Fractionation of Crude Oil Acids by HPLC and Characterization of

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Energy & Fuels 2007, 21, 2816-2826

Fractionation of Crude Oil Acids by HPLC and Characterization of Their Properties and Effects on Gas Hydrate Surfaces Anna E. Borgund,*,†,‡ Kristin Erstad,† and Tanja Barth† Department of Chemistry, UniVersity of Bergen, Alle´ gt. 41, 5007 Bergen, Norway, and Centre for Integrated Petroleum Research, CIPR, UniVersity of Bergen, Alle´ gt. 41, 5007 Bergen, Norway ReceiVed February 26, 2007. ReVised Manuscript ReceiVed May 25, 2007

In this work, acids have been extracted from eight crude oils by ion-exchange extraction and liquid-liquid extraction, and the two methods have been compared. The acid distribution as a function of increasing acid strength is obtained for the different extracts using a recently developed high-performance liquid chromatography (HPLC) procedure based on normal phase separation on a cyanopropyl stationary phase. The fractions are characterized with regard to molecular weights by gel permeation chromatography, functional groups by Fourier transform infrared spectroscopy, and quantitative distributions by HPLC. The results show that the different extraction methods give different yields and compositions. The acid profiles are not necessarily different for biodegraded and non-biodegraded oils, but the yields are much higher in biodegraded oils, as expected from bulk acid determinations like total acid number. The profiles can to some degree be correlated with surface activity of the acidic compounds.

1. Introduction Crude oil is a very complex mixture that contains an extensive range of compounds and molecular species, including functionalized compounds that are surface-active.1 Surface-active components can affect the phase behavior of a crude oil in contact with one or several other phases, like water or solid surfaces. For instance, surface-active components are important for the processing of crude oil in the formation of emulsions2,3 and foam.4 Adsorbed surface-active components also influence the wetting properties of reservoir rocks.5 Thus, knowledge of the surface-active compounds in petroleum phases are of considerable interest in oil production and transport. Several classes of hetero compounds in crude oil show surface activity, but especially the carboxylic acids are important for the interfacial activity of crude oil.6 In addition, biosurfactants formed during the petroleum biodegradation of oil under reservoir conditions have been identified.7,8 Recently, the importance of petroleum acids for the plugging properties of gas hydrates formed in crude oils has been * To whom correspondence should be addressed. Tel.: +47 55583480. Fax: +47 55589490. E-mail: [email protected]. † University of Bergen. ‡ CIPR. (1) Speight, J. G. The Chemistry and Technology of Petroleum, 3rd ed., revised and expanded; Marcel Dekker: New York, 1999. (2) Ese, M. H. Langmuir Film Properties of Indigenous Crude Oil Components. Influence of Demulsifiers. Ph.D. thesis, University of Bergen, Norway, 1999. (3) Sjo¨blom, J.; Aske, N.; Auflem, I. H.; Brandal, Ø.; Havre, T. E.; Sæther, Ø.; Westvik, A.; Johnsen, E. E.; Kallevik, H. AdV. Colloid Interface Sci. 2003, 100-102, 399-473. (4) Vikingstad, A. K. Static and Dynamic Studies of Foam and FoamOil Interactions. Ph.D. thesis, University of Bergen, Norway, 2006. (5) Standal, S. H. Wettabilitty of Solid Surfaces Induced by Adsorption of Polar Organic Components in Crude Oil. Ph.D. thesis, University of Bergen, Norway, 1999. (6) Seifert, W. K.; Howells, W. G. Anal. Chem. 1969, 41, 554-562. (7) Yakimov, M. M.; Timmis, K. N.; Wray, V.; Fredrickson, H. L. Appl. EnViron. Microbiol. 1995, 61, 1706-1713. (8) Vater, J.; Kablitz, B.; Wilde, C.; Franke, P.; Mehta, N.; Cameotra, S. S. Appl. EnViron. Microbiol. 2002, 68, 6210-6219.

demonstrated.9,10 The petroleum acids showed surface activity toward hydrate surfaces. Thus, hydrate plug inhibition can be added to the other important physical effects of petroleum acids related to the production and transport of oil-water-gas fluids, and detailed and precise knowledge of the critical aspects of the acid composition in different crude oils is needed for a better understanding of the phenomena. Acids can be extracted from crude oil by various methods. Several authors report the use of liquid-liquid extractions, where alkaline solutions are used to extract the acidic components from crude oil.6,11-16 An alternative method for extracting the acids from petroleum is using an ion-exchange resin.17-22 Though the extracted acid fractions have a simpler composition than the whole crude oil, the fractions still comprise such a wide range of structures and acid strengths that their further charac(9) Høiland, S.; Borgund, A. E.; Barth, T.; Fotland, P.; Askvik, K. M. Proc. Int. Conf. Gas Hydrate, 5th 2005, 4, 1151-1161. (10) Høiland, S.; Askvik, K. M.; Fotland, P.; Alagic, E.; Barth, T.; Fadnes, F. J. Colloid Interface Sci. 2005, 287, 217-225. (11) Constantinides, G.; Arich, G. Non-Hydrocarbon Compounds in Petroleum. In Fundamental Aspects of Petroleum Geochemistry; Nagy, B., Colombo, U., Eds.; Elsevier: Amsterdam, 1967; pp 109-175. (12) Dzidic, I.; Sommerville, A. C.; Raia, J. C.; Hart, H. V. Anal. Chem. 1988, 60, 1318-1323. (13) Hoeiland, S.; Barth, T.; Blokhus, A. M.; Skauge, A. J. Pet. Sci. Eng. 2001, 30, 91-103. (14) Tomczyck, N. A.; Winans, R. E.; Shinn, J. H.; Robinson, R. C. Energy Fuels 2001, 15, 1498-1504. (15) Lo, C. C.; Brownlee, B. G.; Bunce, N. J. Anal. Chem. 2003, 75, 6394-6400. (16) Barth, T.; Høiland, S.; Fotland, P.; Askvik, K. M.; Pedersen, B. S.; Borgund, A. E. Org. Geochem. 2004, 35, 1513-1525. (17) Fan, T.-P. Energy Fuels 1991, 5, 371-375. (18) Meredith, W.; Kelland, S.-J.; Jones, D. M. Org. Geochem. 2000, 31, 1059-1073. (19) Jones, D. M.; Watson, J. S.; Meredith, W.; Chen, M.; Bennett, B. Anal. Chem. 2001, 73, 703-707. (20) Mediaas, H.; Grande, K. V.; Hustad, B. M.; Rasch, A.; Ruesla˚tten, H. G.; Vindstad, J. E. Soc. Pet. Eng. 2003, paper 80404. (21) Jewell, D. M.; Weber, J. H.; Bunger, J. W.; Plancher, H.; Latham, D. R. Anal. Chem. 2004, 44, 1391-1395. (22) Saab, J.; Mokbel, I.; Razzouk, A. C.; Ainous, N.; Zydowicz, N.; Jose, J. Energy Fuels 2005, 19, 525-531.

