Analysis of PAH Compounds Using LC/Single Quadrupole MS with a Field Free APCI Source

Analysis of PAH Compounds Using LC/Single Quadrupole MS with a Field Free APCI
Source
Avinash Dalmia; Thomas White ; Daniel Pentek; PerkinElmer, Inc., Shelton, CT
1
Atmospheric Pressure Chemical Ionization Source
Overview
Purpose – A study was conducted to compare
sample throughput and detection limits between
conventional reverse phase HPLC/MS and
UHPLC/MS using a novel “field free” APCI source.
The study was performed on a 16 component
mixture of PAHs.
Mass Spectrum of Benzo[k]fluoranthene
Conventional LC/MS Chromatogram of a 16 Component PAH Mixture
13
500 ng injection
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15
16
Naphthalene
[M ]·+
Naphthalene
Acenaphtylene
Acenaphthene
Fluorene
Anthracene
Phenanthrene
Fluoranthene
Pyrene
Benzo [a] anthracene
Chrysene
Benzo [a] pyrene
Benzo [b] fluoranthene
Benzo [k] fluoranthene
Dibenzo [a,h] anthracene
Benzo [g,h,i] perylene
Indeno[1,2,3-cd] pyrene
[M+H]+
15
2
Figure 1a
Figure 1b
Figure 1 : Figure 1a represents a schematic of most commonly used APCI configuration.
Note that the heated gas exiting the vaporizer assembly experiences rapid expansion
minimizing the exposure of the analyte to the corona discharge needle. Figure 1b
illustrates the Field Free APCI configuration where the corona discharge needle is
contained within the grounded vaporizer assembly shielding the needle electric field.
This configuration allows the high electric field in the corona discharge region to be
separated from the ion focusing region into vacuum.
Methods
A field free APCI source was interfaced to a PerkinElmer
Flexar FX-10 UHPLC system and coupled to a Flexar SQ 300
MS detector. A PerkinElmer Brownlee Analytical DB PAH
Column: 3.2 X 150 mm, 5 µm, was used for the conventional
reverse phase LC. A sixteen component mixture of various
PAHs was injected on-column at a solvent flow rate of 1.0
ml/min. A gradient was run from 50% to 60% ACN in water
for 6 minutes, from 60% to 100% ACN over 6 minutes and
then 100% ACN for an additional 6 minutes. For the UHPLC
analysis, a PerkinElmer Brownlee Analytical DB PAH Column:
2.1 X 50 mm, 1.9 µm, was run at a solvent flow rate of 0.6
ml/min. The same PAH mixture was injected and a gradient
was run from 50% to 100% ACN over 5 minutes then held at
100% ACN for 1 minute. The optimized field free APCI source
parameters used were held constant for both the
conventional reverse phase LC and the UHPLC separations.
The corona current was set to 5 µA at the APCI vaporizer
temperature was set to 350°C.
Naphthalene
m/z = 128
Acenaphthylene
m/z = 152
Anthracene
m/z = 178
Phenanthrene
m/z = 178
Benzo [a] anthracene
m/z = 228
Benzo [k] fluoranthene
m/z = 252
Fluorene
m/z = 166
Acenaphthene
m/z = 154
Fluoranthene
m/z = 202
Chrysene
m/z = 228
Benzo [a] pyrene
m/z = 252
Pyrene
m/z = 202
Benzo [b] fluoranthene
m/z = 252
11
Figure 7 : Mass spectrum for benzo[k] fluoranthene. Note that two different species
were detected, one being the molecular radical cation and the other being the
protonated species. For all PAH’s analyzed, the radical cation was the most intense ion
detected. Consequently, the radical cation was used to acquire all SIM data in order to
determine the detection limits and linearity response of the PAH’s.
