Supercritical fluid chromatography for the analysis of oxygenated polycyclic aromatic compounds in unconventional oils

Journal of Chromatography A, 1589 (2019) 162–172

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Supercritical fluid chromatography for the analysis of oxygenated polycyclic aromatic compounds in unconventional oils Josephine S. Lübeck, Linus M.V. Malmquist, Jan H. Christensen ∗ Analytical Chemistry Group, Department of Plant & Environmental Sciences, University of Copenhagen, Thorvaldsensvej DK-1871 Frederiksberg C, Denmark

a r t i c l e

i n f o

Article history: Received 4 September 2018 Received in revised form 21 December 2018 Accepted 24 December 2018 Available online 26 December 2018 Keywords: Supercritical fluid chromatography Unconventional oils Oxygenated polycyclic aromatic hydrocarbon PAH Column screening Modifier additives Method optimisation Homologous series

a b s t r a c t Unconventional oil feeds can be rich in oxygenated organic compounds that will negatively affect the fuel properties if they are not removed during refining. In this study, supercritical fluid chromatography (SFC) was utilised for the combined analysis of polycyclic aromatic hydrocarbons (PAHs) and oxygenated polycyclic aromatic compounds (OPACs). One objective was to chromatographically separate PAHs from OPACs; another to reach a high peak capacity, improved peak shapes and high signal-to-noise ratios (S/N) for OPACs. These objectives were set to establish a non-target analysis method for oxygenated compounds in unconventional oils by SFC hyphenated to a UV detector and a quadrupole time-of-flight mass spectrometer (QTOF-MS) with negative electrospray ionisation (ESI− ). Highest peak capacities were observed with a 2-picolylamine column with methanol as modifier, however, a better resolution and S/N were obtained with ethanol and 0.1% formic acid. The elution order for OPACs on all columns followed mainly the polarity of the analytes: furans < aldehydes ≤ ketones < phenols ≤ carboxylic acids. Best separation between PAHs and OPACs was achieved with the ethylene-bridged silica column. The optimised SFC-UVESI− -QTOF-MS method was tested on a coal tar middle distillate and a pyrolysis oil where a number of homologous series (e.g. hydroxy-naphthalenes and –benzaldehydes) was tentatively identified. © 2019 Elsevier B.V. All rights reserved.

1. Introduction A key priority of the Europe 2020 agenda is to reduce greenhouse gas emissions by at least 20% while renewable energy consumption is to be increased by 20% [1]. This includes a step back from fossil fuels towards alternatives such as pyrolysis oils. However, liquefied fuels and refinery petroleum products, from for example coal tar, are still of interest and will remain so for several decades to come [2]. These unconventional oils need an extensive refining process to be used as transport fuels as they contain high proportions of heteroatomic compounds such as compounds with phenol- and amide/amine-functional groups [3]. The oxygen content in unconventional oils varies substantially depending on the feedstock and process conditions. Comparing an uncut crude oil (0.05–1.5 wt% oxygen), liquefied coal (0.5–6.4 wt%) and woodbased pyrolysis oil (11.2–46.9 wt%), the latter has by far the highest oxygen content [4–7]. Oxygen-containing compounds are reactive, can lead to instability at high temperatures of the feedstock (e.g. bio-oils are unstable above 100–150 ◦ C) and are in general

∗ Corresponding author. E-mail addresses: [email protected] (J.S. Lübeck), [email protected] (J.H. Christensen). https://doi.org/10.1016/j.chroma.2018.12.056 0021-9673/© 2019 Elsevier B.V. All rights reserved.

immiscible with petroleum [8–10]. Chemical characterisation and identification of oxygen-containing species in a feedstock prior to upgrading is therefore useful for optimisation of the upgrading process as different compound classes affect the processing in various ways. As a by-product of coal carbonisation, coal tar is a complex but highly valuable mixture containing a wide range of compounds dominated by polycyclic aromatic hydrocarbons (PAHs) and oxygenated polycyclic aromatic compounds (OPACs) [5,11,12]. The most abundant OPACs in coal tar are phenols (0.065 mol/100 g), methoxylated OPACs (0.035 mol/100 g) and carboxylic acids (0.005 mol/100 g) but also carbonyl compounds (i.e., ketones and aldehydes) and furans have been reported [7,13]. Plant-based biomasses contain large carbohydrate polymers as lignin and cellulose that generate oxygenated poly- and monomers such as methoxyphenols during the pyrolytic process [6,14]. Few standardised and validated procedures for the combined analysis of PAHs and OPACs in various matrices exist in the literature [8,15–18]. Separation of oxygenated compounds from hydrocarbons remains a difficult task due to the large number of structurally related compounds. Gas chromatography (GC) with mass spectrometry (MS) or flame ionisation detection (FID) has been employed most commonly for the analysis of volatile and semi-volatile oxygenated compounds in bio-oils, liquefied

