CRU042 CRU044 CRU045 CRU051 CRU141 CRU148 CRU219

Sample

Peak

are

a

0%

5%

10%

15%

20%

25%

30%

35%

40%

Char

ged/

Tota

l in

%

ChargedTotal%

Figure 10: Results of NACE measurements of ‘CRU’ samples.

23

The amount of charged fraction in the samples is consistent with the colour of

the sample vial. The darker the vial, the higher is the charged fraction. CRU044 and CRU219 do not have any charged fraction at all and also their total peak areas are not high. This would indicate that these oils contain a very low amount of asphaltenes. Most samples which have a charged fraction >20%, also have a relative high asphaltene content.

This would also indicate that the asphaltenes in these samples, CRU41, 042, 045, 051, 148, are more oxidized and/or contain a lot of metal ions and/or more basic than the rest of the crude oils. It is also possible that asphaltenes are more likely to dissolve in the darker oils due to a higher aromatic content in the darker oils. But, at the same time, a (little) perturbation of the solution conditions will allow them to precipitate. This is observed in the sample vials, most notably for CRU041 and 042. When the BGE was added to the sample vial, the whole sample stayed in solution. During the night, since the sample vial isn’t closed off properly, a part of the sample would evaporate. The next day, when new BGE was added, (a lot of) precipitation is seen in the sample vial. However, the precipitates are not affecting the total or the relative peak areas. This was the case with all samples from this series and subsequent series. This suggests that the precipitate is a very small fraction of the asphaltenes or that they still behave more or less the same during NACE as precipitate. This also supports the idea that the charged asphaltenes are more likely to precipitate/aggregate, due to their heteroatom and metal content. Liquid SARA asphaltene series The second batch of crude oils was injected with a pressure of 30 mbar for 4 seconds. Injections of 50 mbar for 4 seconds were too concentrated. BGE used was 12 mM LiClO4 in 20% ACN. Results are displayed in Figure 11.

% Charged of total, CO series

0

200

400

600

800

1000

1200

SH011569

SH019345

SH019308

SH019307

SH008120

WTC#9115

SH019408

SH019409

SH019309

Sample

Peak

are

a

0%

10%

20%

30%

40%

50%

60%

Char

ge/T

otal

in %

ChargedTotal%

Figure 11: Results of NACE measurements of liquid SARA asphaltenes.

24

The amount of charged fraction is consistent with the viscosity of the samples.

Although all the samples were viscous, those who have a low charged fraction, less than 40%, were much more liquid than the others. There is also a difference in colour of the diluted samples, although much less pronounced than in the CRU series. Overall, the samples containing high amounts asphaltenes do have higher percentages of charged fraction, although CO_A has the lowest asphaltene content but has one of the largest charged fractions.

The percentage of charged asphaltene is higher in these SARA asphaltenes than those in crude oils. It seems that in crude oils, there are (more) species present to inhibit charge formation and/or residency on the asphaltenes. This could be in the form of natural antioxidants in the crude oils. The major part of these antioxidants resides in the heavier fractions.29 By removing the SAR-fractions from the crude oil, the antioxidants could be made more available/susceptible to oxidation. Also it is more likely that charged asphaltene aggregates are less covered by resins and/or other aromatic or aliphatic species after fractionation and thus increasing the effective charge. This can explain why a higher asphaltene content also results in a higher charged:neutral ratio.

25

Solid SARA asphaltene series The solid SARA samples were injected with a pressure of 30 mbar for 4 seconds, except for SH013448, which was injected with 50 mbar for 4 seconds. BGE used was 12 mM LiClO4 in 20% ACN. Each measurement is done in quadruplicate with new buffer vials for each sample.   350  nm  charged   350  nm  neutral   %  charged  vs  total  SH019130   193  

191  230  222  

 

0  0  0  0  

 

100%  100%  100%  100%  

 

SH010563   239  277  338  315  

 

165  0  0  0  

 

59%  100%  100%  100%  

 

SH0105631C   246  342  330  324  

 

193  0  0  0  

 

56%  100%  100%  100%  

 

SH0105632C   249  331  309  301  

 

179  0  0  0  

 

58%  100%  100%  100%  

 

SH019308   291  396  379  381  

 

0  0  0  0  

 

100%  100%  100%  100%  

 

SH013448   821  1172  1126  1189  

 

613  0  0  0  

 

57%  100%  100%  100%  

 

SH019307   182  225  231  240  

 

0  0  0  0  

 

100%  100%  100%  100%  

 

Ultrapure   51  99  90  95  

 

0  0  0  0  

 

100%  100%  100%  100%  

 

Table 5: Results of NACE measurements of solid SARA asphaltene samples.

26

Electropherogram of ultrapure asphaltene

-100

0

100

200

300

400

500

600

700ab

s in

mAU

-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5260 nm350 nm

Figure 12: Electropherogram of ultrapure asphaltene.

