Proﬁling of Polycyclic Aromatic
Hydrocarbons in Crude Oil with theAgilent 1290 Inﬁnity 2D-LC Solution

Application Note
Energy and ChemicalsAuthors

Abstract

Gerd Vanhoenacker, Frank David,

The Agilent 1290 Inﬁnity 2D-LC Solution was used to proﬁle the polyaromaticand Pat Sandrahydrocarbon (PAH) fraction from mineral oil using comprehensive two-dimensional

Research Institute for Chromatographyliquid chromatography (LCxLC). The complexity of this fraction, consisting ofKennedypark 26

nonsubstituted PAHs, alkyl-substituted PAHs, and heterocyclic PAHs, largely

B-8500 Kortrijk

exceeds the peak capacity of a one-dimensional LC separation.

Belgium

A combination of a cyanopropyl column in the ﬁrst dimension and a dedicated PAH

Udo Hubercolumn in the second dimension provided good orthogonality, resulting in higher

Agilent Technologies, Inc.

peak capacity. Detection was performed by parallel diode-array and ﬂuorescence

Waldbronn, Germany

detection. This Application Note shows the potential of 2D-LC for proﬁling thepolyaromatic fraction of mineral oils.

Introduction
Polycyclic aromatic hydrocarbons(PAHs) are well-known contaminants inthe environment and in food samples.
PAHs mostly originate from natural andanthropogenic combustion processes.
To date, most analytical methods forthe trace-level analysis of PAHs inenvironmental samples (soil, sediment,water, air) and in food samples (mostlyfatty foods), focus on a selected number(typically 16) of nonsubstituted polycyclicaromatic hydrocarbons, such as
ﬂuoranthene, chrysene, benzo(a)pyrene,and benzoﬂuoranthenes. Analyticalmethods are based on GC/MS (including
single quadrupole and triple quadrupoleMS) or on HPLC in combination
with diode-array detection (DAD) or
ﬂuorescence detection (FLD).
Carcinogenity of certain PAHs has beenunequivocally demonstrated and, similar
to polychlorinated dioxins and furans(PCDDs/PCDFs) and polychlorinatedbiphenyls (PCBs), toxicity equivalent
factors (TEF) are used to measure thetotal contamination of a sample by PAHs.
More recently, concerns have been raised
regarding the toxicity of alkyl-substituted
PAHs. Indeed, in petroleum products
such as diesel, mineral oils, and crudeoils, the contribution of substituted
PAHs to the total (poly)aromatic fraction
is much larger than the contribution of
the nonalkylated PAHs that are typicallyanalyzed. This fact is, for instance,
recognized by EFSA in their ScientiﬁcOpinion on Mineral Oil Hydrocarbons inFood1. For the analysis of mineral oil infood and in packaging material, GC-FID
methods are used after a preseparationof the saturated hydrocarbons (mineral oil
saturated hydrocarbons, MOSH) fraction
from the aromatic fraction (mineral oil
aromatic hydrocarbons, MOAH) using
solid phase extraction or normal phase
HPLC.
While measuring the MOSH fraction iswell documented, good characterizationof the aromatic fraction is still lacking.
This is contradictory with the factthat toxicity of the aromatic fraction
is substantially higher than that of thesaturated hydrocarbon fraction.

The aromatic and polycyclic aromatic
fraction contains nonsubstituted two
to six-ring PAHs, alkyl-substituted
PAHs, heterocyclic PAHs (for example,dibenzothiophene), alkyl-substituted
heterocyclic PAHs and, possibly, morepolar derivatives such as hydroxy-PAHs,
amino-PAHs, and nitro-PAHs. Due tothis high complexity, high resolution
separation techniques are needed. MOSH
and MOAH fractions are commonly
analyzed by GCxGC and GCxGC/MS, but
comprehensive LCxLC can be considered
as an excellent complementary technique,especially since the high molecularweight PAHs (six rings) can be easily
analyzed, and selective detection by
ﬂuorescence is very sensitive.
The combination of two separation
modes, such as a ring number separation
with a hydrophobicity separation, canbe particularly useful for proﬁling thearomatic fraction in oils. This Application
Note, illustrates the LCxLC approach
using the Agilent 1290 Inﬁnity 2D-LCSolution.