J.S. Lübeck et al. / J. Chromatogr. A 1589 (2019) 162–172

coal products [7,13,19] and in environmental samples [15,17,18]. Comprehensive two-dimensional GC with FID and MS detection (GC × GC-FID/MS) is state-of-the-art for separation, detection and identification of volatile organic compounds in unconventional oils [7,12,13]. Although, GC × GC improves the peak capacity with a factor of 10–100 compared to GC, still only GC-amendable compounds with a sufficient volatility are covered by the analysis unless a prior derivatization reaction is performed as done by Kristensen et al. [20]. A more complete characterization of OPACs in unconventional oils can be performed by direct liquid injection ultra-high resolution MS (e.g., Fourier transform ion cyclotron – FTICR-MS) with complementary ionization techniques such as electrospray (ESI), atmospheric pressure chemical ionization (APCI) and photoionization (APPI) [21,22]. This method also covers non-volatile compounds but without separation of isobaric compounds unless combined with a reversed phase liquid chromatography (RPLC) separation. Hence, a set of complementary analytical methods are required to fully characterize the chemical complexity of unconventional oils. Meyer et al. [18] combined GC-FID, -MS, and high-performance LC (HPLC)-UV for the characterisation of PAHs and O-, N- and Scontaining PAHs in soils. Comprehensive two-dimensional liquid chromatography (RPLC × RPLC-UV) was used to separate compounds in a partly dehydroxygenated bio-oil detecting more than 120 compounds, but the lack of MS detection made reliable identification impossible [10]. Supercritical fluid chromatography (SFC) in normal-phase mode is complementary to RPLC separations and is especially promising for the separation of non-volatile organic compounds in oily matrices as samples can be injected in non-polar solvents. Furthermore, SFC can provide high peak capacities due to the combination of liquid-like (density) and gas-like (diffusivity and viscosity) properties. It is a highly efficient chromatographic platform allowing for higher linear velocities than in LC due to faster diffusion and low viscosities [23]. Unlike normal-phase LC, SFC can be easily coupled to MS and the use of hazardous solvents such as hexane or chloroform can be omitted. Thus, it has the potential to be employed for hydrocarbon group-type separation and for non-targeted analysis of semi-polar and thermolabile compounds as analyses typically run at temperatures between 25 and 50 ◦ C in SFC [24–26]. Additionally, compounds that do not readily vaporise due to their polarity and/or size can be analysed with SFC in contrast to GC. Recently, a few SFC methods have been developed to separate diverse types of OPACs in petroleum products, coal, bio-oils [24,27,28], and more recently in lignin samples [14]. The objective of this study was to establish a non-targeted SFCUV-ESI− -MS method for oxygenated compounds in unconventional oils. First, a suitable column and modifier combination was determined that would lead to the separation of PAHs and OPACs with a high peak capacity and reasonable peak shapes. Further, the effects of gradient steepness and modifier additives on peak capacity and sensitivity were optimised. Finally, the optimised method was tested on a coal tar middle distillate and a pyrolysis oil to separate and detect homologous series of OPACs.

2. Materials and methods 2.1. Reagents, chemicals and samples In Table 1, PAHs (n = 18) and in Table 2, OPACs (n = 20) are listed including structures and selected properties (see Table S1 for vendors). The PAHs were prepared in dichloromethane (DCM, HPLC grade, Rathburn Chemicals, Walkerburn, Scotland) and mixed (PAH mixture) to obtain a nominal concentration of 4 ␮g/mL of each compound. OPAC stock solutions (T-one: 900 ␮g/mL, the

163

Table 1 Model compounds: PAHs. Log Pa

Label

128.063

3.45

a

C12 H8

152.063

4.26

b

Acenaphthene

C12 H10

154.078

4.19

c

Fluorene

C13 H10

166.078

4.16

d

Anthracene

C14 H10

178.078

4.68

e

Phenanthrene

C14 H10

178.078

4.68

f

Fluoranthene

C16 H10

202.078

5.17

g

Pyrene

C16 H10

202.078

5.17

h

Benz(a)anthracene

C18 H12

228.094

5.91

i

Chrysene

C18 H12

228.094

5.91

j

Benz(k)fluoranthene

C20 H12

252.094

6.40

k

Benz(b)fluoranthene

C20 H12

252.094

6.40

l

Benz(a)pyrene

C20 H12

252.094

6.40

m

Benz(e)pyrene

C20 H12

252.094

6.40

n

Perylene

C20 H12

252.094

6.40

o

Indeno(1,2,3-c,d)pyrene

C22 H12

276.094

6.89

p

Dibenz(a,h)anthracene

C22 H14

278.110

7.14

q

Benz(g,h,i)perylene

C22 H12

276.094

6.89

r

Compound

Formula

Mass [g/mol]

Naphthalene

C10 H8

Acenaphthylene

a

Structure

LogP predictions from ACD labs.

rest: 100 ␮g/mL) were prepared individually in methanol (MeOH, LC–MS grade, Sigma Aldrich, St. Louis, MO, USA). All stock solutions, mixtures and dilutions were stored at −20 ◦ C. Carbon dioxide (50 L with dip tube, purity of 4.5) was provided by AGA (Copenhagen, Denmark). Isopropanol (PrOH), and MeOH ® were LC–MS grade (LC–MS Chromasolv ), ethanol (EtOH) was HPLC ® grade (Chromasolv for HPLC), (≥95%), formic acid (FA) LC–MS grade, all purchased from Sigma Aldrich (St. Louis, MO, USA). MilliQ water was produced in-house with a type I ultrapure water purification system from ELGA-Veolia LabWater (High Wycombe, UK). Haldor Topsøe A/S (Lyngby, Denmark) provided a coal tar middle distillate (0.25 mg/mL) and a pyrolysis oil (1.0 mg/mL), dissolved in DCM:MeOH (1:1). The samples were stored in the dark at −20 ◦ C until analysis. 2.2. Instrumentation The SFC was an Acquity UPC2 , and the MS system was a G2-Si Synapt HRMS, both from Waters Corporation (Milford, MA, USA). During method development, the splitter and MS connection were detached, thus, only UV detection (photo-diode array, PDA, Waters Corporation, Milford, MA, USA) was employed. The outlet from the PDA detector was connected directly to the automated back pressure regulator (ABPR). Backpressure was set to 150 bar and column temperature to 40 ◦ C. The analysis was run at 2 mL/min with 3 ␮L injection volume. The following parameters were optimised in this study and are described in the following section: stationary phase chemistry, mobile phase composition, gradient steepness and additive type. For the sample analysis, the MS was connected to the SFC via a double T-piece splitter which introduced MeOH as a make-up solvent at a flow rate of 0.3 mL/min and split the total flow before it entered the ion source. MS detection was conducted in scan mode (centroid) with a scan time of 0.5 s and a scan range between 50 and 1200 Da. Additionally, MS/MS spectra were collected for 15 parent masses that were tentatively identified. A list of parent ions served as input to the TOF-MRM mode of the mass analyser (Table S2).