Linearity Response for Benzo[k]fluoranthene (0.05 ng-500 ng)
1E+09
100000000
UHPLC/MS Acquisition Parameters
Analyte
Ion
Type
Ion (m/z)
Capillary Exit
Voltage/V
Dwell
Time/ms
Retention
Window/mi
n
Retention
Time/min
Naphthalene
M+
128
120
50
0-3.2
1.23
Acenaphthylene
M+
152
200
50
0-3.2
1.48
Acenaphthene
M+
154
110
50
0-3.2
1.85
Fluorene
M+
166
110
50
0-3.2
1.95
Anthracene
M+
178
170
50
0-3.2
2.21
Phenanthrene
M+
178
170
50
0-3.2
2.43
Fluoranthene
M+
202
180
50
0-3.2
2.73
Pyrene
M+
202
180
50
0-3.2
2.90
Benzo[a] anthracene
M+
228
180
75
3.2-6.0
3.57
Chrysene
M+
228
180
75
3.2-6.0
3.66
Benzo [a] pyrene
M+
252
200
75
3.2-6.0
4.26
Benzo [b] fluoranthene
M+
252
200
75
3.2-6.0
4.47
Benzo [k] fluoranthene
M+
252
200
75
3.2-6.0
4.66
Benzo [ghi] perylene
M+
276.1
200
75
3.2-6.0
5.30
Indeno [1,2,3-cd] pyrene
M+
276.1
200
75
3.2-6.0
5.43
Dibenzo [a,h] anthracene
M+
278.1
180
75
3.2-6.0
5.10
Conventional LC/MS Acquisition Parameters
Ion
Type
Ion (m/z)
Capillary Exit
Voltage/V
Dwell
Time/ms
Retention
Window/min
Retention
Time/min
Naphthalene
M+
128
120
100
0-8.2
4.83
Acenaphthylene
M+
152
200
100
0-8.2
5.80
Acenaphthene
M+
154
110
100
0-8.2
7.40
Fluorene
M+
166
110
100
0-8.2
7.75
Anthracene
M+
178
170
100
8.2-16.5
8.61
Phenanthrene
M+
178
170
100
8.2-16.5
9.32
Fluoranthene
M+
202
180
100
8.2-16.5
10.02
Pyrene
M+
202
180
100
8.2-16.5
10.49
Benzo[a] anthracene
M+
228
180
100
8.2-16.5
11.87
Chrysene
M+
228
180
100
8.2-16.5
12.15
Benzo [a] pyrene
M+
252
200
100
8.2-16.5
13.09
Benzo [b] fluoranthene
M+
252
200
100
8.2-16.5
13.51
Benzo [k] fluoranthene
M+
252
200
100
8.2-16.5
13.98
Benzo [ghi] perylene
M+
276.1
200
100
8.2-16.5
15.32
Indeno [1,2,3-cd] pyrene
M+
276.1
200
100
8.2-16.5
15.74
Dibenzo [a,h] anthracene
M+
278.1
180
100
8.2-16.5
14.68
Figure 3 : Acquisition parameters used for the conventional HPLC/MS separation. In
order to find the retention times of the 16 PAH compounds the mass spectrometer was
operated in scan mode.
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0.5
5
50
500
Benzo [k] fluoranthene Amount/ng
Figure 8 : 4 orders of magnitude linear response for benzo[k]fluoranthene is
demonstrated with the SQ 300 MS operating in SIM mode.
On-Column Detection Limits
5
9
1000
Analyte
HPLC SIM Detection
Limit (ng)
UHPLC SIM Detection Limit
(ng)
Naphthalene
15.015
0.459
Acenaphthylene
2.492
0.390
Acenaphthene
4.144
0.285
Fluorene
4.288
1.502
Anthracene
1.502
0.258
Phenanthrene
0.291
0.111
Fluoranthene
0.601
0.099
Pyrene
0.180
0.060
Benzo[a] anthracene
0.366
0.075
Chrysene
0.655
0.105
Benzo [a] pyrene
0.661
0.150
Benzo [b] fluoranthene
0.372
0.123
Benzo [k] fluoranthene
0.021
0.006
Benzo [ghi] perylene
0.051
0.021
Indeno [1,2,3-cd] pyrene
0.111
0.036
Dibenzo [a,h] anthracene
0.114
0.048
Decrease in Detection
Limit
32.7 X
6.4 X
14.5 X
2.9 X
5.8 X
2.6 X
6.1 X
3.0 X
4.9 X
6.2 X
4.4 X
3.0 X
3.5 X
2.4 X
3.1 X
2.4 X
Figure 9 : The SIM detection limits for HPLC/MS and UHPLC/MS analyses are
presented. Note that increased sensitivity was achieved with increasing molecular
weight.
