Research Article

Received: 23 July 2013 Revised: 1 October 2013 Accepted: 18 October 2013 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2014, 28, 200–208

200

Large-volume injection combinedwith gas chromatography/isotoperatio mass spectrometry for the analysis of polycyclic aromatichydrocarbons

Anna J. Buczyńska1*, Benny Geypens1, Rene Van Grieken2 and Karolien De Wael21Joint Research Centre – European Commission, Institute for Reference Materials and Measurements (IRMM), Retiesewg 111,B-2440 Geel, Belgium2University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium

RATIONALE: Compound-specific stable isotope analyses of carbon require relatively large amounts of sample forreliable analyses. Commonly applied injections of 1 μL may thus be insufficient for samples with low concentrationsof pollutants (e.g. air particulate matter) or when the amount of a sample is limited.METHODS: A Large-Volume Injection (LVI) method for carbon stable isotope ratio analysis of Polycyclic AromaticHydrocarbons (PAHs) was optimized in this study. Gas chromatography/combustion/isotope ratio mass spectrometry(GCCIRMS) and ion trap mass spectrometry (ITMS) were used for the determination of stable carbon isotope ratios andquantification of compounds, respectively.RESULTS: The optimized method resulted in very good reproducibility, even for the most volatile PAH, naphthalene,when a small amount of higher boiling co-solvent was used. No significant fractionation of isotope ratios could be seenand the recoveries of analytes were similar to or better than that of a splitless cold injection.CONCLUSIONS: Injection of 100 μL, instead of the commonly used 1 μL, increases the detection limit for PAHssignificantly and/or simplifies the sample preparation step. Using our optimized method, stable carbon isotope ratioscan be reliably measured in samples with concentrations of PAHs down to 0.05–0.1 ng μL–1. Copyright © 2013 JohnWiley & Sons, Ltd.

(wileyonlinelibrary.com) DOI: 10.1002/rcm.6769

Large-Volume (LV) Programmed Temperature Vaporization(PTV) injection is a technique, which allows the introductionof large volumes of samples (such as tens to hundreds ofmicroliters) to the injector of a gas chromatograph. Inconventional injection techniques, e.g. on-column injection,split or the most commonly applied splitless injection,the introduction of more than a few microliters (typically1–2 μL) of solvent causes band broadening and/or inadequatequantitative performance of the chromatographic system.[1]

In addition to PTV, other techniques have been appliedfor large-volume injection, i.e. vapor-overflow, loop-typeinjection or on-column injection.[2] Using Large-VolumeInjection (LVI), not only large amounts of organic solvents canbe introduced to the injector, but also aqueous solutions.[3–5]

LVI is useful especially for environmental trace analysis,such as the analysis of polycyclic aromatic hydrocarbons(PAHs), where enrichment of the analytes is necessary toobtain the required limits of the detection.[3] PAHs aresuspected carcinogens, widely spread in the environment.The US EPA (US Environmental Protection Agency) hasidentified 16 PAHs as priority pollutants.[6] Stable carbonisotope ratio analyses, which can help in source identification

* Correspondence to: A. J. Buczyńska, Joint Research Centre –European Commission, Institute for Reference Materials andMeasurements (IRMM), Retiesewg 111, B-2440 Geel, Belgium.E-mail: abuczynska@interia.pl

Rapid Commun. Mass Spectrom. 2014, 28, 200–208

and apportionment of PAHs,[7] require a relatively largeamount of sample (generally close to 1 nmol of carbonper compound[8]) for reliable results. The concentration ofPAHs present in air is, however, usually very low, in therange of ng per m3.[9] Increasing the sample amount byapplying flows greater than those commonly used duringhigh volume sampling may increase volatilization lossesof compounds from the substrate used for sampling,whilst longer sampling times may increase exposure ofPAHs to oxidants.[10] Combining filters for extraction fromdifferent sampling locations or time intervals couldcomplicate the interpretation of data obtained from stableisotope ratio analyses.

LVI increases the method sensitivity for analytes with lowconcentrations by injecting a larger volume of a sample;it may also simplify the sample pretreatment by reducingor shortening the sample evaporation step.[9,11,12] Theprinciple of LVI involves the injection of a sample, duringwhich the split exit is open and high flow of gas (typically75–250 mL min–1) passes through the injector to enhance thefast evaporation of the solvent. The temperature of theinjector is usually kept below the solvent boiling point,[12]

thus greatly reducing the discrimination of less volatilecomponents of a sample.[14] Moreover, thermal degradationis minimized, since the residence time of the analytes atelevated temperatures is reduced compared with that in theconventional hot injection.[15] Discrimination of analytes inthe injector, apart from leading to low recoveries, may also

Copyright © 2013 John Wiley & Sons, Ltd.