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Table 2 Model compounds: OPACs. H-bond capacitya

Log Pb

156.056

1

2.87

C15 H10 O

206.073

1

4.10

3

C12 H6 O3

198.032

3

1.75

Phen-one

4

C15 H8 O

204.058

1

3.96

T-one

5

C10 H10 O

146.073

1

2.61

Acenaphthoquinone

AceQ

6

C12 H6 O2

182.037

2

2.28

9,10-Anthraquinone

AntQ

7

C14 H8 O2

208.052

2

3.38

9-Oxo-9H-fluorene-1-carboxylic acid

F-one-CA

8

C14 H8 O3

224.047

4

3.26

1-Naphthoic acid

Na-CA

9

C11 H8 O2

172.052

2

3.13

1-Pyrenecarboxylic acid

Pyr-CA

10

C17 H10 O2

246.068

2

4.85

3-Chrysene carboxylic acid

Chry-CA

11

C19 H12 O2

272.084

2

4.57c

Compound

Abbreviation

Label

Formula

Mass [g/mol]

1-Naphthaldehyde

Na-hyde

1

C11 H8 O

9-Phenanthrene-carbaldehyde

Phen-hyde

2

1,8-Naphthalic anhydride

Na-dride

4H-cyclopenta[def]-phenanthren-4-one 1-Tetralone

Structure

2-Carboxycinnamic acid

Cin-diCA

12

C10 H8 O4

192.042

6

2.09

Salicylic acid

Hy-Sa-CA

13

C7 H6 O3

138.032

5

2.06

1-Hydroxy-2-naphthoic acid

Hy-Na-CA

14

C11 H8 O3

188.047

5

3.29

1-Hydroxy-naphthalene

Hy-Na

15

C10 H8 O

144.058

2

2.71

9-Hydroxyphenanthrene

Hy-Phen

16

C14 H10 O

194.073

2

3.94

1-Hydroxypyrene

Hy-Pyr

17

C16 H10 O

218.073

2

4.43

1,7-Dihydroxynaphthalene

1,7Dihy-Na

18

C10 H8 O2

160.052

4

1.94

2,3-Dihydroxynaphthalene

2,3Dihy-Na

19

C10 H8 O2

160.052

4

2.11

Dibenzofuran

Dbf

–

C12 H8 O

168.058

1

4.12

Values retrieved from a PubChem, b ACD labs, c ChemAxon.

Capillary voltage was set to 1.5 kV, cone voltage to 15 V, scan time to 0.5 s, and the collision energy was ramped from 10 to 40 V. 2.3. Method development Three chromatographic columns were screened with modifiers of varying eluent strength to investigate the retentive behaviour of PAHs and OPACs in SFC. The Waters Acquity UPC2 columns (3.0 × 100 mm, 1.7 ␮m, Waters, USA) used, included: ethylenebridged bare silica (BEH); Torus 2-picolylamine (2-PIC) and Torus high-density diol (DIOL). The BEH column was supplied with an in-line pre-column filter and the 2-PIC and DIOL columns with VanGuard columns (2.1 × 5 mm) of the same stationary phase (Torus 2-PIC and DIOL, respectively). First, a 15-min isocratic run at 100% CO2 was run on all columns. Second, a scouting gradient was tested with 2 min isocratic hold at 100% CO2 , 2 min linear gradient to 20%B, hold for 6 min at 20%B, return to starting conditions within 0.5 min and hold for 4.5 min (run time: 15 min). Three organic modifiers were tested: MeOH, EtOH and PrOH (polarities: ␧◦ MeOH = 0.73 > ␧◦ EtOH = 0.68 > ␧◦ PrOH = 0.60 [29]). Experiments were run in triplicates in randomised order with mixtures of PAHs (4 ␮g/mL) or OPACs (10 ␮g/mL). 2.4. Method optimisation 2.4.1. Effect of gradient steepness After the chromatographic screening, several measures were taken to optimise the method for OPAC analysis. The modifier proportion (here: EtOH) was increased to 25%. In order to keep the system pressure below the upper pressure limit of 414 bar, the flow rate was reduced to 1.5 mL/min. The starting composition was

changed to 99:1 CO2 :EtOH to shorten equilibration times and to reduce retention time shifts of early-eluting compounds. The injection solvent was changed from MeOH to DCM:MeOH (1:1) to ensure the solubility of all components in oil samples and to reduce the polarity of the injection solvent and the competition for active sites of the stationary phase. For the method optimization, the injection volume was reduced to 2 ␮L. Three levels of gradient steepness were tested: 10, 2 and 1%B/min. The run time was extended accordingly (Figure S1). The experiments were run in duplicates (OPAC mixture: 5 ␮g/mL). 2.4.2. Effect of mobile phase additives Here, 5% water, 0.1% FA and a combination of both in EtOH were tested and compared to EtOH elution. The maximum %B was increased to 30% as well as the isocratic hold leading to a total run time of 40 min with a rate of 1%B/min. Analyses were performed in triplicates on the OPAC mixture (5 ␮g/mL). 2.5. Equations The following equations were employed for calculating selectivity (separation factor ˛), column plate number N, resolution R, summed peak capacities nsum [30,31] and peak production rates [32]. Seperation factor : ˛ =

kfirst−eluting OPAC klast−eluting PAH

(1)

where kfirst-eluting OPAC and klast-eluting PAH are the retention factors for the first-eluting OPAC and last eluting PAH, respectively. Column plate number : N = 16(

tR 2 ) w

(2a)

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165

Table 3 Final chromatographic method for the analysis of unconventional oils. Parameter

Value

Run time Mobile phase (A:B) Flow Rate Gradient (A:B)

40 min CO2:EtOH (0.1% FA) 1.5 mL/min Initial: 99:1 10.0 m in. 96:4 23.0 m in. 70:30 35.0 m in. 70:30 35.1 m in. 99:1 1.0 ␮L DCM:MeOH (1:1) 2-PIC (3 × 100 mm, 1.7 ␮m) 40 ◦ C 170 bar MeOH 0.3 mL/min ESI− 0.5 sec per scan

Injection Volume Injection Solvent Column Column Temperature ABPR Make-up solvent Make-up flow rate MS Ionisation type Scan time

N = 5.54(

Fig. 1. Log-normalised retention factors (log k) of PAHs on the 2-PIC (circles) and DIOL (triangles) columns compared to log k for the BEH (diamonds) column in 100% CO2 (isocratic conditions).

tR 2 ) w1/2

(2b)

where tR is the retention time and w the average peak width at the base (Eq. 2) and w1/2 at full width at half maximum (Eq. 2).



