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Review

Pros and Cons of Separation, Fractionation and Cleanup for Enhancement of the Quantitative Analysis of Bitumen-Derived Organics in Process-Affected Waters—A Review

by
Ralph Hindle
1,2,
John Headley
3 and
Douglas G. Muench
1,*
1
Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada
2
Vogon Laboratory Services, Cochrane, AB T4C OA3, Canada
3
Environment and Climate Change Canada, Watershed Hydrology and Ecology Research Division, National Hydrology Research Center, 11 Innovation Blvd., Saskatoon, SK S7N 3H5, Canada
*
Author to whom correspondence should be addressed.
Separations 2023, 10(12), 583; https://doi.org/10.3390/separations10120583
Submission received: 29 July 2023 / Revised: 30 October 2023 / Accepted: 13 November 2023 / Published: 24 November 2023
(This article belongs to the Topic Advances in Spectroscopic and Chromatographic Techniques)
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Abstract

:
Oil sands process-affected water (OSPW) contains a diverse mixture of inorganic and organic compounds. Naphthenic acids (NAs) are a subset of the organic naphthenic acid fraction compounds (NAFCs) and are a major contributor of toxicity to aquatic species. Thousands of unique chemical formulae are measured in OSPW by accurate mass spectrometry and high-resolution mass spectrometry (MS) analysis of NAFCs. As no commercial reference standard is available to cover the range of compounds present in NAFCs, quantitation may best be referred to as “semi-quantitative” and is based on the responses of one or more model compounds. Negative mode electrospray ionization (ESI-) is often used for NAFC measurement but is prone to ion suppression in complex matrices. This review discusses aspects of off-line sample preparation techniques and liquid chromatography (LC) separations to help reduce ion suppression effects and improve the comparability of both inter-laboratory and intra-laboratory results. Alternative approaches to the analytical parameters discussed include extraction solvents, salt content of samples, extraction pH, off-line sample cleanup, on-line LC chromatography, calibration standards, MS ionization modes, NAFC compound classes, MS mass resolution, and the use of internal standards.

1. Introduction

The oil sands in Canada’s northern Alberta region represent the third-largest proven oil reserve in the world. Oil sands production is expected to grow from 2.91 million barrels per day (bpd) in 2018 to 4.25 million bpd by 2035 [1]. Mining and processing of oil sands involves a hot water bitumen extraction process that results in the production of large volumes of oil sands process-affected water (OSPW) that contains a mixture of organic and inorganic compounds. These compounds include salts, trace metals, BTEX (benzene, toluene, ethylbenzene, and xylene), polyaromatic hydrocarbons (PAHs), and naphthenic acids (NAs) [2,3]. NAs have been identified as a main contributor of OSPW toxicity [4,5,6,7]. The molecular speciation of NAs using accurate mass spectrometry and high-resolution mass spectrometry is required to generate and confirm chemical formulae to effectively characterize the organic compounds and monitor their removal during remediation experiments. Additionally, off-line sample cleanup and liquid chromatography (LC) separations can help reduce variable ion suppression and improve quantitative evaluations of OSPW.
There are three common classifications used to describe the organic chemical species in OSPW. The first are the classical NAs (cNAs) that have the general molecular formula CnH2n+zO2 where “n” is the number of carbon atoms and “z” is either zero or a negative even integer representing the hydrogen deficiency resulting from saturated alicyclic ring structures (Figure 1) [8]. Each value of z = −2 represents an additional ring or double bond and is referred to as a double bond equivalent (DBE). The double bond found in the carboxylate moiety is included in references to DBE, but it is excluded when referencing the z-value of NAs. The second classification group is NAs that include O3, O4 and higher oxygenated species, and are referred to as oxy-NAs. While cNAs are the most abundant species in freshly produced OSPW, relatively high abundances of O3 and O4 species may also be present in OSPW and may be temporal indicators of transformations. Naphthenic acid fraction compounds (NAFCs) are the third general classification group and is a broad, all-inclusive definition of OSPW organics. NAFCs are the acid extractable organics that include cNAs, oxy-NAs, and compounds with aromatic rings, unsaturated groups, nitrogen, and/or sulphur atoms [9]. Common chemical species identified in NAFCs include SO2, SO3 and various NO-containing compounds.
Effective OSPW detoxification strategies [10,11,12,13,14,15] rely on a thorough understanding of the organic compounds that contribute to toxicity [5,7]. Mass spectrometry (MS) is the most common method of detection for organic compounds in OSPW, with the simplest and least expensive approach being unit mass resolution quadrupole MS. Quadrupoles with unit mass resolution have limited applications due to low resolution. For example, such instruments cannot differentiate between the masses at 32 amu arising from either two oxygens (2 × 16 amu = 32 amu) and a single sulfur (32 amu) in a given ion. The complexity of OSPW components can be better determined using high-resolution accurate mass (HRAM) instruments. Fourier transform ion cyclotron resonance (FT-ICR), Orbitrap, and time-of-flight (TOF) mass spectrometers are capable of mass accuracies of four or more decimal points. This resolution is sufficient to determine or confirm molecular formulae in the mass range that is typical of NAs and able to speciate elemental composition (e.g., O2 = 31.9898; S = 31.9721).
A previous review article [16] evaluated eight areas for consideration in designing a “semi-quantitation” approach to MS analysis of NAFCs in OSPW (Table 1). Semi-quantitative methods do not have authentic reference standards available for all target compounds. Therefore, they use the response factors of structurally similar compounds to estimate concentrations. In this review, we revisit these eight areas to highlight the pros and cons of the critical parameters for NAFC MS analysis, with a focus on chromatographic clean up and separations prior to mass spectrometry detection of OSPW and environmental samples. We also expand the discussion to ten key parameters, including separation effects of salt content, the benefits of sample clean up, and a chromatography step prior to MS. Optimization and control of these parameters can lead to robust methods of analysis, while seemingly reasonable but variable settings can lead to irreproducible and ambiguous results. A good example of conflicting results was reported previously, where OSPW extracts were prepared, split, and identical samples were distributed to two laboratories. Each laboratory analyzed the extracts by measuring ions under either acidic or basic conditions [17]. Large differences were evident for the O2 class depending on whether acidic or basic conditions were employed, regardless of the instrument used by the two laboratories (Figure 2). Of note, the most toxic O2 class would be considered a minor component if only acidic conditions were used. In contrast, separation under basic conditions indicated that the O2 class is by far the most abundant NAFC. While the observed differences can be explained by pH effects on separation of ions (discussed later in this review), the apparent results can lead to confusion about the levels of principal toxicants in the OSPW. Such discrepancies can pose difficulties to end-users of the data for toxicity assessment.
Separations 10 00583 g001
Figure 1. Examples of classical mono-carboxylic naphthenic acid (cNA) structures [18].
Figure 1. Examples of classical mono-carboxylic naphthenic acid (cNA) structures [18].
Separations 10 00583 g001