Large-volume injection for stable carbon isotope analysis of PAHs

be a potential source of isotopic fractionation artifacts.[16,17]

After solvent evaporation, the temperature of the injector isincreased and the analytes transferred to the chromatographiccolumn in the splitless mode.[18] The quantitative retention ofanalytes with low boiling points may be improved with theuse of a packed liner.[1]

Although general rules for PTV performance can be drawnfrom published optimizations, experimental adjustment ofvarious parameters is needed in each particular case.[19]

The following factors need to be taken into account:inlet pressure, initial liner temperature, purge flow,speed of the sample introduction, sample volume andthe physicochemical properties of the solvent. [20] Theanalysis of PAHs with the use of a PTV injector has beenpreviously adopted by Ramos et al.,[21] who used a systemsimilar to ours with the same packed liner for a LVI butused a different injection volume (50 μL) and solvent, thetwo crucial parameters for which the LVI needs to beoptimized. The sample type included soils and sediments.A multiple injections technique was, in turn, accommodatedfor PAHs extracted from air and sediment samples,[9] orPAHs and nitro-PAHs from urban particulate matter.[10]

Other authors have used LVI in combination with varioustypes of liners and injection volumes (10–100 μL) forsample matrices such as urban dust and diesel particulatematter Standard Reference Materials (SRMs),[22] airborneparticles,[23] water and beverages.[24] Large-volume injectionin analyses of PAHs was also recently reported by someother authors, i.e., Gómez-Ruiz et al.,[25] Fernández-Gonzálezet al.,[26] Prieto et al.,[27] Evagelopoulos et al.,[28] and GarridoFrenich et al.[29]

It should be mentioned that all the above publicationsdealt with the application of LVI for mass spectrometrydetection while applications of LVI for isotopic ratio massspectrometry[30,31] approaches are scarce. The aim of this studywas to optimize the large-volume injection of 16 EPA PAHs forstable carbon isotope ratio analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GCCIRMS).Subsequently, this method was implemented in our laboratoryfor air particulate matter extracts containing PAHs.

20

EXPERIMENTAL

The gas chromatograph (Trace GC Ultra; Thermo Scientific,Bremen, Germany) employed in the study was equippedwith an OPTIC 3 PTV injector (ATAS GE International B.V.,Eindhoven, The Netherlands). An HT-5MS capillary column(60 m length, 0.25 mm i.d. and 0.25 μm film thickness)was used for chromatographic separation. An injector linerfilled with packing type A, which can retain up to the150 μL of at-once injected (100-150 μL s–1) sample, wasused. Packing A is composed of deactivated Tenax, whichis highly stable at temperatures up to 350 °C. The packingof the liner serves to retain the sample liquid and preventssample losses via the split exit. This increases the rate ofsolvent elimination significantly due to the increase insolvent-purge gas contact area and, thus, the degree ofsaturation of the purge gas with solvent vapors.[20] Otheradvantages of a packed liner are the minimization oflosses of volatiles and the prevention of the transfer ofthe high boiling components from the matrix to the

Copyright © 2013 JoRapid Commun. Mass Spectrom. 2014, 28, 200–208

analytical column.[13,21] Non-volatile matrix componentsremain deposited in the liner, which, if necessary, can beeasily exchanged.

An ion trap (IT) mass spectrometer (Thermo Scientific), towhich about 10% of the column effluent was directed, wasused in this study. The ion source temperature was 250 °C,and the transfer line was kept at 300 °C. The spectrometerwas operated in total ion count (TIC) mode using electronionization (250 μA emission current). The mass scanningranged between m/z 40 and 550. Molecular ions were usedfor quantitation of PAHs (peak areas). The remaining 90%of the effluent was directed to the combustion oven(ceramic tube filled with Pt, CuO and NiO and operated at940 °C) and further to the DELTAplus XP isotope ratio massspectrometer (Thermo Scientific) for carbon stable isotoperatio measurements.

Standards used included a solution of 16 EPA PAHs(Dr. Ehrenstorfer GmbH, Augsburg, Germany) in tolueneand four solid PAHs (Dr. Ehrenstorfer GmbH or Alfa Aesar,Karlsruhe, Germany) at a purity of at least 98.5%.

RESULTS AND DISCUSSION

The optimization of LV injections in n-hexane

Parameters previously optimized for the LVI (100 μL) ofPAHs in n-pentane[31] were initially adopted for injectionsin n-hexane. The organic solvent was changed to n-hexanebecause of its higher boiling point, which facilitates thecomplete removal of dichloromethane (DCM) during thesample concentration step. DCM is used to elute PAHs inour sample cleanup procedure (Solid-Phase Extraction, SPE);unfortunately, it may dissolve the deactivation coating ofthe liner packing. The optimization was carried out byvarying the injection parameters (Table 1). The transfertemperature (300 °C) was fixed in all our experiments.

Naphthalene was not considered during the initialoptimization experiments as its recovery was always verylow due to its relatively low boiling point compared with thatof the solvent (roughly 150 °C difference). In addition,airborne naphthalene is almost exclusively present in thegas phase[23] and the focus of this work was the air particulatematter rather than the gas phase.

Preliminary optimizations

As a first step in the optimization, the influence of the venttime (90–300 s) was assessed while applying 150 mL min–1

vent flow. Characteristic pre-peaks (peak splitting) wereobserved for acenaphthylene, acenaphthene and fluorene.The reason for their occurrence was probably that duringthe solvent venting, which lasts longer for n-hexane thann-pentane, a small part of sample enters the chromatographiccolumn. The longer the venting time the larger were the areasof the pre-peaks. The chromatogram showing peak splittingis supplied as Supporting Information. Fluorene onlyshowed pre-peaks for higher venting times than 115 s.Pre-peaks were not observed for other PAHs. Godulaet al.[19] reported the occurrence of peak splitting andattributed it to the high volume of a solvent thataccumulated in the inlet. Similarly, peak splitting may occur

wileyonlinelibrary.com/journal/rcmhn Wiley & Sons, Ltd.