Peak capacity (sum) : nsum =

 +

¯ 0.5 N ln 4



tR,last,(III)

¯ 0.5 N ln 4





tR,last,(I) t0,(I)



+

tG ¯ w (3)

t0,(III)

¯ is the average column plate number, tR,last,(x) the retention where N time of the last-eluting analyte in the first isocratic hold (I), gradient (II) or second isocratic hold (III); t0 is dead time of the particular region; tG is gradient time. Correction factor : y = nsum (1 +

amiss −1 ) atotal

(4)

where nsum is the sum of peak capacities, amiss the maximum number of missing analytes (i.e., the worst case scenario where analytes did not elute with PrOH as the organic modifier) divided by the number of analytes in total, atotal (n = 19). Resolution : R =

tR2 − tR1 0.5(w1 + w2 )

(5)

where tR1 /w1 and tR2 /w2 are the retention times and FWHM of peak 1 and 2, respectively. Peak production rate : PPR =

n tR,last − tR,first

(6)

2.6. Final method The final method that was employed for the non-targeted analysis of unconventional oils was summarised in Table 3. 3. Results & discussion 3.1. Chromatographic screening 3.1.1. Elution of PAHs Log-normalised retention factors (log k) of PAHs in 100% CO2 are shown in Fig. 1 for all columns. In general, retention was lower on the BEH column (log kfirst =−0.66 to log klast = 0.25) as compared to the DIOL (log kfirst =−0.46 to log klast = 1.26) and 2-PIC column (log kfirst =−0.32 to log klast = 1.63). The retention on the two latter was similar to each other, however, with slightly stronger retentions on the 2-PIC column (Fig. 1). The elution order on all columns followed

Fig. 2. Base peak wavelength chromatograms for PAHs with a MeOH gradient of 0–20% from 2 to 5 min. The gradient is shown by the dashed line in the chromatogram for the 2-PIC column. Peaks: a – naphthalene, b – acenaphthylene, c – acenaphthene, d – fluorene, e –anthracene, f – phenanthrene, g – fluoranthene, h – pyrene, i – benzo(a)anthracene, j – chrysene, k – benzo(k)fluoranthene, l – benzo(b)fluoranthene, m – benzo(a)pyrene, n – benzo(e)pyrene, o – perylene, p – indeno(1,2,3-c,d)pyrene, q – dibenzo-(a,h)anthracene, r – benzo(g,h,i)perylene.

the increasing number of aromatic rings: 2- < 3- < 4- < 5- < 6-rings (Fig. 2) which is due to an increase in solute polarizability [33]. West et al. classified stationary phases for ultra-high performance SFC based on the linear solvation energy relationships (LSER) [33].They showed that the BEH, 2-PIC and DIOL column – and in general, polar stationary phases – are characterised by dipole-dipole interactions (positive e and s coefficients) and by hydrogen-bonding with acidic and basic molecules (positive a and b coefficients). According to West et al., the e coefficient values are higher for both 2-PIC and DIOL compared to the BEH column. That implies a stronger retention of molecules with an increasing number of aromatic rings and charge transfer interactions on the former two columns opposed to the latter. Lesellier separated 16 US EPA priority pollutant PAHs

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by coupling two RP-columns in series and using methanol or acetonitrile as organic modifier [34]. The same elution order of PAHs was described in the study by Lesellier, however, this was ascribed to the increasing degree of dispersion forces with the increase in molecular size [34]. Band broadening of late-eluting PAHs was also observed on the 2-PIC and DIOL columns when running isocratic at 100% CO2 (not shown). This was reduced by using a gradient (grey line in Fig. 2). The addition of an organic modifier in a linear fashion substantially decreased the retention of 5- and 6-ring PAHs as expected, e.g. log kisocratic = 1.63 vs. log kgradient = 1.15 for benzo(g,h,i)perylene using MeOH as modifier. The effect was larger with increasing polarity and eluent strength of the organic modifier, viz. MeOH > EtOH > PrOH. An example is given in Figure S2 which displays the decrease in retention on the DIOL and 2-PIC column when changing from PrOH to EtOH and/or to MeOH. Interestingly, the decrease in retention was also observed during the isocratic hold of 100% CO2 in the beginning of the run (peaks i to l and g to j on the DIOL and 2-PIC column, respectively, Figure S2). An explanation could be that modifier residues were not sufficiently removed from the column despite an equilibration time of 4.5 min at 100% CO2 in the end of each run. Replicates with the same modifier, however, did not express any retention shifts which implies reproducible results and that the effect is most probably due to the type of modifier affecting the retention of the target analytes. An opposite retention behaviour was observed for indeno(1,2,3c,d)pyrene (peak p) on the DIOL column (Figure S2) when changing from MeOH to PrOH. An explanation has yet to be found.

3.1.2. Elution of OPACs The elution of OPACs was studied on all columns and organic modifiers. Fig. 3 (Table S3 for retention times) shows the chromatographic behaviour of OPACs on the three columns, here with MeOH as modifier running the same gradient as for PAHs. Overall, the retention of OPACs was influenced by the analyte compound class, the modifier and the stationary phase. The elution order of the compound classes on the columns can be described as the following: furans ≤ aldehydes ≤ ketones < phenols ≤ carboxylic acids (Figure S3). Dibenzofuran (Dbf) was tested on the BEH column at a later stage of this study and was therefore not included in Fig. 3. It eluted at the same time as the co-eluting PAHs acenaphthylene and acenaphthene (tR = 0.7 min, peaks b and c in Fig. 2), representing the compound class of non-alkylated furans to elute before aldehydes. The elution order of OPACs with the same functionality followed the same pattern as for PAHs with respect to the number of aromatic rings, e.g. log k(Hy-Na) < log k(Hy-Phen) < log k(Hy-Pyr) (peaks 15, 16 and 17 in Fig. 3). Retention factors increased with higher hydrogen-bond capacities (Fig. 3), especially when comparing mono- with di-functional groups such as hydroxyl (e.g., Hy-Na, peak 15; 1,7Dihy-Na, peak 18) and carboxyl groups (e.g., NaCA, peak 9; Cin-diCA, peak 12). Furthermore, the isomers 1,7- and 2,3Dihy-Na (peaks 18 and 19) were retained very differently on all columns: while 1,7Dihy-Na displayed a good peak shape, 2,3DihyNa produced a broad and badly tailing peak (Fig. 3). It is speculated that the position of the hydroxyl groups on the same site results in very strong specific interactions opposed to hydroxyl groups that are positioned far away from each other. While the most polar compounds with hydrogen-bond capacities ≥5 (viz., peaks 12, 13 and 14, Cin-diCA, Hy-Sa-CA and Hy-Na-CA, respectively) eluted on the BEH column, they did not elute or were not observed on the DIOL and 2-PIC columns in the tested setup (except for Cin-diCA on the DIOL column). This observation may be explained by a strong retention and excessive band broadening on these columns. In general, this was described by the LSER parameters in West et al., i.e., that polar stationary phases like 2-PIC or