2. Critical Analytical Parameters

2.1. Choice of Extraction Solvent

The solvent used for NAFC extraction from water should be based on the chemical properties of target compounds. For example, in the case of relatively non-polar PAHs, lipophilic solvents that are immiscible with water are used. Cyclohexane and dichloromethane are common choices for neutral compounds and are directly amenable to GC analysis following a drying step to remove residual water, typically by passing the solvent through a column of anhydrous sodium sulfate. NAs have surfactant properties, with hydrophobic ring structures and carbon side chains, as well as hydrophilic carboxylic acid groups. Extraction and concentration of NAFCs from OSPW is carried out to lower detection limits, much like that performed for PAH analysis but with additional consideration of aqueous pH that takes advantage of the ionic and neutral forms of the acids. Use of high pH (between 10 and 12) during partitioning will ionize NAs for their retention in the water phase, while non-polar compounds are extracted into the organic solvent and discarded. Lowering of pH to ~2 will then neutralize NAFCs, so they partition into the organic phase and are dried, and concentrated.
The choice of extraction solvent can also align with the solvent used to prepare the sample for MS analysis. For example, a procedure was reported where an NAFC extract concentrate was solvent-adjusted to prepare a series of fractions for nanospray infusion into the ESI source (Figure 3) [18]. The NAFC solution was acidified to pH 2.5, then the neutral NAFCs were extracted into dichloromethane (DCM), concentrated, and the solvent was exchanged into acetonitrile. The acetonitrile extract was then amended with five solvents used separately or in various combinations: Milli-Q water (9.0 polarity index, PI)/acetonitrile (ACN; 5.8 PI); ACN; methanol (MeOH; 5.1 PI)/ACN; DCM (3.1 PI)/ACN; and 1-octanol (<3.9 PI)/ACN. Ammonia solution (35% v/v in water) was added to each solution to improve deprotonation of NAs in the MS source. Of the five solvents, water/ACN, ACN, and MeOH/ACN were similar in their respective spectra. The DCM/ACN and 1-octanol/ACN solvents, however, showed marked differences in intensity and molecular weight distribution compared to the other three, particularly when plotted as carbon number vs. z-values and broken out as monocarboxylate O2 vs. dicarboxylate O4 species (Figure 4, ACN and MeOH/ACN not shown). The authors attributed the difference in NA characterization as an effect of greater solubility of higher-molecular-weight acids in the lipophilic solvents DCM and 1-octanol. Researchers using a more polar solvent system would, therefore, reach a very different conclusion about the NAFC characterization of the same OSPW sample.
More recent work has shown the effects of the apparent pH of the transfer solvent (mobile phase), where identical OSPW extracts were analyzed by Orbitrap and FT-ICR-MS in different laboratories using both acidic and basic mobile phases on each instrument [17]. In this particular application, neither the Orbitrap (flow injection analysis) nor the FT-ICR-MS (infusion) used chromatographic separation before ionization in the source of the mass spectrometer. A flow injection experiment would result in a mixture of the mobile phase and the solvent in the sample vial when the sample reaches the source within seconds, but infusion contains the vial solvent alone. These solvent differences would result in a different apparent pH in the source and therefore different spectra. On-line chromatography will diffuse the sample vial solvent to a greater extent by the time the sample arrives at the source, with the apparent pH being that of the mobile phase alone.
Syncrude researchers describe their routine method for NAFC analysis using DCM extraction and FT-IR detection based on the carbonyl stretching bands at ~1740 cm−1 and ~1703 cm−1 for the monomers and dimers, respectively [20]. Extraction efficiency compared to DCM was 36% for tetrachloroethylene (TCE) and 49% for hexane. The higher extraction rate for DCM was attributed to its higher polarity. This was further borne out by extracting OSPW with a mixture of 1:1 DCM:TCE. When measured gravimetrically, the extract contained twice as much material as a DCM extraction alone, but FT-IR analysis showed that the concentration of NAFC was the same. Therefore, DCM alone is sufficient to extract the acidic components while not extracting less polar compounds.