Instrumentation
An Agilent 1290 Inﬁnity 2D-LC Solution
with the following conﬁguration was used
for the experiments.

•

Agilent 1290 Inﬁnity Binary Pump,for ﬁrst dimension (G4220A)•

Agilent 1290 Inﬁnity Binary Pump,
for second dimension (G4220A)•

Agilent 1290 Inﬁnity Autosampler(G4226A)•

Agilent 1290 Inﬁnity Thermostat(G1330A)•

Agilent 1290 Inﬁnity Thermostatted
Column Compartment (G1316C)•

Agilent 1290 Inﬁnity Diode Array
Detector with standard ﬂow cell(G4212A)•

Agilent 1260 Inﬁnity Fluorescence
Detector (G1321B)•Agilent 1290 Inﬁnity Valve Drive(G1170A)•

Agilent 1290 Inﬁnity
2-position/4-port duo-valve for
2D-LC (G4236A)

Experimental
Samples and sample preparation
The standard solution containing 16 PAHs
in acetone/benzene at a concentrationof 2 mg/mL each (PAH Mix 25, Dr.
Ehrenstorfer, Augsburg, Germany) wasdiluted to 10 µg/mL in acetone.
From a crude oil sample, the polyaromatic
fraction was isolated using a liquid-liquid
partitioning between hexane andnitromethane.
A sample of 100 mg of crude oil
was dissolved in 5 mL hexane. Afterdissolution, 5 mL of nitromethane wasadded and a liquid-liquid extraction wasperformed. The upper hexane fraction
contained the saturated hydrocarbons
bulk fraction. The lower nitromethane
layer, containing the more polar aromatic
fraction, was collected for analysis. For acrude oil sample, the aromatic fraction istypically 5 to 30 % of the total sample1.

2Software•Agilent OpenLAB CDS
ChemStation Edition software,version C.01.07, with 1290 Inﬁnity
2D-LC software, version A.01.02•GC Image LCxLC Edition softwarefor 2D-LC data analysis (GC Image,LLC., Lincoln, NE, USA)

MethodFirst dimension
ColumnAgilent ZORBAX SB-CN, 2.1 × 150 mm, 5 µm (p/n 883700-905)

Solvent A

Water

Solvent B

MethanolFlow rate

100 µL/min

Gradient

40 %B at 0 minutes
100 %B at 80 minutes
100 %B at 85 minutes

Posttime

10 minutes at 40 %B

Column temperature
Second dimension

40 °C

ColumnAgilent ZORBAX RRHD Eclipse PAH, 3.0 × 50 mm, 1.8 µm (p/n 959757-318)

Solvent A

Water

Solvent BAcetonitrile

Flow rate

2 mL/minIdle ﬂow rate

0.3 mL/min

Initial gradient

50 to 70 %B from 0 to 0.35 minutes
70 %B from 0.35 to 0.40 minutes
50 %B at 0.41 minutes

Gradient modulation

50 %B at 0 minutes to 100 %B at 70 minutes
70 %B at 0.35 minutes to 100 %B at 55 minutes

Column temperature
Modulation

40 °C

Modulation on

7 to 85 minutes

Loops

Two 60-µL loops, cocurrent conﬁguration

Modulation time
Injectiona

0.50 minutes

Volume1 µL (injection program, mixed with 1-µL water plug )Needle wash
Detection DADb

5 seconds ﬂush port (methanol/acetone)

Wavelength

Signal 220/10 nm

Data rate
Detection FLDb80 Hz

Wavelength

Multi-emission mode
Signal A: Ex 260 nm/Em 350 nm
Signal B: Ex 260 nm/Em 430 nm
Signal C: Ex 260 nm/Em 500 nm

