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Available online at www.sciencedirect.com

Journal of Chromatography A, 1173 (2007) 129–138

Increasing analytical space in gas chromatography-differential mobilityspectrometry with dispersion field amplitude programming

M. Basanta a, D. Singh a, S. Fowler a, I. Wilson b, R. Dennis c, C.L.P. Thomas d,∗a The School of Medicine, The University of Manchester, Manchester, UK

b AstraZeneca, Alderley Park, Cheshire, UKc GlaxoSmithKline, Stevenage, Hertfordshire, UK

d Department of Chemistry, Loughborough University, Loughborough, Leicestershire LE11 3TU, UK

Received 14 May 2007; received in revised form 17 September 2007; accepted 19 September 2007Available online 11 October 2007

bstract

Enhancing the analytical space of differential mobility spectrometry with dispersion field amplitude programming was proposed. Six volatilerganic compound candidate breath markers, 1,3-butanediol, butanone, ethylbenzene, heptan-2-one, nonanal, and o-xylene were used to characterisehe effect of programming the amplitude of the dispersion field on the sensitivity, and resolution of the responses observed. Sensitivity followedwo patterns of behaviour. Sensitivity to heptan-2-one and 1,3-butanediol increased to a maximum at approximately 20 kV cm−1, attributedo dissociative ionisation effects. The remaining four compounds’ responses were dominated by wall-loss phenomena resulting in a constanteduction in sensitivity as dispersion field amplitude was increased. The effect of the dispersion field on analytical space was pronounced. At a fieldtrength of 18 kV cm−1 protonated monomers and proton-bound dimers could be observed within the chromatographic responses for the carbonylompounds. Dissociative ionisation products were also discerned for 1,3-butanediol and butanone. The ion chemistry of the two hydrocarbons wasot affected by the dispersion field amplitude. Resolution of the product ions and their separation from the reactant ion peaks increased significantlyith increasing dispersion field amplitude. With a range of behaviours observed. Peak resolutions increased from the range 0 to 1.2 to 1.2 to 7,

hile resolving power increased from 0 (at low dispersion field amplitudes) to the range 0.2–6 at 20–24 kV cm−1. The effect of programming

he dispersion field amplitude on a “real-life” application was demonstrated with replicate breath samples obtained from a subject with chronicbstructive pulmonary disease. 2007 Elsevier B.V. All rights reserved.

field o

tq

doffot

eywords: Diffential mobility spectrometry; Gas chromatography; Dispersion

. Introduction

Recent descriptions and embodiments of differential mobilitypectrometry (DMS) indicate that it may be used as a tuneableelective ion detector for complex gas chromatography (GC)pplications [1–3]. Consequently research is underway to estab-ish and develop DMS methodologies for monitoring biogenicolatile organic compounds (VOCs) in humans; ultimately to besed for in-clinic or for at-patient monitoring. An important early

tage in these studies is the generation of GC-DMS data sets fromamples provided by subjects with accurately characterised clin-cal conditions. These data are input into chemometric models

∗ Corresponding author.E-mail address: paul.thomas@manchester.ac.uk (C.L.P. Thomas).

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pc

021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2007.09.082

ptimisation; Biovolatiles

o enable the identification of biogenic VOCs for the in vivouantitative assessment of clinical and morbid states.

A series of papers have presented the concepts underlyingifferential mobility spectrometry and the related approachesf field asymmetric ion mobility spectrometry and, radio-requency ion mobility spectrometry [4–6]. A fundamentalactor in the operation of all these techniques is the strengthf the dispersion field used to induce the dispersion velocity ofhe ions within the detector. As this study exploits the disper-ion field to increase the analytical space available, it is helpfulo review the relationship between the dispersion field strengthnd the ion mobilities of the species as they undergo separation

n a differential mobility spectrometer.