1

Table

1.The

parametersof

n-pe

ntan

eLVan

dsp

litless

cold

injections,ran

gean

dop

timized

values

ofn-he

xane

LVIan

dtheexplan

ationof

softwaresp

ecificterm

s

Parameter

Leg

end

LVIn-pe

ntan

e(100

μL)

Splitless

cold

injection(1

μL)

Ran

gestud

iedLVI

n-he

xane

(100

μL)

Optim

ized

LVIva

lue

n-he

xane

(100

μL)

Initialinjector

tempe

rature

(°C)

4545

45–6

045

Ven

tflow

rate

(mLmin

–1 )

The

flow

rate

ofga

sthroug

htheinjector

duringthesolven

tve

ntingwiththesp

litop

en

150

-80

–250

230

Samplesw

eepcolumn

flow

rate

(mLmin

–1 )

The

columnflow

rate

during

thesolven

tve

nting

1.8

-0.6–

1.8

0.6

Tran

sfer

columnflow

rate

(mLmin

–1 )

The

flow

rate

duringthetran

sfer

ofan

alytes

tothecolumn(splitless

state)

3.6

3.6

2.5–

3.6

3.6

Tempe

rature

ramp(°C

s–1 )

The

rampof

tempe

rature

afterthe

ventingoffthesolven

t(splitless

state)

3030

5–30

5

Injectionsp

eed(μLs–

1 )100

15–100

20

Solven

tve

ntmod

e

Three

mod

esarepo

ssible:"Fixedtime"

uses

fixedtime,

afterwhich

theinjector

switc

hesto

splitless

state;

"Solve

ntleve

l"us

esthefunc

tion

ofsolven

tmon

itor

sensor,

theleve

lcan

bead

justed

toacertain

percen

tage

ofasolven

tpe

ak;

"Thresho

ld",he

realso

thesensor

isus

edan

dthethresholdva

luemay

besetby

theus

er(num

erical

valueinsteadof

percen

t)

Solven

tleve

l(30%

solven

tpe

ak)

-Ven

ttime

(50–300s)

Solven

tleve

l:30%

solven

tpe

ak

Fina

ltem

perature

(°C)

300

300

300

300

Tran

sfer

time(s)

9090

90–140

140

Colum

nflow

rate

(mLmin

–1 )

1.8

1.8

1.8

1.8

A. J. Buczyńska et al.

wileyonlinelibrary.com/journal/rcm Copyright © 2013 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2014, 28, 200–208

202

Large-volume injection for stable carbon isotope analysis of PAHs

as a result of a part of a liquid sample entering the GCcolumn when the amount of solvent injected is too high.In such a case all the chromatographic peaks would showpre-peaks. This did not seem to be the reason in our case asworse results would be obtained with shorter venting times,the reverse of what was observed in our experiments.For higher boiling PAHs, generally, middle range venting

times 115–200 s seemed most suitable; less pronounceddifferences were seen between different venting times thanfor more volatile PAHs. In GCCIRMS chromatograms duringthese optimizations the peaks of acenaphthylene andacenaphthene were not visible in any of the injection methods(the amount injected was 20 ng per compound). The highestvolatile PAH/benzo(a)pyrene ratios were obtained whenminimal tested vent times were used (90 s). On the otherhand, when applying short vent times, higher GCCIRMSbackground was visible in the chromatograms. It was thusdecided to use the venting option of ’30% solvent monitor’(Table 1) which in the case of n-hexane corresponds toapproximately 115 s of solvent venting. Next, the influenceof a sample sweep column flow (the GC column flow rateduring the solvent venting; range tested 1.8–0.6 mL min–1)on pre-peak heights was examined. A significant decrease inthe pre-peak/main peak ratios was visible when the samplesweep column flow was lowered. The lowest studied samplesweep column flow rate (0.6 mL min–1) was adopted forfurther experiments. Pre-peaks were, however, visible at alltimes and more volatile PAHs were lost to a great extent.As a next step, the starting temperature of the GC method

was lowered (in order to improve re-focusing of the analytesat the beginning of the GC column). The temperature adoptedfrom the method of n-pentane injections[31] was 90 °C. Thetested temperatures in this experiment were 80 °C to 50 °C.No pre-peaks were visible when the temperature programstarted at 70 °C and lower. The narrowest widths of peaksat half-heights of acenaphthylene and acenaphthene andalso the best separation from the small peaks of impuritieswere obtained when the starting temperature of 50 °C wasused. This temperature, therefore, was chosen as optimal.Godula et al.[19] found the distortion of peaks (splitting)eluting in the middle part of the chromatogram when using50 °C as the starting temperature of the GC temperatureprogram. The results were significantly improved when thestarting temperature was raised to 90 °C. The authors usedtoluene as an injection solvent and raising the temperature

Figure 1. The influence of various LVIfigure: vent flow, temperature ramp durspeed) on the recovery of low molecularat 0.13 ng μL–1.