2.2. Salt Content of the Sample

OSPWs may contain high levels of natural salts from some oil sands formations or from high levels of water reuse in the bitumen extraction process. Salting-out effects in MS analysis have been noted for crude oil acids in sea water, and the same may be true in OSPW with high salt content [21]. Salinity values over 2800 mg/L have been reported in OSPW samples [22], and these concentrations may affect the partitioning behaviour and separation of NAFCs by the formation of ion-pair complexes between the salt cations and NA anions.
A comparison of NAFC extracts in a DCM/ACN solvent mixture showed significant spectral differences when a saturated salt solution was added [21]. Intensities were enhanced for shorter carbon chain acids with lower z-values for CnH2n−zO2, CnH2n−zO3, and CnH2n−zO4 species but suppressed for longer chain, higher z-value acids. The authors attributed this decrease in response to salting-out effects. High sodium content can lead to the formation of anionic sodium dimers [23], with masses corresponding to [2M-2H + Na]−, where M is the mass of the neutral compound. The mass range would be approximately double the expected value for the [M-H]− anion monomer. The acids forming the dimer may not be the same species, which further complicates spectral evaluation. A possible remedy to these sodium-bridged dimers would be to reduce the sodium in the source of the MS when the acids are present by injecting the sample onto an LC column rather than infusing the sample directly into the source. Sodium ions would be unretained on the column and directed to waste. Alternatively, off-line extraction of NAFCs from water can be carried out using solid phase extraction (SPE) by adjusting the pH to <3 for SPE loading, removing salts with the subsequent wash step, followed by elution of the NAs with organic solvent [24]. Sodium adducts in positive mode ESI can be reduced by adding ammonium to the mobile phase, or by decreasing source temperatures. The same may be true in negative mode. After dimer formation in the source, increasing the TOF fragmentor voltage may also be used to fragment dimers into their monomers. Care must be exercised when using this technique as it can lead to unwanted fragmentation of ions, reducing the abundance of precursor ions.

2.3. pH of Extraction

Bitumen-derived NAFCs possess a wide range of polarities and pKa values for neutral and ionic compounds, respectively. As a result, the choice of extraction solvent may skew the analytical data and could result in selective fractionation or separation of NAs based on solvent polarity and pH. Extractions from a Fluka NA standard containing predominantly O2 species showed that maximum NA solubility was achieved at pH 7 [24], with a 37% increase in signal intensity from pH 7 to 9, but with the same MS spectrum profile. The authors attributed this to a dependence on pH for total solubility, correlating the effect to refining scrubbing processes. A pH-dependent OSPW extraction study investigated the influence of pH on O2, O3, and O4 compounds using LC/TOF [25]. In this study, the pH of the OSPW (pH 8.5) was adjusted to pH 12.4 and extracted with cyclohexane to remove non-polar compounds. The OSPW was then adjusted to pH 10 and re-extracted with DCM, and the extract was dried with sodium sulfate and weighed. Further extraction cycles were performed by lowering the pH in the water sample by 1 unit in each cycle, down to pH 2. The total mass of O2, O3, and O4 NAs was 23.5%, 6.6%, and 3.5%, respectively, and these NAs were preferentially extracted in pH ranges of 3–5, 5–7, and 6–8. A mathematical model was developed to estimate the pKa values of the oxy-NAs to help understand their distribution in wastewater treatments. This revealed that, as oxygen numbers increased in the O2, O3, and O4 series, pKa also increased (4.2, 6.2, and 7.7, respectively). Increases in pKa indicate that the O3 NAs are hydroxylated, and the O4 NAs are more likely to be di-hydroxylated rather than di-carboxylic acids, which otherwise would have resulted in lower pKa values (e.g., propanoic acid pKa = 4.87; propanedioic acid pKa1 = 2.87).
In an ideal situation, the MS detector response for a given number of ions would be the same regardless of the mass-to-charge (m/z) value. In that case, any compound that was fully ionized could be used as a reference and its response factor determined directly when using a known concentration. Comparative analysis [17] provides insight into relative response factors between transfer solvents in the mobile phase. Table 2 shows a 111-fold increase in the response for biphenyl-4-carboxylic acid (B4CA) with ammonium hydroxide added to pH 8.91 compared to formic acid to pH 3.52. The authors confirmed that the formic acid pH, expected to be ~2.7, was the result of a blend of formic acid and OSPW, which had a pH of 8.1. The percent ionization changed rapidly with pH such that a weak acid will effectively be fully neutralized (99.85%) or fully ionized at 2 pH units below and above its pKa, respectively.
This change in ionization is described by the Henderson–Hasselbalch equation relating pH to the dissociation of weak acids (Equation (1)), where [HA] is the concentration of the neutral acid and [A−] is the concentration of the anion. When solution pH equals the pKa, an acid is 50% ionized.
p K a − p H = l o g 10 [ H A ] [ A − ]
Equation (1). Henderson–Hasselbalch equation describing the dissociation of weak acids in solution.
Because the acidic mobile phase pH (3.52) is below the pKa of B4CA (4.19), B4CA will be 17.6% ionized in solution but fully ionized at a basic pH of 8.91. The dependence on solution pH indicates that ions already present in solution may be more readily detected than non-ionized neutral molecules which only ionize in the ESI source. The dissociation rule is further supported by the relative intensities of anthraquinone-2-carboxylic acid (A2CA), which has a pKa of 3.42. This pKa value is closer to the acidic mobile phase pH (3.68) and is more highly ionized in solution (59.7%) and, therefore, gives a higher response, as did trimesic acid (Table 1).