Fig. 3. Extracted wavelength chromatograms (EWC) for OPACs with a MeOH gradient of 0–20% from 2 to 5 min. The gradient is shown by the dashed line in the chromatogram for the 2-PIC column. Zoom-in on densely populated areas (grey boxes) between 3.5 and 5.0/5.5 min for BEH/ DIOL column. Peaks: 1 – Na-hyde (UV absorption maximum: 239 nm), 2 – Phen-hyde (245 nm), 3 – Na-dride (227 nm), 4 – Phen-one (229 nm), 5 – T-one (240 nm), 6 – AceQ (221 nm), 7 – AntQ (246 nm), 8 – F-one-CA (254 nm), 9 – Na-CA (220 nm), 10 – Pyr-CA (239 nm), 11 – Chry-CA (262 nm), 12 – Cin-diCA (265 nm), 13 – Hy-Sa-CA (233 nm), 14 – Hy-Na-CA (220 nm), 15 – Hy-Na (220 nm), 16 – Hy-Phen (244 nm), 17 – Hy-Pyr (238 nm), 18 – 1,7Dihy-Na (220 nm), 19 – 2,3Dihy-Na (220 nm).

DIOL express large a coefficients and thus interact strongly with acidic compounds like OPACs [33]. The effect of adding a modifier was most prominent on the BEH column displayed by the narrow elution window of the OPACs as compared to that of the other columns (Fig. 3). Organic modifiers mainly compete with the solute for active sites of the stationary phase, but also increase the solubility into the mobile phase [35]. Fig. 3 also displays deteriorated peak shapes of some early-eluting peaks (< 2.5 min), e.g. Na-hyde (peak 1) on BEH, Phen-hyde (2) and Phen-one (4) on 2-PIC, and AceQ (6) on both 2-PIC and DIOL. This could be linked to the choice of injection parameters. The bigger the difference in solvent properties between injection solvent and mobile phase starting composition, the more distorted the early-eluting peaks are. This injection solvent effect increases with increasing injection volume [23,29,36]. For example, AceQ (peak 6 in Fig. 3) shows signs of peak splitting on the DIOL column and tailing on the 2-PIC column. The solvent effect may be caused by temporarily adsorption of the injection solvent molecules to the silanol groups creating a local zone of elution strength [29,37]. In this experiment, a starting composition of 100% CO2 was applied. In literature, a mobile phase starting composition with a few percentages (1–3%) of organic modifier or a change in the nature of injection solvent is recommended in order to improve the peak shape of early-eluters [23]. In this study, however, a starting composition of 2% organic modifier had a detrimental impact (viz. earlier elution) on the resolution of the PAHs and OPACs. Therefore, the starting composition was set to 99:1 CO2 :modifier.

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Fig. 4. Average peak capacities with standard deviations (error bars), selectivities (␣) and the number of OPACs that did not elute (italic, in parentheses) for each column-modifier combination.

Carboxylated compounds were overall retained the strongest on all columns (Fig. 3). The large retention factors could be related to the larger hydrogen-bond capacities (as mentioned previously). These peaks were also tailing especially on the DIOL and BEH column despite eluting on the gradient. The pKa values of these OPACs are, e.g. 2.70 (Hy-Na-CA), 3.10 (Na-CA), 3.60 (Pyr-CA) and 3.98 (Chry-CA). These are below, but relatively close to the apparent pH of the mobile phase composition (pH = 4–5) [35]. Thus, these may be present in their dissociated and undissociated state simultaneously which could lead to secondary interactions, viz. ionic interactions with the stationary phase. The use of an acidic additive to lower the apparent pH is therefore recommended (see next section on method optimization). To evaluate the performance of columns and modifiers, peak capacities were calculated based on base peak widths (Eq. 3). This equation is the sum of peak capacities (nsum ) due to the used gradient programme: (I) isocratic hold for 2 min, (II) gradient for 3 min to 20%B and (III) isocratic hold for 6 min. This approach of summing the peak capacities has been used elsewhere [28]. Elution windows and thus information whether an analyte eluted in either one of these regions were based on the actual gradient time (viz. the sum of theoretical gradient time, dwell time, tD , and dead time, t0 ). A detailed description and which compound was used in which part of the calculation is provided in Table S4. Furthermore, a correction factor (Eq. 4) was applied to the calculated peak capacities in order to account for missing analytes since not all analytes eluted in all experimental conditions. Thus, amiss (the number of missing analytes) in Eq. 4 equals 2, 7 and 5 for the BEH, 2-PIC and DIOL column, respectively. Fig. 4 illustrates the results from a two-way ANOVA with replicates. Average base peak widths and peak capacities were based only on compounds that eluted for all types of modifier (viz. MeOH, EtOH and PrOH) on each individual column, i.e., 17 analytes were observed on the BEH column (missing Na-dride and Hy-Sa-CA); 12 for 2-PIC (missing 2,3Dihy-Na, Cin-diCA, Chry-CA, F-one-CA, Hy-Na-CA, Hy-Sa-CA and Pyr-CA); 15 for DIOL (missing 2,3Dihy-Na, Cin-diCA, Hy-Na-CA and Hy-Sa-CA) (Table S5). Some of these compounds (e.g. Pyr-CA on 2-PIC or Na-dride on BEH) were detected only with MeOH as modifier because EtOH and PrOH were not sufficiently strong eluents. However, a lower gradient steepness or higher maximum percentage of organic modifier could be employed to counteract that. Furthermore, the isocratic hold (I) was excluded from the peak capacity calculation because early-eluting analyte in that region featured deteriorated peak shapes (e.g., split peaks), low column plate numbers (N < 500) or base peak width > 0.250 min, e.g., Phen-one, Phen-hyde and AceQ (peak 2, 4, 6) for the 2-PIC and AceQ (6) for the DIOL column (Fig. 3). This analyte