10.1021/ef070100r CCC: $37.00 © 2007 American Chemical Society Published on Web 07/04/2007

Fractionation of Crude Oil Acids

terization by chemical properties and molecular composition is a major challenge.23 Naphthenic acids comprise a large part of the carboxylic acid fraction in crude oils. The naphthenic acids are a complex mixture of alkyl-substituted acyclic and cycloaliphatic carboxylic acids, with the general formula CnH2n+ZO2, where n stands for the number of carbon atoms and Z specifies the hydrogen deficiency. When Z is 0, the formula represents an acyclic fatty acid. An overview of the naphthenic acids can be found in the review article by Clemente and Fedorak24 and references therein. Many methods have been used for the analysis of the naphthenic acid fraction. Fourier transform infrared (FTIR) spectroscopy, gas chromatography (GC), negative-ion electronspray ionization-mass spectrometry, and high-performance liquid chromatography (HPLC) have previously been applied in the quantitative analysis of naphthenic acids.24 Mass spectrometric (MS) methods have also been used to determine the molecular composition of naphthenic acids, and several different MS methods are described in the review by Clemente and Fedorak. GC is often combined with MS in order to separate components in the acids prior to MS analysis.12,25-27 However, some authors12,17 state that GC-MS is not suitable for the analysis of naphthenic acids due to a large unresolved complex mixture (UCM) that appears in the chromatogram. When it is not possible to separate the components, the authors claim that direct MS with no prior separation is just as appropriate for the analysis of the naphthenic acids. High molecular weight and low volatility also make the use of GC difficult, and liquid chromatography has an advantage from this perspective. Green28 used liquid chromatography to separate petroleum acids by acid strength, giving a profile of acids that correlated with their physical effects in the crude oils. The reported method uses a dynamic equilibrium on silica to control the retention of acidic compounds on the column, which makes it difficult to acquire stable retention time values and gives a long equilibrium time. Clemente et al.29 used HPLC for the analysis of naphthenic acids. In this method, the naphthenic acids eluted as a large hump in the chromatogram, and the total amount of naphthenic acids was calculated, though with no separation into subfractions that can be related to the effects of the acids on the bulk oil phase properties. In a recent paper, we presented an HPLC procedure for separating acid extracts into different subfractions.30 This HPLC method uses normal phase chromatography on a cyanopropyl bonded phase column to provide a stable and fast separation of organic acids from crude oils into four well-defined fractions that correspond to the main types of acidic compounds found in the oils: weak acids with no acidic protons, saturated carboxylic acids, phenols, and polyfunctional acids. The method was developed both on an analytical scale for characterization of the acid extracts and on a preparative scale to provide sufficient sample amounts for further analysis by complementary (23) Altgelt, K. H.; Boduszynski, M. M. Composition and Analysis of HeaVy Petroleum Fractions; Marcel Dekker: New York, 1994. (24) Clemente, J. S.; Fedorak, P. M. Chemosphere 2005, 60, 585-600. (25) de Campos, M. C. V.; Oliveira, E. C.; Filho, P. J. S.; Piatnicki, C. M. S.; Carama˜o, E. B. J. Chromatogr., A 2006, 1105, 95-105. (26) Clemente, J. S.; Fedorak, P. M. J. Chromatogr., A 2004, 1047, 117128. (27) St. John, W. P.; Rughani, J.; Green, S. A.; McGinnis, G. D. J. Chromatogr., A 1998, 807, 241-251. (28) Green, J. B. J. Chromatogr. 1986, 358, 53-75. (29) Clemente, J. S.; Yen, T.-W; Fedorak, P. M. J. EnViron. Eng. Sci. 2003, 2, 177-186. (30) Borgund, A. E.; Erstad, K.; Barth, T. J. Chromatogr., A 2007, 1149, 189-196.