2.4. Sample Cleanup or No Cleanup

Sample cleanup has many advantages over direct analysis, but comes at the cost of increased time, expense, potential analyte losses, and waste generation. Cleanup is usually required for complex and ‘dirty’ matrices such as food, soil, petroleum, and biological matrices to separate matrix co-extractives from target compounds. The sample cleanup also serves the purpose of analyte concentration and can include a solvent exchange step suitable to the instrumental technique of choice. For water samples, cleanup may not be necessary if the instrument has sufficient sensitivity to meet detection limit requirements and if the samples are relatively clean (e.g., drinking water, groundwater, and surface water). Complex samples such as OSPW can also be analyzed without prior cleanup by using LC separation before the MS, or by using HRAM MS instruments that can differentiate the masses of target ions from the background. However, ion suppression may occur in these samples and can bias results. The presence of high salt concentrations in OSPW may lead to sodium-bridged dimers and can complicate data analysis. The choice ultimately comes down to having an injectable sample or extract that contains all the compounds of interest at sufficient concentrations to measure with minimum interference and low ion suppression. Comparison of samples with and without cleanup is an important step in method development and is particularly true for the analysis of the thousands of organic compounds present in OSPW [23,26,27].
Selectivity in OSPW cleanup is generally carried out using one of two extraction techniques. Liquid–liquid extraction is a simple procedure where a strong base is added to the samples to ionize the weakly acidic NAFCs and keep analytes dissolved in the aqueous phase while neutrals are partitioned into DCM and discarded, removing much of the dissolved organic content (DOC) [23]. The water is then acidified to pH~1.5 using H2SO4 and re-partitioned to extract the NAFCs into fresh DCM, then dried and concentrated by evaporation. The neutralized acids are reconstituted into a suitable solvent such as MeOH or ACN: water for MS analysis. Neutral compounds that are not ionized in either extraction procedure will be extracted into the final DCM phase based on their Kow coefficients, whereas salts remain in the aqueous waste fraction.
The second cleanup procedure uses SPE (described above) to selectively remove acids from samples using neutral sorbents such as divinyl benzene (DVB) [24]. A study that used a highly selective, mixed-mode polymer with additional anion exchange properties for acidic compounds (Oasis® MAX) [28] required no acidification of the OSPW. Also, for the MAX column cleanup procedure, the organic compounds were separated into two fractions, first using MeOH to elute the non-acidic compounds followed by acidified MeOH to elute neutralized NAs. Methanol alone eluted 10% of the DOC from the total MAX recoveries. The highest DOC recovery (95.4%) was achieved when using universal sorbent divinylbenzene hydrophilic–lipophilic balanced (DVB-HLB) cartridges and MeOH elution. The MAX sorbent only recovered 53.5%, an indication that only half of the organic compounds were acidic.

2.5. On-Line Chromatography vs. Injection or Infusion

Injection or infusion of samples directly into the source of an LC/MS is a fast and convenient method for qualitative characterization of OSPW. Injection or infusion is used more frequently with FT-ICR MS compared to other instrumental methods for two main reasons. First, the ultrahigh-resolution accurate mass feature allows for mass separation from interfering compounds. Second, the acquisition rate of some instruments may be too slow to produce high-resolution spectra when using LC separations [29]. A downside to infusion can be the presence of high salt levels that result in ion suppression and the formation of salt-bridged dimers, both of which can be reduced by using on-line chromatography (LC/MS).
NAs are classified as weak acids, with pKa values calculated to be 3.5, 4.8, and 6.8 for O2, O3, and O4 species, respectively [25]. Under acidic conditions (pH < 1.5), the acids will be present in their neutral form and become more hydrophobic. Condensed phase-membrane introduction mass spectrometry (CP-MIMS) used this property of NAs to calculate the pKa of many compounds of environmental concern, including pharmaceuticals, algal toxin biomarkers, and naphthenic acids [30]. The technique uses a semipermeable polydimethylsiloxane membrane probe immersed into pH-controlled solution. Neutral hydrophobic compounds permeate the membrane and infuse into the MS source with a methanol carrier, leaving salts and other ionic interferences behind. This approach works for both individual compounds in solution at parts-per-billion concentrations as well as complex mixtures such as OSPW. The CP-MIMS technique was extended to monitor the compositional changes in OSPW in a constructed wetland treatment system (CWTS), using unit mass quadrupole MS. The analysis took~15 min per sample and ion suppression effects were corrected through the use of lauric acid-d2 ISTD infused into the methanol stream [31]. Because LC separation is not required, the technique is proposed as an approach to implement in situ monitoring of treatment systems with relatively inexpensive MS systems.
An ISO/IEC 17025-validated method for cNAs using reversed-phase LC separation and TOF analysis showed excellent linearity (R2 = 0.9996) based on average response factors for all detected O2 acids [32]. Many researchers have used reversed-phase chromatography to provide additional cleanup for samples, separation from salts that are concentrated in OSPW, and to increase resolution by compound separation over time [9,28,33,34]. When using reversed phase LC, the mobile phase is typically acidic to neutralize the NAs, allowing retention on the LC column. Negative polarity ionization takes place in the source of the MS. Mobile phase modifiers such as ammonium acetate or ammonium hydroxide can lead to ionization in solution, enhancing ESI signals, although column retention can decrease as a result. Hydrophobic interaction liquid chromatography (HILIC) can be used as an alternative separation technique, where anionic NAs in a high organic mobile phase are selectively retained, reversing elution order, and providing the benefits of ion formation before entering the source, enhancing the response (Hindle et al., unpublished). HILIC requires longer equilibration times between injections and is sensitive to method parameters such as solvent strength in the vial and injection volume (Figure 5).