Copyright © 2013 JoRapid Commun. Mass Spectrom. 2014, 28, 200–208

diminished the amount of re-condensed solvent. Similarlyto our findings, Sanchez et al.[32] found an initial columntemperature of 50 °C during the transfer of solutes to yieldnarrowpeaks for all compounds investigated (organophosphateesters). Other PAHs, with molecular weights higher thanthat of phenanthrene, in our experiments had similar peakwidths in all temperature programs despite the differencesin starting temperature.

The optimization of other injection parameters

Vent flow, temperature ramp and injection speed

A series of tests was conducted in order to find the best ventflow, optimal temperature ramp and injection speed. Theywere studied in combination with each other since interactioneffects among these variables might give rise to an erroneousevaluation of the results if they are optimized by a one-factor-at-a-time approach.[33] The tested ranges and optimizedvalues for all studied parameters are listed in Table 1.Optimization was performed by duplicate injections of the16-PAH standard. The detector response (peak areas) forduplicate injection was generally within 15% (5% on average)difference from each other for more volatile PAHs and evenbetter for PAHs with boiling points higher than that offluoranthene.

In the first instance, flow rates of 80, 150 and 250 mL min–1,temperature ramps of 30, 15 and 5 C s–1, and injection speedsof 100 and 5 μL s–1 were investigated. Vent flow rates of 150and 80 mL min–1 resulted in very low recoveries of morevolatile PAHs compared with a flow rate of 250 mL min–1

(Fig. 1). Similarly, a fast injection (100 μL s–1) resulted in lowerrecoveries of those PAHs than the slow one (5 μL s–1). Thetrend was opposite for PAHs with higher MWs thanfluoranthene (150 and 80 mL min–1) or phenanthrene(250 mL min–1), where fast injection produced higherresponse. In fast injection, the liquid penetrates lower in theliner packing, analytes are located closer to the column andthus the time spent in the injector at high temperatures isshortened.[13,34] High molecular weight PAHs are thought tobe insensitive to the high flows during the solvent ventingbut are sensitive to degradation at high temperatures.Fast injection using n-hexane, however, resulted only in about40% recovery of the lower molecular weight PAHs(acenaphthene through fluorene) compared with injection in

parameters (from the bottom of theing splitless transfer and the injectionweight PAH compounds in n-hexane

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203

A. J. Buczyńska et al.

204

n-pentane. For the temperature ramps investigated, onlysmall differences were seen in the recovery of the analytes.Slightly higher recoveries of high molecular weight PAHswere found for a low temperature ramp (5 °C s–1) and thiswas especially pronounced for middle boiling point rangePAHs (phenanthrene up to chrysene) and thus this rampwas adopted. For further optimizations only flow rates above200 mL min–1 were considered (200, 215 and 230 mL min–1).In addition, an injection speed of 10 μL s–1 was tested, butresulted in lower recoveries of lower boiling analytes than aspeed of 5 μL s–1. Using the vent flow rate of 230 mL min–1

and an injection speed of 5 μL s–1, no difference was seen inthe recovery of analytes compared with 250 mL min–1; thus,this flow rate was chosen as optimal.

Transfer flow

Transfer flow is the flow applied in order to transfer theanalytes to the column from the liner, immediately after thesplit exit has been closed. It is beneficial to apply higherinjector pressure (thus higher column flows) during this stepin order to ensure the efficient transfer and minimal thermaldegradation of the analytes.[1] Four different transfer flowswere tested (2.5–3.6 mL min–1) (results not shown); thehighest peak areas were obtained when 3.6 mL min–1 wasused, and this flow was thus adopted for further analysis.

Initial injection temperature

An initial injection temperature (45 °C) was used in all ourexperiments. The same initial temperature was used byCrimmins et al.[9] for analyses of PAHs using LVI. As thisparameter was found to be crucial in the recovery of injectedcomponents,[9] the influence of initial temperature on PAHrecoveries in n-hexane injection was checked. Four differenttemperatures were tested, 45, 50, 55 and 60 °C. Temperatureslower than 45 °C required long cooling times, as in oursystem, a compressed air flow was used to cool the injector.Best recoveries of low molecular weight PAHs wereobtained with the initial temperature of 45 °C; again aninjection speed of 5 μL s–1 resulted in higher recoveries of

Figure 2. Comparison of the recovery ofn-hexane (0.1 ng μL–1) as solvent, n-hexa0.1 ng μL–1) and splitless cold injected (one standard deviation (±1σ) of five repe

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these PAHs than 100 μL s–1. For other, higher molecularweight PAHs the temperature of the injector during thesample introduction had no significant effect on their recoveries(results not shown).