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methods. In this work, the method has been applied to a sample set of acid extracts from crude oils mostly originated from the Norwegian continental shelf, which include both biodegraded and non-biodegraded oils. Two detector types have been used: an evaporative light scattering detector (ELSD) which can detect all compounds except those that evaporate together with the solvent, for example, certain phenolic compounds, and a UV detector that registers all compounds that have absorption in the UV region, including the phenolic compounds but excluding saturated acids. The fractions from preparative HPLC analysis were further investigated by use of FTIR and gel permeation chromatography (GPC). 2. Experimental Section 2.1. Materials. Acids are extracted from a sample set of eight crude oils, spanning from heavy biodegraded oils enriched in asphaltenes to light non-biodegraded oils. The oils originate from the Norwegian continental shelf and are supplied by Norsk Hydro ASA (seven oils) and Statoil ASA (one oil). The oils are identified by letters, B for biodegraded oils and S for sweet, non-biodegraded oils, followed by a number indicating field and a letter denoting different wells or different batches within one field. The level of biodegradation (Peters and Moldowan scale31) for the different oils has previously been reported by Barth et al.16 In addition, the crude oils in the data set have been thoroughly characterized with respect to compositional properties such as total acid and base numbers, density, and asphaltene content,16 as well as with respect to the wettability of the freon hydrates that are generated by each crude oil.10 The solvents are all of HPLC or p.a. quality. The standards used for HPLC are of p.a. quality. The standards used for GPC are polystyrenes (purity 99.5%) purchased from Polymer Laboratories and stearic acid (purity 99+%) purchased from Sigma. Also, a sample of vanadyl octaethyl porphyrin is available (synthesized by R. Ocampo at the Louis Pasteur University, Strasbourg). 2.2. Extraction of Acids from Crude Oil. Acids are extracted from petroleum by the use of ion-exchange resin described by Mediaas et al.20 QAE Sephadex A-25 is used as a solid-phase material for the ion exchange of the petroleum acids. The capacity of the ion-exchange material is 2.5 mmol acid equivalents per gram of ion-exchange material, calculated from the total acid number (TAN) of the oil. A buffer solution (1 M Na2CO3/NaHCO3) is added to the ion-exchange material and allowed to run slowly through a filter (GF/C, Whatman glass microfiber filter, Sigma-Aldrich), at a ratio of 75 mL of the buffer solution per gram of ion-exchange material. The ion-exchange material is then cleaned with distilled water until the pH is 7. The water is removed by vacuum filtration, and methanol (MeOH) is added, at a ratio of 25 mL of MeOH per gram of ion-exchange material. The ion-exchange material is then transferred to the crude oil and left to stir for 16 h under a gentle nitrogen flow. The oil is filtered, and the ion-exchange material is washed with toluene several times. Toluene aliquots of 5 mL per gram of ion-exchange material are used. The ion-exchange material is then washed with a mixture of toluene and MeOH, 2:1 (v/v), until the filtrate is colorless. The filtration procedure is repeated two times using a filter paper having a smaller pore diameter (GF/C the first time and GF/F the second and third time). The acids from the crude oil are recovered from the ion-exchange material by adding 1 M formic acid (3.5 mL per gram of ion-exchange material) and a mixture of toluene and MeOH, 1:1 (v/v; 50 mL per gram of ion-exchange material). The mixture is left to stir for 3-4 h before the ion-exchange mass is removed by filtration, first through a GF/C filter, and then through a GF/F filter. The ion-exchange mass is washed with 2:1 toluene/methanol (v/v; portions of 10 mL per gram of ion-exchange material). To ensure that all the petroleum acids are recovered, the residue from the filtrations is collected and the (31) Peters, K. E.; Moldowan, J. M. The Biomarker Guide; Prentice Hall: New York, 1993; pp 252-265.

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Table 1. Gradient Program for HPLC Analyses column analytical

semipreparative

time min

hexane % (v/v)

DCM % (v/v)

0 10 20 35 40 50 65 0 4 8 14 18 31 35 42 55

97 97 70 40

3 3 30 55 100 3 3 3 3 10 10 30 55 100 3 3

97 97 97 97 90 90 70 40 97 97

Table 2. Elution Order of Different Compounds types of compounds

analytical column

semipreparative columna

nonpolar sat. carboxylic acids

FA: 0-10 min FB: 10-20 min

phenols polyfunctional

FC: 20-32 min FD: 32-47 min

FA: 3-12.5 min FB1: 12.5-18 and FB2: 18-22.5 min FC: 22.5-29 min FD: 29-40 min

MeOH % (v/v)

5

a The fraction limits for the semipreparative column have been slightly adjusted compared to the reported method in Borgund et al.,30 due to technical reasons.

5

acid recovery procedure is repeated using formic acid (0.5 mL per gram of ion-exchange material) and 1:1 toluene/MeOH (v/v; 50 mL per gram of ion-exchange material). The mixture is stirred and filtered in the same way as described above. The solvents in the filtrates are removed by a rotary evaporator, and the acid extract is redissolved in a small amount of 93:7 dichloromethane (DCM)/ MeOH (v/v). The liquid-liquid acid extraction procedure is described by Constantinides and Arich11 and others.13,16 A total of 100 mL of hexane is added to 100 g of crude oil, and the acids are extracted with a 1 M NaOH solution of 50 vol % ethanol and 50 vol % water. The acid extract is washed with hexane to remove coextracted oil. The petroleum acids are recovered by protonation by adding concentrated HCl until the pH reaches 2. The acids are extracted into a DCM phase, dried with anhydrous Na2SO4, and quantified. 2.3. HPLC Procedure. The HPLC analyses are performed as described in a recent paper by Borgund et al.30 A P680 Dionex HPLC pump and a Rheodyne 7725 manual injector with a 20 µL (analytical column) or 100 µL (semipreparative column) loop are used for the analyses. Two types of detectors are used: a light scattering detector (ELSD; Sedex 55 Light Scattering Detector; operation temperature, 40 °C; nebulizing gas, nitrogen) and a UVdetector (UVD340U Dionex, diode-array detector). Chromatograms from the UV detector at wavenumbers 230, 250, 280, and 300 nm were chosen for the characterization of each sample. The chromatogram from a blank run is automatically subtracted from the sample chromatogram in order to remove the influence from the solvents. The laboratory data system used is Chromeleon (delivered by Dionex Softrun). Two types of BDS Hypersil Cyano columns and guard columns (Thermo Scientific) are used: an analytical column (250 mm × 4.6 mm, 5 µm) with a guard column (100 × 4 mm, 5 µm) and a semipreparative column (250 mm × 10 mm, 5 µm) with a guard column (100 mm × 100 mm, 5 µm). The gradient programs for the two columns are shown in Table 1. The flow rate is set to 0.5 mL/min for the analytical column and 2 mL/min for the semipreparative column. The samples are dissolved in 93:7 DCM/MeOH (v/v) to a concentration of 10 mg/mL, giving a 0.2 mg sample applied to the analytical column and a 1 mg sample applied to the semipreparative column. For some of the runs on the semipreparative column, a concentration of 20 mg/mL is used, giving a 2 mg sample applied to the column. In the preparative analysis, several portions of the acid extract are fractionated, and five fractions are collected. The solvent of the fractions is evaporated under a N2-gas flow, and the fractions are redissolved in 93:7 DCM/MeOH (v/v). The chromatograms are divided into four fractions using the analytical column (FA, FB, FC, and FD), and five fractions using the semipreparative column (FA, FB1, FB2, FC, and FD). The elution order of different compounds is shown in Table 2. Chromatograms from different acid extracts are compared to each other by looking at the amounts of material found in the different