2.6. Calibration Standards

Quantitative analytical methods use authentic reference standards of high purity for each compound to be measured. For example, the Canadian Water Quality Guidelines list twelve PAHs that require monitoring for the protection of aquatic life [36]. PAHs are well defined, limited in number, and commercially available. Many isotopically labelled analogs are also available that can be used as internal standard (ISTDs). The analysis is straightforward: a series of calibration standards is prepared at various concentrations, the response versus concentration is plotted for each compound, and sample concentrations are calculated from the generated calibration curves.
Measuring the concentrations of NAs in OSPW is complex. Approximately 3000 elemental compositions have been found in OSPW samples from the oil sands in northern Alberta using LC with Orbitrap HRAM/MS, including O1-6, NO1-4, SO1-4, NO2S, N, and S [27]. Each chemical formula can further be expanded into a large number of combinatorial structural isomers [37] which do not separate well chromatographically. Commercial mixtures of calibration standards of this complexity are not available, and NA mixtures that are available are not representative of the wide variety of OSPWs. Model compounds representing various chemical classifications may provide a means for semi-quantitative analysis only.
The ‘Naphthenic Acids Strategies Workshop’ (Saskatoon, Canada, 24–25 November 2011) was held with the goal of reviewing the different routine methods used by laboratories for measuring, classifying, and quantitating NAFCs. Researchers from Canadian provincial and federal governments, universities (including one from the United Kingdom), and commercial laboratories were invited. The forum was also used to gain consensus on chemical classification terms for describing what was being measured, i.e., classical naphthenic acids (cNAs) and the naphthenic acid fraction compounds (NAFCs). Each lab gave a presentation outlining their acquisition approach and quantitation method and the findings were published [9]. Most of the review was concerned with the qualitative characterization of NAFCs as the first step in defining what was being measured.
The following observation was made in the workshop review and is still true today, twelve years later: “The need for the development of target analyses using authentic standards is evident as quantification of NAFC expressed as total NAs is currently at best, semi-quantitative” [9]. No OSPW-derived reference standard has yet been produced for general availability. The consensus from the workshop was that the NAFC fraction from OSPW was too complex for a single technique, and that a working group should be formed to develop protocols for standardized analyses, with interlaboratory studies designed to show conformance of results.
In studying the impact of temperature, pH, and salinity changes on partitioning of NAs [22], 55 model NAs were selected based on their identification by other researchers [27,38], including compounds from the following types: thiophene, indane, tetralin, cyclohexane, and adamantane. A comprehensive list of compounds such as this that spans a variety of elemental compositions may provide representative response factors that differ due to chemical structures. However, as noted earlier, OSPW from different sources contains different compounds. A selection of 39 model NAs showed up to 26-fold differences in response factors using LC/TOF [32], indicating that a one-size-fits-all approach to reference standards is problematic. The use of a commercially available Merichem NA mixture was recommended for semi-quantitation of classical NAs, due to the predominance of O2 acids.