Data rate

37.04 HzPMT Gain7

aThe samples were injected together with a water plug to avoid peak broadening/splitting due to thestrong injection solvent.
bA zero-dead volume T-piece was installed at the outlet of the second dimension column to split theﬂow between the DAD and FLD. Red 0.12-mm PEEK tubing was used to connect to the detectors. The
tubing length from the T-piece to the FLD was twice as long compared to the tubing going to the DAD,
resulting in a DAD/FLD split ratio of about 2:1.

3

Results and Discussion
The LC analysis of PAHs is generally
performed using dedicated PAH
columns and DAD or FLD detection.
Using this column chemistry withwater/acetonitrile gradients resultsin complete separation of the 16 mostimportant nonsubstituted PAHs. When
this sample is analyzed on other reversed
phase systems (other columns, othermobile phases), some coelution typicallydoes occur, but the majority of analytes
are separated. The analyses of PAHs
in more complex samples (number ofPAHs or complexity of the matrix) aresigniﬁcantly more challenging and require
more chromatographic selectivity andseparation power and, if possible, better
detection selectivity. A comparison of theone-dimensional analysis of the standard
mixture of 16 PAHs and the crude oil
extract on the ﬁrst dimension ZORBAX
SB-CN column is shown in Figure 1(chromatographic conditions are different
from ﬁnal LCxLC conditions). It is clearthat the complexity of the real sample
is far too high for one-dimensional
separations. The complexity of thesample originates from the fact that thecrude oil contains substituted PAHs next
to the nonsubstituted PAHs, and thatheterocyclic PAHs are also present. The
substituted PAHs are mainly alkylated
PAHs, and make up a large group ofsolutes taking into account variations insubstitution degree, alkyl chain length,branching, and substitute position. All
these result in extreme complexity and nochromatographic technique is available
that would achieve full resolution of all
possible individual compounds. For thecharacterization of the polyaromatic
fraction, it is important to obtain
information on the number of aromaticrings, relative substitution degree, andon the possible presence of more polar
PAHs.

mAU
350
300
250
200
150
100500A Acetone(solvent)
PAH mix 16

1Benzene(solvent)

0mAU
350
300
250
200
150
1005001632
5456

10781511 12,1310 92015 1425Time (min)B
Crude oil extract

0510152025

Time (min)Figure 1. Comparison of a one-dimensional analysis of the PAH standard mix and sample extract. Column:
Agilent ZORBAX SB-CN, 2.1 × 150 mm, 5 µm, Flow rate: 0.3 mL/min, Gradient: 40–100 % methanol inwater from 0–40 minutes. Peak identities: see Table 1.

Table 1. Peak identities of the 16 PAHs (peaknumbers are assigned according to the expectedelution order on a PAH column).
Peak no.

Compound1

Naphthalene

2

Acenaphthylene

3

Acenaphthene

4

Fluorene

5

Phenantrene

6

Anthracene

7

Fluoranthene

8

Pyrene9

Benzo(a)anthracene

10

Chrysene11

Benzo(b)ﬂuoranthene

12

Benzo(k)ﬂuoranthene

13Benzo(a)pyrene

14Dibenzo(ah)anthracene

15Benzo(ghi)perylene

16

Indeno(1,2,3-cd)pyrene

On the selected ﬁrst dimension SB-CN
column, an interesting separation isobtained. The elution order for the16 PAHs under the applied conditions
differs signiﬁcantly from the normal
elution order in a classical RP-LC PAH
analysis (as in Table 1). This opens
perspectives for orthogonality in the2D-LC setup.
4Adapted chromatographic conditions andswitching to UHPLC equipment could
improve the resolution for the crude oil
extract, but to drastically increase peakcapacity, comprehensive 2D-LC will be
more effective. A combination was madeof the ZORBAX SB-CN column using awater/methanol mobile phase in the ﬁrst
dimension, with a classic PAH analysis
setup in the second dimension (EclipsePAH column with a water/acetonitrile
mobile phase). On the dedicated PAH
column, better separation was obtained
within a group with equal ring number(for example, benzoﬂuoranthene isomers).