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behaviour was not necessarily linked to the column performance. A combination of a high injection volume (3 ␮L) and high eluent strength of the injection solvent most likely were the source of the band broadening and could be improved. Including this region in the peak capacity calculations would lead to overoptimistic results, thus, it was not factored in to yield a more fair comparison of the columns. The number of missing analytes that did not elute or were not detected was added to Fig. 4 in parentheses. A separation factor (␣, Eq. 1) > 1 reveals a complete group-type separation of OPACs and PAHs. Separation factors < 1 express a partial separation of the two group types. Statistically significant differences for the corrected peak capacities (viz. f-value > fcritical -value; p-value <0.05) were observed between the three columns (p = 1.28 × 10−20 ) and modifiers (p = 2.88 × 10-15 ). Although almost all tested OPACs eluted on the BEH column with any modifier (between 0 and 2 OPACs did not elute in that setup), peak capacities were comparably lower with 15.9 ± 0.1 (MeOH), 14.3 ± 0.8 (EtOH) and 16.2 ± 0.0 (PrOH) for the different modifiers. This is due to the small elution window wherein all peaks eluted (time ranged between 2.51–4.29 min for MeOH to PrOH, Fig. 4). The 2-PIC column outperformed the other columns using any modifier with peak capacities ranging from 26.4 ± 1.3 for PrOH, 27.3 ± 0.6 for EtOH to 44.4 ± 1.2 for MeOH (Fig. 4). Here, the elution window was significantly larger with many analytes eluting first during the isocratic hold at 20% organic modifier. The higher retention factors could be linked to additional analytecolumn interactions with the 2-picolylamine ligand. Furthermore, the number of OPACs that did not elute was the largest for the 2PIC column (absence of 4 to 7 peaks) which may be mastered by increasing the proportion of modifier or by adding an appropriate additive (e.g., formic or trifluoroacetic acid) to the organic modifier. All peaks eluted in the first isocratic hold (I) or on the gradient (II) on the DIOL column. Peak capacities increased from 16.4 ± 0.4 for PrOH to 18.4 ± 0.1 for EtOH and 22.8 ± 0.5 for MeOH. An overlap of retention between PAHs and OPACs with carbonyl moieties was observed for both the 2-PIC and the DIOL column with ␣ = 0.05-0.06 and ␣ = 0.08-0.09, respectively (Fig. 4). A clear separation was observed on the BEH column between PAHs and OPACs for all modifiers with ␣-values between 1.44 and 1.51. Therefore, the BEH column could potentially be used for pre-fractionation and thus for the separation of hydrocarbons and oxygenated compounds in coal tar and pyrolysis oils. This straightforward and simple column screening does not do justice to each column. With the current gradient steepness, the 2-PIC column performs best overall despite the missing analytes. However, the use of a shallower gradient (e.g. 1%B/min) might have led to higher peak capacities on both the BEH and DIOL column. Likewise, increasing the maximum %B could potentially result in the elution of more compounds on the 2-PIC column. It would be interesting to extend the experiments on each column in future studies, but this was not the scope herein. Here, the decisive factor eventually was the span of the elution window over the whole analytical run due to diverse retention mechanisms which was largest on the 2-PIC column. Hence, the 2-PIC column was chosen and used for optimization to ensure the elution of late-eluting OPACs. 3.2. Method optimisation These first experiments were designed for a quick screening not accounting for the diversity and complexity of unconventional oils. Therefore, several measures were undertaken to optimise the method for complex samples. A splitter device (double T-coupling) using MeOH as a make-up solvent with 0.3 mL/min flow rate was installed. A first test run of a pyrolysis oil on the 2-PIC using ESI− MS, however, revealed a better resolution of early-eluters with the

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Fig. 5. Randomly chosen EICs of pyrolysis oil for MeOH (dashed red line) vs. EtOH (black solid line) as modifier. R1 to R6 represent calculated resolutions (Eq. 5) of the indicated critical peak pairs (identity unknown) for both modifiers (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 6. The relative changes [%] of analysis time (T), peak capacity (n) and peak production rate (PPR) as a function of gradient steepness (normalised to the individual responses at 1%B/min). EtOH was used as modifier with a maximum modifier content of 25%.

same m/z value (tR < 5 min) with EtOH rather than MeOH as organic modifier (Fig. 5). Therefore, EtOH was chosen as the modifier whilst increasing the gradient to 25%B. Furthermore, effects of gradient steepness and modifier additives were tested. To evaluate the performance of the experiments, a subset of ionisable OPACs (5 ␮g/mL) was used, including ten analytes: Hy-Na, Hy-Phen, Hy-Pyr, 1,7Dihy-Na, Na-CA, Chry-CA, Pyr-CA, F-one-CA, Hy-Sa-CA and Hy-Na-CA. Cin-diCA and 2,3DihyNa were excluded as they were not detected or were tailing under any condition. 3.2.1. Effect of gradient steepness A gradient steepness of 1, 2 and 10%B/min was tested (Fig. 6). Retention times and peak widths at FWHM were determined for the nine OPACs in the test mixture and used to calculate the effective peak capacity (Eq. 3, here, tG equals T = tR,last -tR,first , i.e., the difference between the retention time of last and first analyte), elution window (T) and peak production rate (PPR, Eq. 6) for each gradient steepness. Hy-Na-CA was excluded from these calculations as it did not elute with the lowest gradient steepness (1%B/min) due to strong retention. Longer gradient times tend to yield higher peak capacities which has been described elsewhere for RPLC [32] and was confirmed herein. Overall, peak capacities

and elution windows decreased, whereas the peak production rate first increased from 1 to 2%B/min and subsequently decreased from 2 to 10%B/min (Fig. 6). The peak capacity of 125.0 ± 0.6 at 1%B/min was reduced to 97.5 ± 5.9 and 43.9 ± 4.7 with 2 and 10%B/min, respectively. The peak production rate ranged between 6.8 ± 0.1, 7.3 ± 0.4 and 4.7 ± 0.4 peaks per minute, and elution windows spanned from 18.4 ± 0.2 min over 13.4 ± 1.5 min to 9.4 ± 1.7 min for 1, 2 and 10%B/min, respectively. Apart from the time saving aspect and eluting strongly retained compounds such as Hy-NaCA, a gradient steepness of 10%B/min delivered comparably the worst results in regards to peak capacity and peak production rate. Although 1%B/min produced the highest peak capacity, the peak production rate was lower as the one obtained with 2%B/min (Fig. 6). Additionally, as aforementioned, Hy-Na-CA did not elute within the investigated run time (Figure S1) with 1%B/min because the isocratic hold at 25% organic modifier was not sufficient to elute this compound from the column. There appears to be a trade-off between running at a shallow gradient steepness (1 or 2%B/min) by either extending the run time, increasing the maximum %B or adding an additive in order to achieve elution of the strongly retained compounds. 3.2.2. Effect of mobile phase additives In this study, 5% water, 0.1% formic acid (FA) and a combination of both in EtOH were tested as additives and compared to pure EtOH as modifier. By increasing the maximum %B to 30%, Hy-NaCA could be eluted even at 1%B/min and thus, ten ionisable OPACs could be investigated. The effect on individual compounds for peak width and S/N are shown in Fig. 7 as normalised to the response obtained with EtOH as modifier without any additive. Table S6 provides additional information for each OPAC such as retention time and full width at half maximum. The beneficial effects of additives on peak shapes in SFC have been described in detail elsewhere [23,38]. Briefly, additives aid the elution of polar compounds by competing for active sites on the stationary phase, changing the mobile phase polarity, and affecting selectivity. In this study, 5% H2 O and 0.1% FA had a positive effect on peak widths compared to no additives as peak widths improved on average by 10.0 ± 5.6% for 0.1% FA, 21.5 ± 13.6% for 5% H2 O and 24.7 ± 19.9% for 5% H2 O + 0.1% FA (Fig. 7.a). The improvement in peak width of carboxylated and hydroxyl-carboxylated compounds