The influence of toluene addition on the recovery of PAHs

In order to improve the recovery of low molecular weightPAHs, a higher boiling solvent was added to the standards’solutions. Such a ’co-solvent’ or ’keeper’ forms a stable liquidfilm in the liner within which components can be effectivelyretained. Dodecane was for example used by Christensenet al.[22] The authors focused on PAHs with boiling pointshigher than that of phenanthrene. Toluene (added to thesample before the injection) was chosen in our study. Threelevels of toluene concentration were investigated, 1.7%,3.34% and 5% (v/v). The addition of the highest toluenevolume studied (5%; v/v), gave the best recoveries incombination with the slow injection speed (5 μL s–1). Anincrease in recovery of up to twelve times was seen fornaphthalene and about two times for acenaphthylene andacenaphthene compared with the injection method ofMikolajczuk et al.[31] For other PAHs, with volatility lowerthan that of the fluorene, no difference was seen.

Figure 2 shows the comparison of the optimized injectionmethod, splitless cold injection of 1 μL (concentration ofPAHs was 0.1 ng μL–1 in LVI and matched the amountintroduced in splitless injection) and the LV injection withoutthe addition of a ’co-solvent’. The mean detector response(ITMS, peak areas) is shown and the 1 SD of the mean(n= 5) is given as error bars. The precision (relative standarddeviation, RSD) of the optimized injection method rangedbetween 8.2% for naphthalene and 1.1% for benzo(ghi)-perylene, being 2.7% on average for all PAHs. For none ofthe studied PAHs did the PTV splitless cold injection (1 μL)result in higher recoveries than the LVI method with theaddition of a co-solvent. Actually, lower recoveries were seenfor more volatile PAHs and for lower boiling PAHs, whilstmiddle-range PAHs (anthracene to pyrene) were comparablein all injection methods. Those PAHs are influenced the leastby different injection parameters. It should be mentioned that

PAHs (peak areas) injected with purene with 5% addition of toluene (v/v,10 ng μL–1). The error bars representtitions.

ley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2014, 28, 200–208

Table

2.δ1

3 CVPDBmeanva

lues,m

etho

ddetection

limitan

dcorrelationcoefficien

tsforinve

stigated

PAHsin

theconc

entrationrang

e0.025–1.50

ngμL

–1

GCCIRMS

EAIRMS

Com

poun

dAve

rage

δ13 C

VPDB(‰

)(n

=20–2

3)S.D.

Correlation

coefficien

t(R

2 )a

Amplitud

emass44

attheMDL(m

V)

S.D.

Con

centration

atMDL(ngμL

–1 )

Ave

rage

δ13 C

VPDB(‰

)(n

=3)

S.D.

Anthracen

e–24.23

0.22

0.9990

(0.9993)

496

700.1

–24.48

0.15

Fluo

ranthe

ne–24.72

0.29

0.9994

(0.9995)

245

270.05

–24.60

0.06

Pyrene

–25.48

0.33

0.9995

(0.9996)

234

200.05

–25.47

0.19

Ben

zo(b)fluo

ranthe

ne–24.76

0.31

0.9994

(0.9988)

378

170.1

–24.71

0.06

a Correlation

coefficien

tcorresp

ondsto

thelin

earregression

oftheITMS(peakareas)in

theconc

entrationrang

eof

0.025–1.50

ngμL

–1 ;thecoefficien

tinbracke

tsrefers

tothe

peak

areasob

tained

bycombu

stionof

PAHsan

dsu

bseq

uent

IRMSmeasu

remen

t.

Large-volume injection for stable carbon isotope analysis of PAHs

20

the conditions used for 1 μL injections were not optimizedfor the best recovery of the analytes. It was assumedthat, since the solvent vent step is omitted in this method,the transfer efficiency for different conditions duringthe sample transfer would not differ significantly. Thelower recovery of higher boiling PAHs in the splitlessmethod than in LVI was nevertheless probably causedby rapid heating rate of the injector (30 °C s–1) while lowerefficiencies for lower boiling PAHs could perhaps beattributed to the differences in the liners (empty singlebaffled liner was used for 1 μL injection).

Comparison of EAIRMS and GCCIRMS analyses

Measurement trueness[35]

The trueness of the optimized LVI method for themeasurement of stable carbon isotope ratios was determinedby comparison of the δ13C values with those obtained byElemental Analyser (EA)IRMS. These tests were required tocheck whether any isotope fractionation occurs during thelarge-volume injection since the evaporation anddiscrimination of analytes in the injector are possible sourcesof isotopic fractionation artifacts.[16,17] Four solid PAHcompounds, measured by EAIRMS (n= 3) againstinternational standards (NBS19 and L-SVEC) and thusnormalized to a VPDB scale, were dissolved in n-hexaneand injected into the GCCIRMS system (n= 6; concentrationrange of 0.25–0.75 ng μL–1). The delta values of PAHswere calculated using n-alkanes previously analyzed byEAIRMS; thus the identical treatment principle forcalibration was applied. The results were in very goodagreement (difference of merely 0.01–0.1‰ between thetwo techniques), proving that the LVI does not introduceany significant isotope fractionation. The precision (1 SD)ranged between 0.06 and 0.19‰ for EAIRMS and from0.05 to 0.33‰ in GCCIRMS.