2.7. Ionization Mode for Detection of the Analytes

Selection of the mass spectrometer ion source parameters is based on the chemical nature of the targeted NAFCs for any particular experiment. NAFCs and sulfonic acids in OSPW are typically analyzed with negative polarity due to the ready formation of anionic precursor ions, while many nitrogen-containing compounds can be preferentially ionized with positive polarity due to the basic nitrogen within the molecule. In some cases, more than half of the NAFCs extracted from OSPW at pH 1.5 are not classified as naphthenic acids [23]. Thus, multiple ionization techniques are needed for full NAFC characterization.
Three common ionization sources are available to many analytical laboratories: (1) electrospray (ESI); (2) atmospheric pressure chemical ionization (APCI); and (3) atmospheric pressure photoionization (APPI). Polar compounds such as carboxylic acids and amines are typically ionized using ESI, where the compounds are ionized either in the mobile phase due to selective pH, or within the ion source, often in the presence of mobile phase additives to enhance the ionization process. Non-polar organics such as ketones and aldehydes can be analyzed by APCI [38], where a charge is induced on protic solvent molecules such as methanol, which then transfer the charge as a reagent gas to the analytes. APPI can be used for the analysis of non-polar aromatic compounds such as PAHs by using photoionization to directly ionize compounds when their ionization potential is lower than the energy of the APPI lamp [39,40]. When the ionization potential of compounds is higher than the lamp, either the solvent or added dopant molecules (e.g., acetone or toluene) can be ionized, and this is followed by charge transfer to the target compounds. Both APCI and APPI can produce radicals as well as charged adducts [38].
To demonstrate the need for additional ionization techniques and polarity, Barrow et al. compared both positive and negative polarities using ESI and APPI sources (Figure 6) [26]. The negative ion mode showed that most ions are in the 190–450 amu range, while the positive ion mode included ions up to 700 amu. The distribution within those ranges also changed along with the number of peaks. A greater number of peaks was observed in the positive-ion mode than negative-ion mode, and by APPI than ESI. The APPI positive-ion mode showed approximately 19,000 peaks.
Some overlap and distinct differences exist in the ionization efficiencies of compounds using different ionization methods and polarities. Pereira et al. showed that compounds with the same empirical formula eluted with different chromatographic retention times in positive and negative mode ESI [27], indicating the presence of different compounds. Using ESI+, 162 and 85 compounds were identified in OSPW produced from surface mined and in situ extracted bitumen, respectively, that were not detected by ESI-. The O2+ species were determined to be dihydroxy, diketo, or ketohydroxy groups based on MS2 and MS3 fragmentation and similar behaviour of model compounds. The authors noted that some sex hormones, such as testosterone, androstenedione, and 5-androstenediol, belong to these chemical classes and that OSPW has endocrine-disrupting effects. ESI does not ionize these functional groups in either positive or negative polarities. Thus, the analysis of OSPW for possible toxic compounds should include additional ionization techniques. Matrix effects, especially ion suppression, are a well-known phenomenon of ESI. Ion suppression with reverse-phase chromatography is most evident in samples containing high salts and other hydrophilic compounds. LC separations can help reduce salt-induced ion suppression compared to infusion by retaining organic compounds on the column while the non-retained salts are directed to waste. However, suppression effects are often evident due to other co-eluting compounds. APCI and APPI are less prone to ion suppression than ESI.

2.8. Naphthenic Acids vs. Acid Extractable Organics

Ultrahigh-resolution electrospray ionization FT-ICR MS was used to characterize various OSPWs that contained between 1 and 7% sulfur, as determined by elemental analysis [23]. MS spectra contained up to 1880 peaks, with over 99% of the ions between 145 and 600 amu. Of these, less than 20% were determined to be cNAs, and less than half of the total abundance was due to cNAs plus oxy-NAs, indicating that >50% of the extracted material was not naphthenic acids. The authors, therefore, proposed replacing the term “naphthenic acids” with “oil sands tailings water acid-extractable organics (OSTWAEO)” which has subsequently been replaced by the simpler phrase acid-extractable organics (AEO) or NAFCs [9].

2.9. Mass Resolution of the Mass Spectrometer Instrument

Mass resolution is defined as the mass of an ion divided by the mass width at half height of the spectral peak (Equation (2)) and is a measure of how well two similar masses are separated in a spectrum. Accurate mass instruments such as Orbitraps and TOFs provide much higher mass accuracies than unit mass quadrupoles due to their higher mass resolutions.
R = m / ∆ m
Equation (2) is a formula for determining mass resolution of a mass spectrometer, where m is the mass of an ion, and ∆ m is the mass width at half height.
Mass resolution for TOF instruments using the formula in Equation (2) is defined when two peaks have equal height and width. When the peak height ratio is 100:1, the resolution may need to be 10× higher than calculated, since the mass at lower abundance will otherwise be included in the base of the more abundant ion [29]. High-resolution accurate mass (HRAM) instruments such as FT-ICR, TOF, and Orbitrap are, therefore, better suited to chemical characterization of NAFCs than unit mass quadrupoles. Their relatively high cost of purchase, maintenance, and operation preclude them from use in many labs. They are, however, required for elemental speciation and separation of OSPW into compound classes due to their ability to produce accurate mass assignments to the fourth or fifth decimal place. Mass accuracy at this level is required for unambiguous assignment of chemical formulae to unknown compounds. Correct formula assignments typically require mass accuracies of ±5 ppm when restricting the formula to C, H, O, N, and S in the molecules. This forward calculation between two chemical formulae provides straightforward mass error calculations. However, since mass spectrometers measure accurate masses, the calculation must be carried out in reverse, i.e., start with an accurate mass and use it to determine the chemical formula. Constraints for the type and number of elements that are included in the calculation are used to limit the number of possible formulae. For example, if the mass of an ion was measured to be 281.1763 (mass error −1.7 ppm for C16H26O4), there are six possible formulae when C, H, O, N, and S are allowed, with mass error up to ±10 ppm, (Table 3). Using an MS with mass accuracy known to be ±5 ppm, only the top three formulae would be possible. The third ion formula (C17H21N4) would not be forbidden but is unlikely to be an anion. If mass accuracy was known to be ±2 ppm, neither of the first two formulae could be dismissed but the third would be eliminated. Adding an LC separation to the analysis and using model compounds with known formulae and retention times may separate isobaric compounds (i.e., compounds with the same unit mass) and add more confidence to otherwise ambiguous compound assignments.