fractions. The percentage of each area in the chromatogram is used to calculate the amount of the fractions in the crude oil. This is performed on the basis of the amount of acids extracted from the corresponding oil. These calculations are based on an assumption that all the compounds present in the samples have identical response factors. Thus, the results given are estimated distributions for a comparison of samples and as such are semiquantitative. Uncertainties caused by some peaks going out of range for the detectors are also not taken into account. 2.4. FTIR Analysis. An FTIR analysis is performed on a Nicolet Protege 460 FTIR spectrometer with a diamond attenuated total reflection (ATR) Dura sampler cell (from SensIR). The samples are dissolved in 93:7 DCM/MeOH (v/v). A small amount of the sample (one droplet) is placed on the ATR diamond, and the solvent is evaporated before the spectra are recorded. The spectra are recorded from 600 to 4000 cm-1, using 32 scans and a resolution of 4 cm-1. The five fractions attained from preparative HPLC for three of the acid extracts are analyzed on the FTIR spectrometer. 2.5. GPC Analysis. The molecular weights of acid extracts and HPLC fractions are determined by the use of GPC. The GPC analyses are carried out with conventional HPLC equipment described in section 2.3, using ELS detection at 40 °C. The column used for GPC is packed with a polystyrene/divinylbenzene copolymer (PLgel 3 µm MiniMix-E 4.6 mm × 25 cm, from Polymer Laboratories), and the molecules are separated according to molecular size and shape. The mobile phase is distilled tetrahydrofuran (THF), stabilized with KOH pellets. The flow rate is set to 0.2 mL/min. Calibration of the relationship between retention times and molecular weights is performed using nine polystyrene standards covering the molecular weight range between 580 and 19 880 g/mol. In addition, two other standards in the low-molecularweight range are used (vanadyl octaethyl porphyrin, 599.6 g/mol, and stearic acid, 284.49 g/mol). Samples and standards are dissolved in THF. When a sample injection volume of 20 µL is used, concentrations between 0.3 and 0.6 mg/mL are suitable for the standards, while the fraction samples are prepared in concentrations varying from 1 to 30 mg/mL. The molecular weights are calculated from the retention times corresponding to the maximum intensity of the chromatographic peaks. Table 3. Amount of Acids Extracted from Oilsa oil

amount of acids mg/g oil

extraction method

TAN mg KOH/g oil

biodegr. level

B1c B2b B2c B4a B4c S3b S7b B4a B4d

16.1 18.6 5.3 9.1 11.8 1.1 0.2 3.0 3.2

ion exchange ion exchange ion exchange ion exchange ion exchange ion exchange ion exchange liquid-liquid liquid-liquid

2.18 2.66 3.18 1.02 1.4 0.16 0.024 1.02b n.m.

2b 6 5 8b 2 0 0b 8b n.m.

a The oils labeled B are biodegraded oils, and the oils labeled S are nonbiodegraded oils. The acidity (TAN) of the crude oils is given in the fourth column, and the level of biodegradation on the Peters and Moldowan scale31 in the fifth column (mostly reported by Barth et al.16). b Measured on a previous batch of oil. n.m. not measured



Figure 1. Chromatograms of acid extracts from the oil B4a using the analytical HPLC column and the ELS detector. Upper chromatogram: ion-exchange extraction. Lower chromatogram: liquid-liquid extraction.

2.6. TAN Determination. TAN values are determined according to the standard method ASTM664-8932 and are also described in Barth et al.16

3. Results 3.1. Acid Extraction. The amount of acids extracted from the different crude oils is shown in Table 3. The B4c oil has been extracted with the ion-exchange method several times, and an average from six extractions is shown in Table 3. The standard deviation is calculated to 0.2 mg/g. Most of the other oils are extracted at least two times, and deviations of 0.030.9 mg/g have been found. The amount of acids extracted from the ion-exchange method shows linear correlation to the TAN value of the corresponding oil. The oil B2c deviates from the correlation, but when the oil B2c is disregarded, the correlation coefficient (R2) is calculated to 0.98. The reason for the oil B2c being so different with regard to the correlation is not known at this time. 3.2. Comparison of Extraction Methods. The results from the extractions show that generally the amounts extracted are much larger when using the ion-exchange method compared to when using the liquid-liquid extraction method. The oil B4a has been extracted by both methods, and the amount of acids extracted by the ion-exchange method is 3 times the amount that was extracted by the liquid-liquid method, see Table 3. The HPLC chromatograms show that the composition of the acid fractions extracted using liquid-liquid extraction is different from that of acid fractions using the ion-exchange method, see Figure 1. In these chromatograms, the acid extracts have a concentration of 10 mg/mL, and a direct comparison of the relative amount of the different compound types can be made. The acid extract from the liquid-liquid method contains a relatively larger amount of nonpolar components compared to the acid extract using the ion-exchange method. The amount of saturated carboxylic acids is much smaller, and the relative (32) Standard Test Method for Acidic Number of Petroleum Products by Potentiometric Titration. Annual Book of ASTM Standards; American Society for Testing Materials: Philadelphia, PA, 1989; Section 5.

