## 2 Critical Analytical Parameters
### 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 O\({}_{2}\) 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 O\({}_{2}\), O\({}_{3}\), and O\({}_{4}\) 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 O\({}_{2}\), O\({}_{3}\), and O\({}_{4}\) 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 pK\({}_{\rm a}\) values of the oxy-NAs to help understand their distribution in wastewater treatments. This revealed that, as oxygen numbers increased in the O\({}_{2}\), O\({}_{3}\), and O\({}_{4}\) series, pKa also increased (4.2, 6.2, and 7.7, respectively). Increases in pK\({}_{\rm a}\) indicate that the O\({}_{3}\) NAs are hydroxylated, and the O\({}_{4}\) NAs are more likely to be di-hydroxylated rather than di-carboxylic acids, which otherwise would have resulted in lower pK\({}_{\rm a}\) values (e.g., propanoic acid pKa = 4.87; propanediolic acid pK\({}_{\rm a1}\) = 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 (B4 CA) 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 \(\sim\)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 pK\({}_{\rm a}\), 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 pK\({}_{\mathrm{a}}\), an acid is 50% ionized. \[\mathrm{pKa-pH=log10\frac{[HA]}{[A-]}} \tag{1}\]  
Equation (1). Henderson-Hasselbalch equation describing the dissociation of weak acids in solution. Because the acidic mobile phase pH (3.52) is below the pK\({}_{\mathrm{a}}\) of B4 CA (4.19), B4 CA 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 (A2 CA), which has a pKa of 3.42.