Method detection limit (MDL)

To determine theMDL for stable carbon isotope measurementsusing optimized LVI, four PAH compounds, at nineconcentration levels ranging from 0.025 to 1.5 ng μL–1, wereanalyzed in triplicate. An approach similar to that ofJochmann et al.[16] was used. The mean delta value of thethree highest concentrations measured was calculated anda ±0.5‰ uncertainty interval was set around this value. Thisinterval is thought to include both the reproducibility oftriplicate injection and the accuracy of the measurement withrespect to the international standard.[16] Subsequently, a newmean value was calculated including lower concentration.This was repeated as long as the mean value for lowerconcentration was within the ±0.5‰ of the interval and thestandard deviation at this level was below ±0.5‰. The lastconcentration for which both conditions were met wasdefined as the MDL. Triacontane (δ13CVPDB = –32.9‰) wasused as the calibration standard for δ13CVPDB calculations.The results are given in Table 2. Lower concentrationsresulted in higher standard deviations and/or delta valueswhich are outside the set thresholds. The MDLs (amplitudeof the m/z 44 signal) were 496 mV, 245 mV, 234 mV and378 mV for anthracene, fluoranthene, pyrene and benzo(b)-fluoranthene corresponding to 0.1, 0.05, 0.05 and 0.1 ng μL–1,

wileyonlinelibrary.com/journal/rcmCopyright © 2013 John Wiley & Sons, Ltd.Rapid Commun. Mass Spectrom. 2014, 28, 200–208

5

Figure 3. Mean δ13CVPDB (‰) of 16 EPA priority PAHs (n= 4–5) for LVinjections in n-hexane, n-hexane with 5% (v/v) toluene (100 μL; 0.1 ng μL–1)and splitless cold injection (1 μL; 10 ng μL–1). Starting from the left: naphthalene(Nap), acenaphthylene, acenaphthene (Ace), fluorene, phenanthrene (Phe),anthracene, fluoranthene (Flu), pyrene, benz(a)anthracene (B(a)A), chrysene,sum of benzo(b)- and benzo(k)fluoranthenes (B(b+k)F), benzo(a)pyrene, indeno-(1,2,3-cd)pyrene (Ind), dibenz(a,h)anthracene and benzo(ghi)perylene (B(ghi)P);the error bars represent ±1σ of the mean.

A. J. Buczyńska et al.

206

respectively. Excellent linearity was obtained for thestudied range (0.025–1.5 ng μL–1) both in the ITMS and theIRMS detectors.

Precision of δ13CVPDB determination for 16 EPA priority PAHs

The precision of the determination of δ13CVPDB values forother EPA priority PAHs was assessed at the concentrationlevel close to the limit of the δ13CVPDB determination(0.1 ng μL–1). No reference delta values were available forall compounds; thus, comparison with the values obtainedby splitless cold injection was performed as a check fortrueness. The results are graphically presented in Fig. 3.The data for naphthalene were only available in splitlesscold injection and when toluene was added (5%; v/v) inthe LVI. The error bars represent 1 SD of the mean of 4–5analyses. Generally, higher standard deviations of the meanδ13C values were found than previously, which wasexpected taking into consideration the relatively lowconcentration. They were, however, within 0.5‰ for mostof the compounds, thus fulfilling the condition for MDLdetermination. The highest standard deviations wereobtained for components that were not resolved completely,i.e. benz(a)anthracene and chrysene, benzo(b)- and benzo(k)-fluoranthene, as well as indeno(1,2,3,-cd)pyrene and dibenz(a,h)-anthracene. Thiswas significantly improvedwhen theunresolvedcomponents were integrated together. A similar approachwas taken by Mazeas and Budzinski[36] in their analyses ofunresolved PAHs. High boiling compounds (MW >252 Da)showed in general higher standard deviations at this concen-tration level (up to 1‰).For some of the compounds, viz.fluorene and acenaphthylene,

co-elution with an unknown peak was observed. Theseimpurities were not seen in previous experiments. Thedownside of the LVI technique is that not only are analytesconcentrated in the inlet liner but also impurities present inthe solvent or originating from the vial septa. Norlocket al.[8] also noticed ghost peaks in their LV injections ofPAHs originating from GC vial septa. The contribution ofthe impurities is generally less pronounced at higherPAH concentration levels. Following this finding, care wastaken that the septum of the vial was pierced only once(samples were placed in 200 μL vial inserts) and eachreplicate was injected using a separate vial. In addition,

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column conditioning was performed frequently, as well aschanging of the injection liner in order to minimize thecontribution of impurities.

There was no significant difference (t-test, 95% confidencelevel) in the δ13C values of PAHs between the splitless andLV injections except for two compounds (phenanthrene andpyrene, Fig. 3). The difference between means was, however,only 0.40 ‰ for phenanthrene and slightly higher for pyrene,0.94 ‰. No significant fractionation of more volatile PAHsduring the solvent venting step was seen.

CONCLUSIONS

A method for large-volume injection (LVI) of PAHs wasoptimized and applied for compound-specific stable carbonisotope analysis. Addition of a small volume (5%, v/v) of ahigher boiling solvent resulted in an improvement of therecoveries of most volatile PAHs. Injecting larger volumesof sample than those commonly applied in conventionalinjectors considerably improves the limit of detection and isespecially useful for samples with low concentration ofpollutants or when the amount of a sample is limited. Thisinjection technique is also very promising for other stableisotope ratio analyses, such as for example hydrogen, whichrequires even larger amounts of sample than carbon.Moreover,injecting a sample at low temperature (45 °C) reducesthe discrimination of less volatile components, therebyeliminating a possible source of isotopic fractionation. Nosignificant isotopic fractionation was observed for thisinjection method compared with splitless injection of 1 μLand with the results obtained by EAIRMS. Using ouroptimized method, stable carbon isotope ratios can bereliably measured in samples with concentrations of PAHsdown to 0.05–0.1 ng μL–1.