amount of phenolic compounds seems to be larger compared to that from the ion-exchange method. UV spectra of fractions FB and FC of the acid extracts from B4a are shown in Figure 2. The spectra show that acid extracts from the liquid-liquid extraction have more absorbance in the region above 250 nm in the phenolic fraction (FC) than the ionexchange extract, while the response in the saturated carboxylic acid fraction (FB) is much stronger for the ion-exchange extract. This shows that the liquid-liquid extract may contain some components in the FC fraction that are not present in the ionexchange extract from the same crude oil and confirms that a lower yield of saturated carboxylic acids is obtained. 3.3. Comparison of Biodegraded and Non-Biodegraded Oils. The extraction yields show significant differences between the biodegraded and non-biodegraded oils. Comparing the profile from HPLC for a non-biodegraded oil (Figure 3) with that for a biodegraded oil (Figure 1), both having a concentration of 10 mg/mL, shows that the non-biodegraded oil contains a relatively larger amount of nonpolar compounds. The nonbiodegraded oil also contains relatively less saturated carboxylic acids. The relative amount of compounds in the polyfunctional peak of the chromatogram seems to be larger in the chromatogram from the non-biodegraded oil. 3.4. Quantitative Distribution of the Acid Extracts. Quantitative estimates are based on the use of the ELS detector, which is close to universal in response to organic compounds. The UV detector can give additional information about, especially, the phenolic compounds, but the very variable response factors for different molecular structures makes quantification difficult without specific calibration for each compound type. An example of the same acid extract (from the oil B4c ion-exchange method) analyzed with the ELS detector and the UV detector is shown in Figure 4. The largest difference between the chromatograms can be seen in the FB fraction. The large amount of saturated carboxylic acids found in this fraction using the ELS detector is not observed using the UV detector, because the aliphatic carboxylic acids cannot be detected. In the FC fraction, we can see a larger relative amount detected by the UV detector than by the ELS


Borgund et al.

Figure 2. UV spectra of fractions FB (upper) and FC (lower) from using the analytical cyano column of acid extracts B4a extracted by the ion-exchange method (left) and liquid-liquid method (right).

Figure 3. Chromatogram of the acid extract from the oil S7b (ion-exchange extraction) using the analytical HPLC column and the ELS detector.

detector. Still, no clear peaks are found in the phenolic part of the chromatogram. For the purpose of comparing the samples, uniform and constant response factors have been assumed. On this basis, the quantitative distributions of the acid extracts are compared to each other on the basis of chromatograms from both the ELS and the UV detector, see Figures 5 and 6, respectively. Seven acid extracts from biodegraded oils are shown to the left, and two acid extracts from non-biodegraded oils are shown to the right. The amount of material found in the non-biodegraded oils is much lower than that in the biodegraded oils, so the y axes are shown with different scales. The results from the ELS detector, in Figure 5, show that four of the biodegraded oils extracted by the ion-exchange method (B1c, B2b, B4a, and B4c) have a quite similar acid distribution, with the largest amount of saturated carboxylic acids and more polyfunctional compounds than phenols. The other biodegraded oil extracted by the ion-exchange method (B2c) has a different distribution, containing more nonpolar components and smaller amounts of saturated carboxylic acids. The two non-biodegraded oils shown to the right in Figure 5 are not similar to each other. The relative composition of the S3b oil is similar to the relative acid composition of most of the biodegraded oils. The S7b oil is completely different from all the other extracts, in the way that it holds a larger amount of nonpolar compounds and contains small amounts of saturated carboxylic acids.

Of the biodegraded oils extracted by the ion-exchange method, the results from the UV detector, in Figure 6, show that oils B2b and B4c have a relatively similar composition with regard to the FB, FC, and FD fractions. The B2c extract is different from the other oils, with equal amounts in all the fractions. The B1c extract has a large amount of the FC fraction compared to the other extracts. The extracts from the nonbiodegraded oils have a different distribution than the other extracts (to the right in Figure 6). These extracts have relatively smaller amounts of saturated carboxylic acids compared to the acids from the biodegraded oils. UV spectra of fraction FC of some of the acid extracts are shown in Figure 7. The spectra show that the ion-exchange acid extracts from B1c and S3b have a higher absorbance in the region above 250 nm than the B2b and B4a ion-exchange extracts. These results indicate that fraction FC from the acid extracts from B1c and S3b might contain components that are not present in the B2b and B4a extracts. 3.5. FTIR Analysis of HPLC Fractions from Preparative HPLC. FTIR analyses of the HPLC fractions from three different acid extracts, B1c, B4a, and B4c (ion-exchange method), have been performed, and the results show very little variation between extracts from different crude oils. The FTIR spectra from the HPLC fractions of the acid extract from B4a



Figure 4. Chromatograms of the acid extracts from the oil B4c (ion-exchange extraction). Upper: ELS detector. Lower: UV detector.