2.10. Use of Internal vs. External Standard

Internal standards (ISTDs) are compounds of similar chemical structure to the target compounds that are not present in the sample matrix. Preferred ISTDs are isotopically labelled analogs of target compounds that contain isotopes such as 2H (D, deuterium), 13C, 15N, and/or 17O. ISTDs can correct for a multitude of instrumental variances and their use increases the robustness of analytical methods. They are often used to correct for ion suppression in LC/MS applications, where co-eluting compounds may suppress the response of target analytes relative to their response in pure solvent and are very effective when each target has its corresponding ISTD analog. However, in the case of complex mixtures such as OSPW, this is not possible. Figure 7 illustrates that using a single ISTD may only correct for ion suppression within a small portion of a chromatographic run (R. Hindle, Vogon Labs, unpublished data). The Total Ion Chromatogram (TIC) is the sum of all MS responses at a given time. The spectrum captured at 4.12 min, the apex of the TIC, is much more complex than the spectrum at 6.85 min, where myristic acid-d27 elutes. As greater ion suppression is assumed at the earlier retention time, the single ISTD does not make a proportional correction for all components. A single ISTD may be more useful as a measure of instrument performance [33] between injections within a batch and over a longer period of time [41].
When comparing external standardization (ESTD) to ISTD within the same set of experiments, Huang et al. showed that ESTD required the use of liquid–liquid extraction (LLE) at high pH to remove the interfering matrix before quantitation [43]. However, this resulted in an underestimation of higher Ox compounds that partitioned into DCM. Addition of myristic acid-13C1 as an ISTD avoided the necessity of LLE cleanup prior to analysis and showed that either technique was suitable for cleaner matrices such as groundwater. For ESTD, calibration curves are prepared by analyzing a series of different concentrations of the target compounds, either in pure solvent, an analyte-free matrix (matrix blank) that has been taken through all the method steps, or a simulated matrix when no suitable matrix blank is available. Sample background interferences must be considered closely so that cleanup methods remove as many interferences as possible.

3. Conclusions and Recommendations

Ensuring consistency of data among laboratories has always been a cornerstone of effective analytical methods. The methods do not necessarily have to be prescriptive, where specified columns, mobile phases, or analytical instruments are mandatory for a given end use. Performance based methods allow a wider range of choices by individual laboratories while still providing confidence in the data as long as critical objectives such as compound specificity, sensitivity, and reproducibility are met. In the case of the complex mixtures of NAFCs, seemingly different data from the same extract can be obtained unless due care is given to key factors [17]. Although thousands of structures are present in OSPW, the focus should be the classical NAs as these components are implicated as primary toxicants [7]. However, further studies are warranted to determine the fractions within the classical NAs that are most bioavailable and toxic according to carbon number and double bond equivalence. Such studies are needed to help guide the development of NA guidelines for the protection of aquatic environments. In the case of labs providing classical NA data for regulatory purposes, the following recommendations are made for the listed categories that were detailed in this review paper:
  • Choice of extraction solvent: If using liquid–liquid extraction, adjust the aqueous pH to ≤1.5 and extract with DCM.
  • Salt content of sample: Partition cNAs into a water-immiscible solvent such as DCM or use a wash step with SPE cleanup to avoid salt in the final extract. Use LC and discard the early eluant to waste before directing column eluant to the MS source.
  • pH of extraction: Classical NAs have pKa values that range from 3.5 to 6.8. When partitioning into the organic phase, use pH ≤ 1.5. When partitioning into the aqueous phase, use pH ≥ 8.8. A narrow range will reduce the extraction of additional unwanted compounds.
  • Sample cleanup: Perform LLE or SPE cleanup prior to MS analysis to reduce interferences and minimize ion suppression.
  • On-line chromatography: Use LC separation to reduce interferences, minimize ion suppression, and add a time dimension to compound resolution.
  • Calibration standards: Use Merichem® mixture or concentrated NAFC extract from OSPW in acetonitrile.
  • Ionization mode: For cNAs, use electrospray negative-ion mode.
  • NAs versus NAFCs: Classical NAs are a subset of NAFCs. Use method parameters to focus on recovering and measuring only cNAs.
  • Mass resolution: The minimum resolution should be 25,000 when using an LC separation. The infusion should be 50,000 for previously characterized OSPW, and 100,000+ for unknowns.
  • Use of internal standard: use an ISTD to monitor instrument performance.

Author Contributions

Writing—original draft preparation, R.H.; writing—review and editing. R.H., D.G.M. and J.H. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Genome Canada through a Large Scale Applied Research Project (LSARP) grant (grant number 18207), in partnership with Genome Alberta and Genome Quebec.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest

The authors declare no conflict of interest.