AcknowledgementsThe authors would like to thank four anonymous reviewersfor critical constructive comments and valuable suggestionsthat improved the clarity of this paper.

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Large-volume injection for stable carbon isotope analysis of PAHs

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REFERENCES

[1] H. G. J. Mol, H.-G. Janssen, C. A. Cramers, U. A. Th.Brinkman. Large-volume injection in gas chromatographictrace analysis using temperature-programmable (PTV)injectors. Trends Anal. Chem. 1996, 15, 206.

[2] Z. Vačeřa, A. Bartošíková, J. Sklenská, P. Mikuška. A largevolume injection procedure for GC-MS determination ofPAHs and PCBs. Chromatographia 2005, 61, 197.

[3] J. Teske, J. Efer, W. Engewald. Large-volume PTV injection:new results on direct injection of water samples in GCanalysis. Chromatorgaphia 1997, 46, 580.

[4] J. Teske, J. Efer, W. Engewald. Large volume injection:comparison of direct water injection and in-vial extractionfor GC analysis of triazines. Chromatographia 1998, 47, 35.

[5] M. Forcada, J. Beltran, F. J. López, F. Hernández.Multiresidue procedures for determination of triazine andorganophosphorus pesticides in water by use of large-volume PTV injection in gas chromatography.Chromatographia 2000, 51, 362.

[6] Environmental Protection Agency (EPA) of the UnitedStates of America, Compendium Method TO-13A, EPA,Cincinnati, OH, USA, 1999.

[7] A. J. Buczyńska, B. Geypens, R. Van Grieken, K. De Wael.Stable carbon isotopic ratio measurement of polycyclicaromatic hydrocarbons as a tool for source identificationand apportionment – a review of analytical methodologies.Talanta 2013, 105, 435.

[8] J. T. Brenna, T. N. Corso, H. J. Tobias, R. J. Caimi. High-precision continuous-flow isotope ratio mass spectrometry.Mass Spectrom. Rev. 1997, 16, 227.

[9] F. M. Norlock, J.-K. Jang, Q. Zou, T. M. Schoonover, A. Li.Large-volume injection PTV-GC-MS analysis of polycyclicaromatic hydrocarbons in air and sediment samples. J. AirWaste Manage. Assoc. 2002, 52, 19.

[10] B. S. Crimmins, J. E. Baker. Improved GC/MS methods formeasuring hourly PAH and nitro-PAH concentrations inurban particulate matter. Atmos. Environ. 2006, 40, 6764.

[11] E. Hoh, K. Mastovska. Large volume injection techniques incapillary gas chromatography. J. Chromatogr. A 2008, 1186, 2.

[12] Y. Li, J. S. Whitaker, C. L. McCarty. New advances in large-volume injection gas chromatography-mass spectrometry.J. Liq. Chromatogr. Related Technol. 2009, 32, 1644.

[13] H. G. J. Mol, M. Althuizen, H.-G. Janssen, C. A. Cramers.Environmental applications of large volume injection incapillary GC using PTV injectors. J. High Resolut. Chromatogr.1996, 19, 69.

[14] A. Covaci, J. De Boer, J. J. Ryan, S. Voorspoels, P. Schepens.Determination of polybrominated diphenyl ethers andpolychlorinated biphenyls in human adipose tissue bylarge-volume injection-narrow-bore capillary gaschromatography/electron impact low-resolution massspectrometry. Anal. Chem. 2002, 74, 790.

[15] H. G. J. Mol, H.-G. M. Janssen, C. A. Cramers, J. J. Vreuls,U. A. Th. Brinkman. Trace level analysis of micropollutants inaqueous samples using gas chromatography with on-linesample enrichment and large volume injection. J. Chromatogr.A 1995, 703, 277.

[16] M. A. Jochmann, M. Blessing, S. B. Haderlein, T. C. Schmidt.A new approach to determine method detection limitsfor compound-specific isotope analysis of volatileorganic compounds. Rapid Commun. Mass Spectrom.2006, 20, 3639.

[17] W. Meier-Augenstein, P. W. Watt, C.-D. Langhans. Influenceof gas chromatographic parameters on measurement of13C/12C isotope ratios by gas-liguid chromatography-combustion isotope ratio mass spectrometry. I.J. Chromatogr. A 1996, 752, 233.

Copyright © 2013 JoRapid Commun. Mass Spectrom. 2014, 28, 200–208

[18] F. J. Señoráns, J. Tabera, J. Villén, M. Herraiz, G. Reglero.Variables affecting the introduction of large sample volumesin capillary gas chromatography using a programmed-temperature vaporizer. J. Chromatogr. 1993, 648, 407.