Figure 5. Estimated distribution of acids in the fractions from different oils, using the analytical HPLC column and the ELS detector. The oils marked with “ion” have been extracted by the ion-exchange method, and the oils marked with “liq” have been extracted by the liquid-liquid extraction.

are presented in Figure 8. The FTIR interpretations are based on works from Williams and Fleming33 and Coates.34 In fraction FA, the spectrum is dominated by bonds from C-H stretch vibrations around 3000 cm-1, and in the region below 1500 cm-1, C-H bending and rocking vibrations bonds are observed, indicative of linear (unbranched) long-chain aliphatic backbone structures. The location of the carbonyl absorption bond (CdO) at 1707-1708 cm-1 is characteristic of ketone functionalities present in the sample. There seems to be an absence of aromatic compounds in fraction FA, as the characteristic bond around 1600 cm-1 (aromatic ring stretch) is not present. The most obvious feature with the spectrum of fraction FB1, compared with that of fraction FA, is the increased intensity of (33) Williams, D. H.; Fleming, I. Spectroscopic Methods in Organic Chemistry, 5th ed.; The McGraw-Hill Companies: London, 1995. (34) Coates, J. Interpretation of Infrared Spectra, A Practical Approach. In Encyclopedia of Analytical Chemistry; Meyers, R. A., Ed.; John Wiley & Sons Ltd.: Chichester, U.K., 2000; pp 10815-10837.

the CdO bond (1703-1706 cm-1) compared to the C-H bond at around 3000 cm-1. For the B4a and B4c HPLC acid fractions, this bond is even more intense than the C-H stretching bond. Also, in fraction FB1, the shift of this bond to some lower frequencies indicates carboxylic acids in preference to ketones. This interpretation is supported by the presence of the broad band spanning from approximately 3300 to 2500 cm-1 (overlapping the C-H region) and a secondary absorption band close to 2600 cm-1, both associated with hydrogen-bonded O-H from carboxylic acids. A band at approximately 940 cm-1 can come from out-of-plane O-H bendings, which also indicates the presence of carboxylic acids. The spectrum in fraction FB2 is very similar to the FB1 spectrum, but it is distinguished by the presence of a bond (weak intensity) at 1598-1602 cm-1, attributed to the CdC-C aromatic ring stretch. This bond is not observed in the FA and FB1 spectra. Fraction FC contains low concentrations of organic material, which results in an FTIR spectrum with high noise-to-signal


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Figure 6. Estimated amount in the fractions of the acid extracts from different oils using the analytical HPLC column and the UV detector. The oils marked with “ion” has been extracted by the ion-exchange method, and the oils marked with “liq” have been extracted by the liquid-liquid extraction.

Figure 7. UV spectra of fraction FC from using the analytical cyano column of acid extracts.

ratios. Hence, a complete interpretation of this spectrum is difficult to achieve, and only C-H and CdO absorption are clearly seen. A tendency for a wide peak in 1200 to 1250 cm-1 is also seen in most samples, and this may represent C-O in phenolic functional groups. From the retention time of model compounds, the compounds expected to be found in this fraction are of a phenolic type. Obviously, the content of these components are very low in the extracts compared to the carboxylic acids, and their presence cannot be confirmed by using IR. In fraction FD, polyfunctional compounds are expected to be found. The spectrum shows that the intensity of the CdO bond is weaker than the C-H signals. A significant increase in aromaticity compared to all the other fractions is observed by the enhanced intensity of the bonds at 1602 cm-1. The bond located at around 1455 cm-1 (methyl/methylene bending vibrations) does not show the splitting pattern which is observed in the FA, FB1, and FB2 spectra. This is indicative of shorter chain lengths in the aliphatic structures. In the region below 1500 cm-1, peaks are overlapping, indicative of increased complexity of the molecules in the mixture.

3.6. GPC Analysis of Whole Extract. The GPC molecular weight range is determined for the two extracts B4c and S3b and is calculated to have a maximum of approximately 500 g/mol for both extracts. A chromatogram of the acid extract from the crude oil B4c (ion-exchange method) is shown in Figure 9, with a symmetrical peak shape corresponding to a molecular weight of 500 g/mol at the maximum intensity. 3.7. GPC Analysis of Acid Fractions from Preparative HPLC. GPC results for the acid fractions collected from the preparative HPLC of three acid extracts, B1c, B4a, and B4c (ion-exchange method), are presented in Figure 10. The given values are calculated from the maximum intensity of the peak in the chromatograms and are averages of 2-4 runs of each individual sample. The experiments are fairly reproducible (the calculated deviations varied between 3 and 42 g/mol). The molecular weights of the maximum in the chromatograms in the acid fractions were found to range from approximately 400 to 1100 g/mol, and there seems to be a somewhat different distribution of the molecular weights in HPLC fractions. The FB2 fractions hold the lowest-molecular-weight compounds, and the FC fractions contain compounds in the higher-molecular-



Figure 8. FTIR spectra of HPLC fractions from the acid extract from B4a (ion-exchange method). In the spectra of fractions FC and FD, the CO2 peak has been removed.

weight range. Only small variations in average molecular weights are observed between the acids from the three oils in fractions FB1 and FB2, while in fractions FA, FC, and FD, the variations within the fractions for the three acid extracts are larger. Fraction FA contains compounds with molecular weights (at maximum intensity) ranging from 700 to 900 g/mol, and the compounds in the B4c extracts seem to have lower molecular weights than the B1c and B4a extracts. Fractions FB1 and FB2 have molecular weights ranging from 400 to 600 g/mol in the investigated oils. The molecular weights of the compounds in fraction FC are high, especially for the B4a extract (approximately 1100 g/mol). In the FD fraction, the molecular weights at the maximum of the peaks range from 700 to 800 g/mol for the three extracts. 3.8. GC-MS analysis of acid fractions from preparative HPLC. Some of the fractions from preparative HPLC are analyzed by GC-MS using the procedure described in Borgund

et al.35 The chromatograms show large UCMs and are difficult to interpret. Some alkanes are found in the FA fraction, and fatty acids with 16 and 18 carbon atoms are identified in the FB2 fraction. In general, GC-MS is not considered to be a good method for analysis of the acid extracts, due to the intense UCM and the high molecular weights of the samples. Discussion 4.1. Quantification of Total Extract. The results from the extraction of acids from crude oil show that more organic material is extracted using the ion-exchange method than using the liquid-liquid extraction. Earlier work by Barth et al.16 has shown that the liquid-liquid extraction method has a low recovery of acids in the extract. The ion-exchange procedure, on the other hand, has a very good recovery.20 These results confirm that ion exchange is a far more effective method, and this method was chosen for most of the samples. (35) Borgund, A. E.; Høiland, S.; Barth, T.; Fotland, P.; Askvik, K. M. Appl. Geochem. Submitted October 2006.