mAU

A

1160
1401203100
8060

274054201615 1486010The LCxLC contour plots obtained withDAD using the SB-CN/Eclipse PAH
combination is shown in Figure 2B,
and can be compared to Figure 2A,
showing the 1D separation on theSB-CN column. Better separation wasobtained for phenanthrene/anthracene,benzo(a)anthracene/chrysene, andbenzoﬂuoranthenes. Only benzo(ghi)perylene and indeno(1,2,3-cd)pyrene werestill not completely separated. Next, thearomatic fraction of the mineral oil wasanalyzed using the same conditions. The
LCxLC contour plot is shown in Figure 2C.

1113/1210 9203040
Time (min)5060

B1310118316451215 1614972C86319457Figure 2. Comparison of a 1D-LC run of the standard mix (A), LCxLC run of the standard mix (B), andLCxLC run of the sample extract (C). Signal: DAD, 220 nm.

5Figure 3, Figure 4, and Figure 5 show
the LCxLC results from FLD using
different emission wavelengths. Some
additional series of compounds (probablysubstituted PAHs) are clearly detected.
As an example, there is a series ofcompounds that elutes prior to thenonsubstituted PAHs on the second
dimension column. This is clearly visible
with FLD. There is also a considerable
number of compounds that are more orless scattered around the PAHs present
in the standard mix. Identiﬁcation of theadditional compounds in the mineral oil
extract will require further investigation
with, for example, hyphenation to
MS using atmospheric pressure
photoionization (APPI), but from theirrelative elution pattern it can be predictedthat these are alkyl-substituted PAHs.
These results clearly illustrate the highcomplexity of the PAH fraction of crudeoil.

LU5A
16141210486103142678010203040Time (min)5060B

3Although many compounds remain
unidentiﬁed, the results clearly
demonstrate the potential of theAgilent 1290 Inﬁnity 2D-LC Solution forthis type of analysis.

84545110C

81Figure 3. Comparison of a 1D-LC run of the standard mix (A), LCxLC run of the standard mix (B), andLCxLC run of the sample extract (C). Signal: FLD, Ex 260 nm/Em 350 nm.

6Conclusion
This Application Note shows thepotential of the Agilent 1290 Inﬁnity
2D-LC Solution for proﬁling PAHs in thearomatic fraction of mineral oils using
parallel diode-array and ﬂuorescence
detection. The method is useful forproﬁling PAHs in crude oils, bitumen, andother mineral oils. The combination of thepresented 2D-LC method with MS using
APPI ionization should facilitate further
structure elucidation of the detected PAH
compounds.

Reference
1. EFSA Journal 2012; 10(6):2704,
European Food Safety Authority
http://www.efsa.europa.eu/fr/search/doc/2704.pdf

LU

13/12A806040

611159208750102030

101440Time (min)5060

B13151012865149117C
131210568911Figure 4. Comparison of a 1D-LC run of the standard mix (A), LCxLC run of the standard mix (B), andLCxLC run of the sample extract (C). Signal: FLD, Ex 260 nm/Em 430 nm.

7LU
1211A

10
13/12
8616

7
4

26
10

0
10203015

940Time (min)

5060

B
1316
12
116

97

C

126

9117

www.agilent.com/chem
This information is subject to change without notice.
© Agilent Technologies, Inc., 2015
Published in the USA, March 1, 2015
5991-5619ENFigure 5. Comparison of a 1D-LC run of the standard mix (A), LCxLC run of the standard mix (B), andLCxLC run of the sample extract (C). Signal: FLD, Ex 260 nm/Em 500 nm.