[19] M. Godula, J. Hajšlová, K. Maštouska, J. Křivánková.Optimization and application of the PTV injector for theanalysis of pesticide residues. J. Sep. Sci. 2001, 24, 355.

[20] J. Staniewski, J. A. Rijks. Solvent elimination rate intemperature-programmed injections of large samplevolumes in capilllary gas chromatography. J. Chromatogr.1992, 623, 105.

[21] L. Ramos, J. J. Vreuls, U. A. Th. Brinkman. Miniaturisedpressurised liquid extraction of polycyclic aromatichydrocarbons from soil and sediment with subsequentlarge-volume injection-gas chromatography. J. Chromatogr.A 2000, 891, 275.

[22] A. Christensen, C. Östman, R. Westerholm. Ultrasound-assisted extraction and on-line LC-GC-MS for determinationof polycyclic aromatic hydrocarbons (PAH) in urban dustand diesel particulate matter. Anal. Bioanal. Chem. 2005,381, 1206.

[23] V. Yusà, G. Quintas, O. Pardo, A. Pastor, M. de la Guardia.DeterminationofPAHs in airborneparticles byaccelerated solventextraction and large-volume injection-gas chromatography-massspectrometry. Talanta 2006, 69, 807.

[24] R. Rodil, M. Schellin, P. Popp. Analysis of polycylicaromatic hydrocarbons in water and beveragesusing membrane-assisted solvent extraction in combinationwith large volume injection-gas chromatography-massspectrometric detection. J. Chromatogr. A 2007, 1163, 288.

[25] J. A. Gómez-Ruiz, F. Cordeiro, P. López, T. Wenzl.Optimisation and validation of programmed temperaturevaporization (PTV) injection in solvent vent mode for theanalysis of the 15 + 1 EU-priority PAHs by GC-MS. Talanta2009, 80, 643.

[26] V. Fernández-González, E. Concha-Graña, S. Muniategui-Lorenzo, P. López-Mahía, D. Prada-Rodríguez. Amultivariate study of the programmed temperaturevaporization injection-gas chromatographic-massspectrometric determination of polycyclic aromatichydrocarbons. Application to marine sediments analysis.Talanta 2008, 74, 1096.

[27] A. Prieto, S. Schrader, M. Moeder. Determination of organicpriority pollutants and emerging compounds in wasterwaterand snow samples using multiresidue protocols on the basisof microextraction by packed sorbents coupled to largevolume injection gas chromatography-mass spectrometryanalysis. J. Chromatogr. A 2010, 1217, 6002.

[28] V. Evagelopoulos, T. A. Albanis, El. Kodona, S. Zoras.Particle-associated polycylic aromatic hydrocarbons nearpower plants as determined by large volume injection –GC/MS. Chemosphere 2010, 80, 235.

[29] A. Garrido Frenich, R. Martínez Ocaña, J. L. Martínez Vidal.Determination of polycyclic aromatic hydrocarbons inairborne particulate matter by gas chromatography-triplequadrupole tandem mass spectrometry. J. AOAC Int. 2010,93, 284.

[30] A. Smirnov, T. A. Abrajano, A. Smirnov, A. Stark.Distribution and sources of polycyclic aromatichydrocarbons in the sediments of Lake Erie, Part 1. Spatialdistribution, transport, and deposition. Org. Geochem. 1998,29, 1813.

[31] A. Mikołajczuk, B. Geypens, M. Berglund, P. Taylor. Useof a temperature-programmable injectior coupled to gaschromatography-combustion-isotope ratio mass spectrometryfor compound-specific carbon isotopic analysis of polycylicaromatic hydrocarbons. Rapid Commun. Mass Spectrom. 2009,23, 2421.

wileyonlinelibrary.com/journal/rcmhn Wiley & Sons, Ltd.

7

A. J. Buczyńska et al.

208

[32] C. Sanchez, M. Ericsson, H. Carlsson, A. Colmsjö.Determination of organophosphate esters in air samples bydynamic sonication-assisted solvent extraction coupled on-line with large-volume injection gas chromatography utilizinga programmed-temperature vaporizer. J. Chromatogr. A 2003,993, 103.

[33] J. Villén, F. J. Señoráns, M. Herraiz, G. Reglero, J. Tabera.Experimental design optimization of large volume samplingin a programmed temperature vaporizer. Application infood analysis. J. Chromatogr. Sci. 1992, 30, 261.

[34] J. Zrostlíková, J. Hajšlová, M. Godula, K. Maštovská.Performance of programmed temperature vaporizer, pulsedsplitless and on-column injection techniques in analysis ofpesticide residues in plantmatrices. J. Chromatogr. A 2001, 937, 73.

wileyonlinelibrary.com/journal/rcm Copyright © 2013 John Wi

[35] International Vocabulary of Metrology – Basic and generalconcepts and associated terms (VIM), (3rd edn.). JointCommittee on Guides for Metrology (JCGM), 2008.

[36] L. Mazeas, H. Budzinski. Polycyclic aromatic hydrocarbon13C/12C ratio measurement in petroleum and marinesediments. Application to standard reference materials anda sediment suspected of a contamination from the Erika oilspill. J. Chromatogr. A 2001, 923, 165.

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