Borgund et al.

Figure 9. GPC chromatogram of the acid extract from the crude oil B4c (ion-exchange method).

Figure 10. Average molecular weights of HPLC fractions from three acid extracts, measured by GPC. The values are calculated from the retention times that correspond to the maximum intensity of the chromatographic peaks.

The biodegraded oils contain larger amounts of acids than the non-biodegraded oils. The importance of biodegradation for the acid composition of crude oil has previously been discussed by Barth et al.16 and Tomczyk et al.14 Meredith et al.18 indicate that biodegradation is the dominant process that produces high concentrations of carboxylic acids. Clemente et al.36 have studied the biodegradation of commercial naphthenic acids and conclude that the presence of some carboxylic acids after biodegradation results from introduction of the acids with the inoculum from the enrichment culture. Thus, the observed increase in content of carboxylic acids may have contributions from both functionalizations of petroleum hydrocarbons and degradation of the microbial biomass. The correlation between the amount of acids extracted from the ion-exchange method and the TAN values for most of the oils supports the claim that the extracted acids comprise the acid amount as determined by TAN. Saab et al.22 have also found a strong correlation between the carboxylic acids fraction and TAN. Meredith et al.,18 however, found a strong correlation between TAN and the carboxylic acids fraction for many of the oils investigated, but some non-biodegraded oils did not have a clear correlation, indicating that some components other than carboxylic acids contribute significantly to the TAN in these oils. The one oil in the present work that does not follow the general correlation, B2c, has a high TAN value compared to the amount of extracted acids. This indicates that it is not only (36) Clemente, J. S.; MacKinnon, M. D.; Fedorak, P. M. EnViron. Sci. Technol. 2004, 38, 1009-1016.

the extractable carboxylic acids that comprise components responsible for the acidity (TAN) in this oil. 4.2. Composition of Acid Extract. The HPLC results show that the two extraction methods give different compositions of the acid extracts. Thus, the variations between the two extraction methods are not just a matter of different amounts of total material but also what kind of material has been extracted. The ion-exchange extract contains larger amounts of the saturated carboxylic acid fraction. Furthermore, the amount of nonpolar components is lower in the ion-exchange extract compared to the liquid-liquid extract. This shows that the extra amount of organic material obtained by the ion-exchange method is not coextracted crude oil, which would be revealed as an increase in the amount of nonpolar compounds. With regard to the distribution of acids in the HPLC fractions, many of the biodegraded oils show similar compositional trends. The one biodegraded oil that deviates from the rest of the data set, B2c, is the same oil that deviates from the correlation with the TAN value. Of the two non-biodegraded oils that are investigated, one of them has a similar relative composition as the biodegraded oils. Thus, the HPLC results show that there are groups of oils with similar acid compositions, but the criteria for which oils group together is not just whether the oils are biodegraded or not. However, all the oils except S7b have saturated carboxylic acids as the major constituents. The exception is the ion-exchange extract of the oil S7b, where the polyfunctional acids have the highest concentration. This oil also has an exceptionally low concentration of acids. Analyses



Table 4. Wettability of the Hydrate Surfaces in Crude Oil/Brine Systema oil wet

intermediate wet

water wet

B2b (0.26) B4a (0.35) B4c (0.38)

B2cb S3b (-0.06) S7b (0.17)

B1cc (-0.32)

a The quantitative values are given in parentheses and are mostly taken from Høiland et al.10 b The oil might contain significant amounts of water. The results may not be trustworthy. c Measured on a previous batch of oil.

of more oil samples are needed to map the range of compositions that may be present. The differences between the ELS and UV detectors for the analysis is clearly shown in the chromatograms. While the ELS detector gives the best overall quantitative estimate, the UV detector is important to get information about the phenolic compounds in the samples, and the results showed that no distinct peaks from phenolic compounds were found. This requires further investigation to determine whether the phenols are either not present in the biodegraded oils or not extracted by the applied procedure, or whether they are lost in the workup procedure. The molecular weights of the ion-exchange extract and fractions are found to be in the range of 400-1200 g/mol, which is in the intermediate range of molecular weights of crude oil compounds. These molecular weights are higher than what is found from using the liquid-liquid method by Barth et al.16 (approximately 300 g/mol). Seifert and Howells,6 who also used liquid-liquid extraction, have found the average molecular weights of the acid extracts to range from 300 to 400 g/mol. Saab et al.,22 on the other hand, who use a similar ion-exchange extraction method as that of the present work, have found the average molecular weight of acid extracts from a crude oil to be 653 g/mol. Thus, the ion exchange seems to extract acids of a higher molecular weight range than the liquid-liquid extraction, which accounts for the increased yields and different profiles. 4.3. Fractionation of Acid Extracts. The FTIR analysis of HPLC fractions confirms the assignations based on the standards of the type of compounds typically found in the different HPLC fractions. The FA fraction has a strong C-H stretch compared to the CdO carbonyl functionality, giving a more nonpolar fingerprint. In fractions FB1 and FB2, strong CdO adsorption bands from carboxylic acids are found. The phenolic fraction FC contains so little material that FTIR analysis does not give specific information, and the presence of phenols cannot be confirmed. The polyfunctional fraction FD shows a lower relative amount of carbonyl (CdO), higher aromaticity, and an increased complexity of the spectrum.