In most measurements there is no neutral peak area to compare with. A valley instead of a peak at 350 nm is the cause of this. In Figure 12 the electropherograms of the ultrapure asphaltene at 260 and 350 nm are plotted. There is a large toluene peak in 260 nm which indicates the time where the neutral peak should appear in 350 nm. This dip in the peak closely resembles the valley observed in the TDA. At first instance it is thought that oxidation of THF was the cause of this, but after measurements with fresh THF it is concluded that this is not the case. A part of this is contributed to the refractive index of the capillary, that is, the placing of the capillary, since detection occurs in the capillary itself. After careful inspection and repositioning of the detection window, the valley in the NACE of solid (SARA) asphaltene samples still appears. This effect can be attributed to the (unusual) aggregation sequence of asphaltenes. Asphaltenes tend to aggregate per molecule, going from a monomer to a dimer, then to a trimer etc. and eventually to nanoaggregates and clusters. A UV-study on the effect of the concentrations of an asphaltene solution finds that the Lambert-Beer law for extinction can’t be applied to asphaltenes due to their aggregation sequence. Herein the UV-absorption at 633 nm drops at the moment dimers are formed. At the same time the Rayleigh scattering at 670 nm is rising.30 These results could explain partly why a valley is observed at the place where the neutral peak should be. First, the aggregates formed are large enough to scatter the light (> 35 nm). Assumed is that the neutral asphaltene also absorbs at 350 nm. At this point, there should be still a decrease in the amount of 350 nm light arriving at the detector and thus result in a peak in the electropherogram. The scattering is not homogeneous in all directions and more intense in the direction parallel of the light path. Since Rayleigh scattering does not change the wave length of the scattered light, it is possible that the scattered light is more pronounced towards the detector and will show a valley instead of a peak.31

Another point to be noticed is that some samples, SH010563, SH0105631C, SH0105632C and SH0134487, have a neutral peak at the first run, only for it to vanish during subsequent runs. As mentioned in the method discussion, the composition of the BGE changes under an electric field since oxidation at the cathode and reduction at the anode takes place. This change ensures that more charged fraction is formed and this is also the case in the crude oil samples. The previous study26 proposed that H+-ions could be the source of the increased charged fraction. As mentioned before, it seems H+-ions are not solely responsible for the increase in charged fraction. Another possibility is oxidation of asphaltenes. At the cathode there

27

will be an excess of perchlorate-ions and if the solvents are not sufficiently oxidizable, oxidation of asphaltenes instead can take place. This oxidation can make the asphaltenes more basic. An increase of injection does not make the valley disappear, as is shown with AS_E. One last possibility could be that there is no neutral fraction to measure, but this would very unlikely.

When the average areas of the charged fractions are plotted in Figure 13, it shows that they do not differentiate much from each sample, except for SH013448, since more is injected. This would suggest that the degree of ionization in organic solvents is more or less the same.

Average peak area last 3 runs

0

200

400

600

800

1000

1200

1400

SH019130

SH010563

SH0105631C

SH0105632C

SH019308

SH013448

SH019307

Ultrapure

Sample

Peak

are

a

Figure 13: Average peak area of each sample, excluding the first run of each sample. One last thing to mention is the extreme low peak areas of the ultrapure asphaltene. This sample was in practice the most difficult one to dissolve in toluene, even after three days of continuous mixing. Hereafter, a lot of solid asphaltene could still be seen on the wall of the vial. This could explain why there is so little asphaltene visible in the first place.

28

Conclusion Asphaltene MW depends on the solvent as well on the concentration LiClO4. The MW increases while decreasing the amount of ACN in THF. Here it increases while decreasing the polarity of the solution, which is a result of an increase in aggregation of asphaltenes. With increasing concentrations of LiClO4 the MW increases non-linear. The Li+-ion could play a role by stabilizing the nanoaggregates and/or clusters.

The size of the charged asphaltene fraction in crude oils is larger when the total asphaltene fraction is larger, although this is not always the case. Also the darker the crude oil sample, the larger the charged fraction is. Determination of the size of the charged fraction in asphaltenes was not possible because a valley instead of a peak appears at the time where the neutral fraction should be. Diverse explanations for this oddity are discussed, most likely it is a combination of the refractive index of the glass capillary and Rayleigh scattering effects.

4 consecutive runs in triplicate with one sample using the same BGE vial shows that the size of the charged fraction is increasing after the first run and the maximum size is reached after the third run. Runs with either an overdose with respect to the asphaltenes of trifluoroacetic acid or formic acid added to the BGE showed an increased charged fraction with the first run but the charged fraction still increases with consecutive runs, although the maximum size remains the same. This indicates that H+-ions are not the direct cause of the increase in charged fraction. It is more likely that H+-ions are promoting the oxidation process of the BGE-components which in turn increases the charge:neutral ration..

The CE method developed is from a qualitative point of view a fast and reliable way to determine asphaltene content in crude oil. Almost no sample preparation is needed and three consecutive sample runs can be done in an hour. Also CE uses very low amounts of solvent, about 10-20 mL per day, which makes the method economical and environmental friendly.

Future Challenges A UV-study of the asphaltene dilutions can be done to investigate the cause of the observed valley. During electrophoresis, the composition of the BGE changes which increases the size of the asphaltene charged fraction. What this change is, is not known, so a study of used BGE is needed to increase the reliability and speed of the method.

29

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