Previous observations show that ions form clusters in the gashase, they have hydration spheres, and entrained within theluster structure are molecules of the carrier gas [7]. The exact

1 atogr. A 1173 (2007) 129–138

satcadi

it

�v

Atpwmctsra

K

Tpicltiam

tai

nftdaooS

be

�

Fig. 1. The effect of a dispersion waveform used in differential mobility spec-trometry. This voltage waveform is applied to an ion filter that consists of twoparallel electrodes. An ion-cluster passing between the two electrodes will con-sequently experience two levels of electric field. For example the high field(E1 = 20kVcm−1) is applied for a short time interval (t1 = 91 ns) and the low-field strength (E2 = 1098 kV cm−1) is applied for a correspondingly longer time(t2 = 909 ns) such that �E1(kV cm−1) × t1(s) = �E2(kV cm−1) × t2(s). The heat-ici

ab

D

Had

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�v�vai

�v

30 M. Basanta et al. / J. Chrom

tructure and composition of an ion cluster is determined byvariety of factors, predominant of which are the concentra-

ion of water in the carrier gas and the temperature of the ionluster. The concentration of the parent ion is important as wellnd many parent ions form stable and observable proton-boundimer clusters as the concentration of the parent neutral speciesncreases [8].

When an electric field, �E(Vcm−1), is applied to the ion clustert accelerates to a limiting velocity �v(cms−1) that is proportionalo its mobility, K(cm2V−1s−1), described by the relationship,

= K �E (1)

ccelerating an ion cluster through a carrier gas will cause ito heat up and this will in turn produce changes in its com-osition. Thus, the mobility of the ion cluster, tends to changeith increasing electric field strength. Typically the ion clusteray undergo partial or complete declustering as a result of the

ollisional heating it experiences as it is driven faster throughhe carrier gas at higher electric fields. In this case it becomesmaller, and consequently its mobility will tend to increase. Theelationship between the collision cross-section of an ion clusternd its mobility is described by the Mason Schamp equation,

= 3q

16N×

√2π

μkT× 1

Ω(2)

his expression indicates that under constant conditions of tem-erature, T (K), and pressure (or number density, N), the mobilitys most strongly determined by the ratio of the charge, q (C), toollision cross-section, Ω (cm2). The reduced mass of the col-ision pair, μ (kg), is a factor with a marginal effect that tendso a constant value as the molecular weight of the ion clusterncreases beyond 100 amu. The Boltzman constant, and its units,re defined by k (cm2 kg K−1 s−2). In this treatment the units forobility are C s kg−1 which may also be cast as cm2 V−1 s−1.Not all ion clusters necessarily undergo heat induced declus-

ering, some may respond differently to collisional heating withconsequent reduction in their ion mobility as the field is

ncreased, see Eq. (2).Differential mobility spectrometry, and other related tech-

iques, exploits the difference in the structures of the ion clustersormed under high electric-field and low electric-field condi-ions. The principal of ion separation is based on inducing aispersion velocity in an ion cluster as it passes between two par-llel electrodes. An asymmetric dispersion waveform consistingf a high electric-field strength segment, �E1(Vcm−1), and anpposite polarity segment at a lower electric field �E2(Vcm−1).ee Fig. 1, is applied to the parallel electrodes.

The dispersion velocity of the ion, �vD(cm s−1), is determined

y the difference between the high electric-field and the lowlectric-field mobilities, K1 and K2, respectively (Eq. (3)).

K = K1 − K2 (3)

T

�v

ng experienced by the ion cluster as it accelerates under high-field conditionshanges its structure causing a change in the mobility of the species that resultn a dispersion velocity.

nd the mark-space ratio of the dispersion waveform, D, giveny Eq. (4),

= t1

t1 + t2(4)

ere t1(s) and t2(s) are the time intervals over which the highnd low fields are applied respectively during each cycle. Theispersion velocity may be defined as

D = �K �E1D (5)

or an ion to be detected by a differential mobility spectrom-ter, the dispersion velocity has to be compensated for by anpposing dc electric field (the compensation field �EC(Vcm−1)).he compensation field induces an equal and opposite velocity

o balance out the dispersion velocity, and so provide a stablerajectory for the ion cluster through the two parallel electrodes.he compensation field is applied continuously to the ion in both

he high electric-field and low electric-field states.In the low electric-field state the compensation velocity,

L(cms−1) may be described by

L = K2 �EC (6)

nd the compensation velocity, �vH(cm s−1) experienced by theon in the high electric-field state is

H = (K2 + �K) �EC (7)

he overall compensation velocity �vC(cm s−1) is therefore

C={K2 �EC(1−D)} + {(K2+�K) �ECD}=�ECK2+�EC�KD

(8)

romatogr. A 1173 (2007) 129–138 131

Fa

�va

�

Aandmss(satcb2

�

Iepstiiotpi

ifiw[pt[

epcfi3ti

oTi

Fig. 2. A plot showing the relationship between compensation field and boil-ing point for twenty three volatile organic compounds studied by microgas chromatography-differential mobility spectrometry [1]. These data wereobtained with a differential mobility spectrometer operating at 373 K with a dis-persion field amplitude of 26 kV cm−1. The numbered data are discussed in themain body of the text, and the compounds are identified in the table below.