The GPC analysis of the different fractions shows that fractions FB1 and FB2 have molecular weights ranging from 400 to 600 g/mol, which corresponds to saturated carboxylic acids with more than 30 carbon atoms. The polyfunctional compounds in the samples, fraction FD from HPLC, are believed to contain the most surface-active compounds, and they have a relatively high molecular weight range from 700 to 800 g/mol. This corresponds well with the molecular weight of some types of biosurfactants. These GPC results generally show that acid extracts mostly contain compounds of intermediate molecular weights. Thus, neither high-molecular compounds such as asphalthenes nor simple, low-molecular petroleum acids and bases are prominent in the acid extracts. 4.4. Relation to Crude Oil Properties. The amounts of organic material found in the different fractions from HPLC are compared to the biodegradation level, the amount of extracted acids, TAN, and the wettability of hydrate particles formed in the crude oils (the values for the amount of extracted acids, TAN, and the wettability were not included for the oil B2c, due to uncertain results). The wettabilities of hydrate particles formed in crude oils have been reported by Høiland et al.10 and are given in Table 4. Here, negative wettability values or wettability values close to zero correspond to hydrate plugging systems, whereas positive wettability values correspond to nonplugging systems. As expected, a positive correlation between the estimated amount of material in each fraction based on responses from the two detectors is found; that is, the amount of material in fraction FA from the ELS detector has a strong positive correlation with the amount of material found in fraction FA using the UV detector (R ) 0.92). This confirms that both detectors give quantitatively relevant and similar results, even if the UV detector cannot be considered quantitative on a general basis. The amounts of material in the largest fractions from the chromatogram (FB and FD from the ELS detector and FB, FC, and FD from the UV detector) have a positive correlation with the amount of extracted acids and the TAN value, as expected, since the total yield provides the basis for calculating the fraction yields. The biodegradation level and the wettability effect of the crude oil acids on hydrate surfaces do not show strong correlations with the amount present in any HPLC fraction. Relative amounts of material in the HPLC fractions are also compared to the other crude oil properties, and the correlations are shown in Table 5. A strong negative correlation between the amount of material found in fraction FC using the ELS detector and the wettability of hydrates formed in the crude oil is found (R ) -0.94), as shown in Figure 11 and Table 5. The negative correlation means that large amounts of phenolic compounds (fraction FC) will give lower values for the wettability. Low values for wettability correlate with water wet

Table 5. Correlation Coefficients (R) by Comparing Crude Oil Properties to the Relative Amounts of Material Found in the HPLC Fractions Using ELS and UV Detector TAN biodegr. TAN acids wettab. ELS_A ELS_B ELS_C ELS_D UV_A UV_B UV_C

0.51

extr. acids ion-meth. 0.56 0.99

wettability

ELS_A

ELS_B

ELS_C

ELS_D

UV_A

UV_B

UV_C

UV_D

0.48 -0.06 -0.01

-0.37 -0.62 -0.65 0.09

0.56 0.56 0.60 0.11 -0.93

-0.63 0.09 0.05 -0.94 -0.14 -0.18

-0.61 -0.50 -0.54 -0.16 0.76 -0.95 0.34

-0.03 -0.70 -0.70 0.24 0.77 -0.50 -0.39 0.20

0.75 0.67 0.73 0.15 -0.86 0.93 -0.19 -0.86 -0.46

0.09 0.40 0.40 -0.67 -0.78 0.62 0.58 -0.48 -0.61 0.59

-0.68 -0.56 -0.62 0.07 0.63 -0.79 0.18 0.87 0.03 -0.86 -0.57


Borgund et al.

Further work is needed to elucidate the factors that cause the correlation and transform them into a description of the mechanism behind wettability changes. More work on the fractionation of extracts and further analysis by LC-MS has the potential to provide molecular information on the critical constituents. 5. Conclusions

Figure 11. Correlation between the wettability and the relative amount of material in the FC fraction using the ELS detector.

particles, and more agglomeration of the hydrates.10 Since this relationship is based on statistical correlation, it is not possible to determine directly whether this is a cause-effect relationship or whether the absence of compounds eluting in the phenolic fraction is indicative of some other compositional factor that determines the wettability. The UV spectra of ion-exchange acid extracts show that crude oils with low wettability values (B1c and B3c) contain components that are not observed in crude oils with higher wettability values (B2b and B4a). This strengthens the hypothesis that the relative amount of material in fraction FC from HPLC based on ELS detection is an indicator for the wettability effect of the crude oil acids relative to gas hydrate surfaces. The liquid-liquid acid extracts (B4a and B4d) have UV spectra indicating that they contain the components present in the crude oils with low wettability values, even though the B4a oil has been shown to form oil wet hydrates (higher wettability value). The liquid-liquid acid extract from B4a has a large relative amount of phenols, but again the wettability values are high. This shows that the system is very complex, and the correlation found in Figure 11 between the relative amount of material in the FC fraction and the wettability most probably is indicative of some other compositional factor that determines the wettability.

In general, the HPLC separation of acid extracts from crude oil gives useful information about the distribution of the compounds. The profiles illustrate the distribution of the acids by polarity and simplifies the further analysis. Of the two acid extraction procedures tested, the ion-exchange method was shown to extract more material than the liquid-liquid extraction, and the HPLC profiles were different for acids extracted by the two methods. The HPLC method was shown to give reproducible and comparable results on crude oil acid fractions. Though the data set used in this work is small, the results show a strong negative correlation between the amount of fraction FC and the wettability. The results presented here are useful with regard to evaluating factors that influence hydrate particle wetting properties, because they place the focus of attention on functionalized fractions of intermediate molecular weight in the oil. Such compounds have perhaps been given the least attention in petroleum analysis because the generally used methods for hydrocarbon analysis are not suitable for their identification due to the low volatility and polar properties. Further work addressing the composition of these fractions of the oil and their properties with regards to modifying the bulk oil properties is thus required and can be envisioned to contribute to a general understanding of problems involving the deposition of diverse solids from crude oil. Acknowledgment. Norsk Hydro ASA, Centre for Integrated Petroleum Research, and the Norwegian Research Council are acknowledged for the financing of this work. Morten H. Strand is acknowledged for titration of the crude oils. EF070100R

Fractionation of Crude Oil Acids by HPLC and Characterization of

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