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Fig. 8. Overlaid EICs of OPACs (5 ␮g/mL) measured with the final method using EtOH with 0.1% FA. Gradient profile (%B) is represented with dashed line. Compound identities: 1 – Hy-Na, 2 – Hy-Phen, 3 – Na-CA, 4 – Hy-Pyr, 5 – 1,7Dihy-Na, 6 – Chry-CA, 7 – Pyr-CA, 8 – 2,3Dihy-Na, 9 – Hy-Sa-CA, 10 – Hy-Na-CA.

be correlated with a more acidic mobile phase composition and facilitated ionisation. 3.3. Sample analysis

Fig. 7. Change [%] with 5% H2 O, 0.1% FA and 5% H2 O + 0.1% FA as mobile phase additive compared to EtOH alone. Data was normalised to EtOH without additive as modifier (set to 0%); (a) Peak width (positive change - narrower peaks); (b) S/N (positive change - higher S/N); (c) peak height (positive change - higher peaks). The axes have to be viewed independently. A gradient steepness of 1%B/min with a maximum modifier content of 30% was used.

was especially significant for the combination of water and FA, e.g., 50.2% and 59.0% for Hy-Sa-CA and Hy-Na-CA, respectively. The picture was more complex for S/N and peak height (Fig. 7b and c). The changes ranged far more and were also negative for some, e.g., Hy-Na, Hy-Phen or Na-CA. For FA, however, there was an overall significant improvement with two orders of magnitude for several analytes, including Hy-Phen (305 and 523% for S/N and peak height, respectively), Hy-Pyr (216 and 562%), Na-CA (483 and 479%), Hy-Sa-CA (625 and 838%) and Hy-Na-CA (386 and 498%). The effect of additives on S/N and peak height can be related to multiple factors. On one hand, additional protons from water and/or FA have an effect on the chromatography by adsorbing to free silanol groups and masking the few but strong sites that give rise to distorted peaks leading to altered retention and slimmer peaks compared to no additives (Fig. 7, Table S6). On the other hand, the ionisation efficiency is affected as protons assist the reduction in negativeion mode ESI. As described by Wu et al. [39], additional protons from acidic additives facilitate the accumulation of excess negative charges on the surface of spray droplets leading to an increase in pH at the surface of the droplet and hence, a favourable local environment for deprotonation of compounds. That was also observed herein with the overall highest responses for FA (Fig. 7). Adding both water and FA shows a comparable response as to having water as the only additive. Thus, the highest S/N and peak height could

The final method is shown in Table 3. A scouting gradient with gradient steepness of 0.3%B/min and 2%B/min using EtOH with 0.1% FA was chosen to extend the elution window of early-eluting and more nonpolar compounds further. The gradient steepness of 2%B/min was chosen over 1%B/min to speed up the analysis time without sacrificing a satisfying peak production rate (Fig. 6). The ABPR was set to 170 bar to improve the stability of the spray in the ion source during the analytical run [40]. Besides the OPAC mixture (Fig. 8), two unconventional oils (pyrolysis oil and coal tar) were analysed (Fig. 9). Fig. 8 shows that the peak shapes of compounds with mixedfunctional groups are broad and tailing (peaks 8–10). The upper system pressure limit of 414 bar did not allow any further increase for the maximum %B with the given flow rate. Decreasing the flow rate however was not tested. The analyte Cin-diCA with two carboxyl moieties was not eluted from the 2-PIC column and/or detected under any of the tested conditions which marks a limitation of the developed method towards compounds that are adsorbed too strongly onto the stationary phase. In future studies, it could be tested whether the addition of a higher concentration of additive and/or a stronger additive (e.g., TFA) and/or merely a higher proportion of organic modifier (e.g., 40–100%) would enable the elution of strongly retained compounds. Perhaps the combination of two different types of modifiers or additives in the same run could increase the elution window further if the instrumentation would allow it. Regarding the analysis of unconventional oil samples, most peaks were observed between 80 and 300 Da and eluted before 20 min (Fig. 9). By visual inspection and MS/MS analysis, some compounds and homologous series (viz. −CH2 -) could be tentatively identified using SIRIUS software 4.0 [41,42] and the database PubChem [43]. The most probable matches were outlined in Table 4. It comes as no surprise that all signals belong to phenols given that ESI− -MS is a highly sensitive ionization source for this compound class. Monoaromatic and polyaromatic phenols were identified for the pyrolysis oil and coal tar, respectively. Identification of the specific isomers was not possible, hence, the indication with ‘Cx - ‘for each peak group was used in Table 4. Methoxy- and hydroxybenzaldehydes (RT1 and RT2 for the pyrolysis oil, Table 4) have been described regularly in biomassbased pyrolysis oils originating from the degradation of lignocel-

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Fig. 9. Two-dimensional plots (tR × m/z) of pyrolysis oil (a) and coal tar (b), processed in Matlab R2016a and binned to nominal mass. The colour scale indicates the intensity (A.U.). Background ions (m/z 113, 132, 187, 219) that evolved from the instrumentation were removed. Homologous series are encircled in red (1–5). Refer to Table 4 for tentative IDs (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Table 4 Tentatively identified structures in pyrolysis oil and coal tar sample. Retention time [min]

Measured m/z [M-H]−

Theoretical m/z [M-H]−

Mass formula [M]

MS2 fragments (relative intensity)

Tentative structures

Tentative compounds

Pyrolysis Oil - RT1 1

1.7

179.0710

179.0708

C10H12O3

164 (1.0), 136 (0.2)