Naphthalene
Acenaphtylene
Acenaphthene
Fluorene
Anthracene
Phenanthrene
Fluoranthene
Pyrene
Benzo [a] anthracene
Chrysene
Benzo [a] pyrene
Benzo [b] fluoranthene
Benzo [k] fluoranthene
Dibenzo [a,h] anthracene
Benzo [g,h,i] perylene
Indeno[1,2,3-cd] pyrene
6
10000
1
0.05
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5
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100000
10
UHPLC/MS Chromatogram of a 16 Component PAH Mixture
150 ng injection
1000000
100
Figure 5 : The acquisition parameters used for the UHPLC/MS separation.
The retention times of the 16 PAH compounds were determined using scan
mode. Note that the retention times for all 16 components are considerably
shorter when compared to the separation using conventional HPLC. In
addition the pulse counting dwell times were shortened to increase the
sampling rate necessary for narrower eluting peaks do to the use of the 1.9
µm particle size column.
Results
Analyte
R² = 0.9999
10000000
Benzo [ghi] perylene Indeno [1,2,3-cd] pyrene Dibenzo [a,h] anthracene
m/z = 276
m/z = 276
m/z = 278
Figure 2 : The chemical structures of the 16 different PAH’s are shown that were
separated by conventional reverse phase HPLC and UHPLC before analysis by a
field free APCI source coupled to a Flexar SQ 300 MS detector.
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Figure 4 : The total ion chromatogram for the conventional HPLC/MS separation is
shown. In this experiment 500 ng was injected on column. A single analysis
required 18 minutes of run time.
Chemical Structures of the 16 Polyaromatic Hydrocarbons
Introduction
Methods for LCMS analysis of polycyclic aromatic
hydrocarbons (PAHs) were developed using conventional
reverse phase UHPLC coupled to a single quadrupole
mass spectrometer featuring a proprietary “field free”
Atmospheric Pressure Chemical Ionization (APCI) source.
PAHs are a group of semi-volatile organic compounds
that consist of one or more aromatic rings. They are
common byproducts of fossil fuel combustion which lead
to atmospheric pollutants. These non-polar compounds
are usually not ionizable by ESI and are of interest since
they have been identified as carcinogenic, mutagenic and
teratogenic. In this work, a comparison between
conventional reverse phase HPLC/MS to UHPLC/MS was
performed to determine the sample throughput and
detection limits for a 16 component mixture of PAHs.
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Response/Counts
Results – Going from a 5 µm to a 1.9 µm
particle size improved the detection limits by an
average factor of 6.5 for the 16 component PAH
mixture and decreased the total run time by a
factor of 3. The detection limits varied from 1.5
ng to 6.0 pg.
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Methods – A recently introduced PerkinElmer
“field free” APCI source was interfaced to a
PerkinElmer Flexar TM FX-10 UHPLC system
coupled to a Flexar SQ 300 MS detector.
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Figure 6 : The total ion chromatogram for the UHPLC/MS separation. Note
that the peak widths are narrower when compared to the conventional
HPLC/MS separation. This allowed a lower injection volume of 150 ng as
compared to 500 ng due to the increase in sensitivity with the UHPLC method.
In addition, the total run time to achieve the separation was decreased by a
factor of 3 increasing the sample throughput time.
Conclusions
For the conventional HPLC/MS analysis, all sixteen PAHs were
separated with detection limits ranging between 15 ng to 21 pg.
To complete this experiment a total run time of 18 minutes was
needed. For the UHPLC/MS analysis the on-column detection
limits achieved were between 1.5 ng to 6 pg. In order to
complete one UHPLC/MS run, a total run time of 6 minutes was
needed. From these experiments the results showed that going
from a 5 µM particle size to a 1.9 µm particle size can lower
detection limits substantially with a significant increase in
throughput. The detection limits were decreased by an average
factor of 6.5 for the 16 components, and the total run time for
the analysis was decreased by a factor of 3.
PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com
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