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Separations 10 00583 g002
Figure 2. O2 DBE plots from ESI- Orbitrap (top) and FT-ICR MS (bottom) data using acidic (brown) and basic (green) conditions [17].
Figure 2. O2 DBE plots from ESI- Orbitrap (top) and FT-ICR MS (bottom) data using acidic (brown) and basic (green) conditions [17].
Separations 10 00583 g002
Separations 10 00583 g003
Figure 3. Summary of liquid–liquid extraction of NAFCs from OPSW.
Figure 3. Summary of liquid–liquid extraction of NAFCs from OPSW.
Separations 10 00583 g003
Separations 10 00583 g004
Figure 4. Effect of solvent on FT-ICR MS abundances of z-series NAs for DCM/acetonitrile, Milli-Q water/acetonitrile, and 1-octanol/acetonitrile for O2-NAs (a,c,e) and O4-NAs (b,d,f) [19].
Figure 4. Effect of solvent on FT-ICR MS abundances of z-series NAs for DCM/acetonitrile, Milli-Q water/acetonitrile, and 1-octanol/acetonitrile for O2-NAs (a,c,e) and O4-NAs (b,d,f) [19].
Separations 10 00583 g004
Separations 10 00583 g005
Figure 5. Effects on chromatography on fatty acid separation (C8 to C24) resulting from reduced injection volume on HILIC LC column [35].
Figure 5. Effects on chromatography on fatty acid separation (C8 to C24) resulting from reduced injection volume on HILIC LC column [35].
Separations 10 00583 g005
Separations 10 00583 g006
Figure 6. Comparison of negative- and positive-ion FT-ICR spectra of OSPW with ESI and APPI ionization [26].
Figure 6. Comparison of negative- and positive-ion FT-ICR spectra of OSPW with ESI and APPI ionization [26].
Separations 10 00583 g006
Separations 10 00583 g007
Figure 7. Total ion chromatogram (TIC; abundance = 6 × 107) of OSPW and myristic acid-d27 ISTD (abundance = 6 × 104) with corresponding spectra (both spectra at the same scale) at apex of peaks [42].
Figure 7. Total ion chromatogram (TIC; abundance = 6 × 107) of OSPW and myristic acid-d27 ISTD (abundance = 6 × 104) with corresponding spectra (both spectra at the same scale) at apex of peaks [42].
Separations 10 00583 g007
Table 1. Factors affecting analytical results of classical NA quantitation methods (annotations refer to original publications) [16].
Table 1. Factors affecting analytical results of classical NA quantitation methods (annotations refer to original publications) [16].
No.FactorConclusions
1Definition of total NAsUse the classical definition of NAs
2Extraction phase, pH, temperatureLiquid–liquid extraction at pH 2 and room temperature with DCM as organic phase, or use ENV + SPE
3Use of surrogate standardsUse isotopically labelled model compounds as surrogate standards
4Minimum resolving power of instrument50,000 at m/z 200, acknowledging that potential interferences contribute to method uncertainty
5Use of derivatizationDo not utilize derivatization
6Polarity and mode of ionizationNegative-ion-mode ESI
7Suitable calibration standard and internal standardUse commercially available Merichem NA mixture and at least one isotopically labelled internal standard
8Use of on-line or off-line fractionation of sampleEmploy on-line chromatography prior to MS detection
Table 2. Spectrum abundances by FT-ICR-MS (ESI-) on model NAs with acidic and basic pH conditions [17].
Table 2. Spectrum abundances by FT-ICR-MS (ESI-) on model NAs with acidic and basic pH conditions [17].
CompoundAdditiveIntensityApparent pH
Biphenyl-4-carboxylic acid0.1% NH4OH5.34 × 10108.91
C13H10O20.1% HCOOH4.79 × 1083.52
Anthraquinone-2-carboxylic acid0.1% NH4OH5.46 × 10109.19
C15H8O40.1% HCOOH3.31 × 1093.68
Trimesic acid0.1% NH4OH2.81 × 10108.75
C9H6O60.1% HCOOH1.86 × 10103.59
Table 3. Mass accuracies of chemical formulae of ions within 10 ppm of m/z measured at 281.1763 and the mass resolution that would be required to separate that chemical species from the observed mass.
Table 3. Mass accuracies of chemical formulae of ions within 10 ppm of m/z measured at 281.1763 and the mass resolution that would be required to separate that chemical species from the observed mass.
Ion Formulam/z (Calc)Diff (ppm)Resolution
C9H25N6O2S281.1765 0.8 1,406,000
C16H25O4281.1758 −1.7562,000
C17H21N4281.1772 3.1 312,000
C11H27N3O3S281.1779 5.5 176,000
C14H23N3O3281.1745 −6.4156,000
C19H23N O281.1785 7.8 128,000
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Hindle, R.; Headley, J.; Muench, D.G. Pros and Cons of Separation, Fractionation and Cleanup for Enhancement of the Quantitative Analysis of Bitumen-Derived Organics in Process-Affected Waters—A Review. Separations 2023, 10, 583. https://doi.org/10.3390/separations10120583

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Hindle R, Headley J, Muench DG. Pros and Cons of Separation, Fractionation and Cleanup for Enhancement of the Quantitative Analysis of Bitumen-Derived Organics in Process-Affected Waters—A Review. Separations. 2023; 10(12):583. https://doi.org/10.3390/separations10120583

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Hindle, Ralph, John Headley, and Douglas G. Muench. 2023. "Pros and Cons of Separation, Fractionation and Cleanup for Enhancement of the Quantitative Analysis of Bitumen-Derived Organics in Process-Affected Waters—A Review" Separations 10, no. 12: 583. https://doi.org/10.3390/separations10120583

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