2

1.8

165.0551

165.0552

C9H10O3

150 (1.0), 122 (0.4)

3

2.0

151.0395

151.0395

C8H8O3

136 (1.0), 108 (0.2), 92 (0.2)

Pyrolysis Oil – RT2 1

9.0

163.0756

163.0759

C10H12O2

C3-Hydroxybenzaldehyde

2

9.0-9.4 ␣

149.0602

149.0603

C9H10O2

3

9.3-9.6 ␣

135.0446␤

135.0446

C8H8O2

4 Coal tar – RT1 1 2

9.7

121.0291␤

121.0290

C7H6O2

120 (0.1), 93 (1.0), 92 (0.6) 149 (0.6), 107 (0.4), 106 (1.0); 149 (0.6), 120 (0.4), 92 (1.0) 106 (0.1); 135 (0.8), 120 (0.3), 92 (1.0) 92 (0.4)

6.8-8.5 ␣ 6.9-8.8 ␣

185.0967 ␤ 171.0811 ␤

185.0966 171.0810

C13H14O C12H11O

C3-Hydroxynaphthalenes C2-Hydroxynaphthalenes

3 4 Coal tar – RT2 1

7.3-8.4 ␣ 8.2-8.9 ␣

157.0652 ␤ 143.0504 ␤

157.0653 143.0497

C11H10O C10H8O

170 (0.4-0.8)␥ 156 (0.05-0.9)␥ , 143 (0.05) 129 (0.1) 115 (0.2)

12.4-13.8

221.0971 ␤

221.0966

C16H14O

–

2

13.1-14.1

207.0806 ␤

207.0810

C15H12O

179 (0.1)

␤

193.0653

C14H10O

165 (0.2)

C2-Hydroxyanthracene/phenanthrene C1-Hydroxyanthracene/phenanthrene Hydroxyanthracene/phenanthrene

217.0653

C16H10O

189 (0.08)

3

13.5

193.0649

Coal tar – RT3 1

16.3

217.0667 ␤

␣ ␤ ␥

C2-Hydroxy-methoxybenzaldehyde C1-Hydroxy-methoxybenzaldehyde Hydroxy-methoxybenzaldehyde

C2-Hydroxybenzaldehyde

C1-Hydroxybenzaldehyde Hydroxy-benzaldehyde

C1-Hydroxynaphthalenes Hydroxynaphthalenes

Hydroxypyrene

Retention time window if several peaks were detected for that mass. Parent ion was also base peak ion. Relative intensity range of the detected peaks.

lulosic polymers [14,44,45]. Comparisons with Prothmann et al. [14] show that these monoaromatic structures likely elute in the given elution window on the 2-PIC column, e.g. vanillin (C8 H8 O3 ),

acetovanillone (C9 H10 O3 ), p-hydroxybenzaldehyde (C7 H6 O2 ) and –acetophone (C8 H8 O2 ). Coal tars (and their products) have been shown to be rich in PAHs and heteroatomic PAHs, including OPACs

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[11,13]. Hydroxylated OPACs in the tested sample could partly be confirmed by the elution of the respective standards within close proximity of the peaks in the samples (Table 4), incl. Hy-Na (tR = 8.7 min), Hy-Phen (13.8 min) and Hy-Pyr (16.2 min) (Fig. 8). There are many ways to achieve the best chromatographic separation of unconventional oils. All tested columns could have been employed for different reasons. The BEH column could be used as a fractionation tool to separate hydrocarbons and PAHs from a large fraction of the polar analytes if that was the primary aim of the separation. Using SFC for pre-fractionation before a GC × GC analysis was described elsewhere in detail [25]. However, the use of GC × GC would not enable the analysis of more polar components of unconventional oils. As seen in this study, the DIOL column would be able to elute compounds such as phenolic acids that were too strongly retained on the 2-PIC column with the same gradient steepness and modifier composition. Prothmann et al. [14] came to a similar conclusion showing closely related behaviours of the 2-PIC and DIOL column, but they decided for the DIOL column in the end due to better peak shapes of phenolic acids and a good general resolution. Herein, it was possible to elute aromatic acids as sharp peaks on the 2-PIC column under optimized conditions (viz. with 0.1% FA); and although the elution of phenolic acids (i.e., Hy-Sa-CA and HyNa-CA) was achieved in the current setup, peaks were rather broad. Furthermore, it is unlikely that 30% of organic modifier will wash off all strongly retained compounds even with the current additive at hand. Thus, either one could optimize towards a higher maximum percentage of organic modifier like it was done for pyrolysis oil SFC-UV/MS analysis in [28], add an even stronger and/or higher amount of additive or add a washing step of high proportion of organic modifier at a low flow rate like it was done in [46]. Although the composition of pyrolysis oil and coal tar are very different and although the set of analytes to develop this method was chosen mostly on the basis of what a coal tar contains, it was possible to develop a method that can perform satisfactorily for either matrix. The use of MS/MS adds additional value by allowing the identification of specific constituents and homologues series. 4. Conclusion A SFC-UV/MS method was developed for the simultaneous analysis of PAHs and their oxygenated derivatives. The use of environmentally friendly solvents (CO2 and ethanol), speed and efficiency emphasise SFC as a reasonable chromatographic alternative and partially as a replacement for GC and LC methods. SFC was able to elute PAHs within 5 min on all investigated columns for a concentration of 4 ␮g/mL. The normal-phase like behaviour of SFC with a polar stationary phase (2-PIC or DIOL) led to a strong retention of heteroatomic PAHs and showed the potential for group-type separation between carbonyl- and hydroxyl-/carboxyl-OPACs. Further, a bare silica column (BEH) could be utilised to fractionate PAHs and OPACs in unconventional oils because of its high separation efficiency. The use of an additive (water and/or FA) was shown to improve the signals of OPACs immensely when performing SFCESI− -MS. The optimised method (based on the optimal combination of organic modifier, column type, gradient steepness and additive) was used for the characterisation of oxygenated hydrocarbon species in two unconventional oil samples (pyrolysis oil and a coal tar). Here, homologous series of hydroxylated mono- and polyaromatic compounds were tentatively identified. Acknowledgements The authors would like to thank Victor Abrahamson for his kind support in finalising this paper. Haldor Topsøe A/S (Lyngby, Denmark) is acknowledged for providing samples of coal tar and

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