Compound Boilingpoint

Compound Boiling point

Pentane (2) 309.0 2-Pentanone 375.0Methyl sulfide 311.0 Toluene 384.0Tetrahydrofuran 339.0 4-Methyl-2-

pentanone389.0

Tertbutylethyl ether 346.0 Butylacetate (1) 399.0Ethylacetate (1) 350.0 2 Hexanone 400.0Benzene 353.0 Ethylbenzene 409.0Acetonitrile (3) 354.0 M-xylene 412.0Thiophene 357.0 o-Xylene 417.0Ethyleneglycoldimethylether 358.0 Cyclohexanone 428.02-Ethyl furan 365.0 Bromobenzene 429.03P

imstpFit

iretoairms

M. Basanta et al. / J. Ch

or detection of the ion cluster the compensation field has to bepplied in the opposite direction to the dispersion field, so

D(cm s−1) = −�vC(cm s−1) (9)

nd combining Eqs. (5) and (8) gives

K = − 1

D

�ECK2

( �E1 + �EC)(10)

lthough it is tempting to cast the compensation field scale indifferential mobility spectrum in terms of �K it is helpful toote that the relationship between compensation field and �Kepends on the species involved and that the low electric-fieldobility of each of the species present in a compensation field

pectrum is different precluding the adoption of a single �Kcale. It is helpful however to look at the Mason Schamp equationEq. (2)) in order to relate changes in the compensation fieldpectrum to changes in the structure of the ion clusters involvednd the collisional heating they experience. This enables �Ko be described in terms of the differences in temperature andollisional cross-sections of the ion cluster under high (denotedy subscript 1 in Eq. (13)) and low field (denoted by subscriptin Eq. (13)) conditions (see Eq. (13)).

K = 3q

16N×

√2π

k×

(1

Ω1√

μ1T1− 1

Ω2√

μ2T2

)(11)

t is the interaction between collisional heating and the resultingffect on the structure of the ion cluster that determines the dis-ersion behaviour of an ionised species in a differential mobilitypectrometer. A negative shift in the compensation field requiredo scan an ion through the differential mobility spectrometerndicates that the reduction in the collision cross-section of theon due to collisional heating is greater than the heating effectn the ion’s mobility, while a positive shift in the compensa-ion field indicates that either collisional heating is the dominantrocess or that the collision cross-section of the ion cluster isncreasing.

As molecular size increases so the relative effect onon mobility of declustering phenomena at high electric-eld strengths decreases. (For instance, the loss of a singleater molecule from a protonated heptan-2-one ion cluster

(CH3CH2CH2CH2CH2CH2COCH3)(H2O)n(N2)H3O]+ has aroportionately smaller effect on the collision cross-section thanhe loss of a water molecule from a protonated butanone entity(CH3COCH2CH3)(H2O)n(N2)H3O]+.)

The phenomenon of reducing dispersion with increasing par-nt ion size is discernable when the relationship between boilingoint and compensation field for a range of volatile organicompounds is studied. Fig. 2 shows how the compensationeld decreases with increasing boiling point over the range09–432 K for twenty two compounds used in a landmark studyo characterise the performance of a micro-gas chromatographnterfaced to a differential mobility spectrometer [1].

The reduction of the compensation field maximum for a rangef ethers, ketones and aromatic hydrocarbons is a marked one.he observed responses for ethyl and butylacetate, labelled 1

n Fig. 2, are consistent with dissociative charge transfer form-

Mbas

-Methyl-2-butanone 367.0 Propylbenzene 432.0ropionitrile (3) 370.0

ng a common fragment ion for both species, possibly protonatedethanol. Ionised pentane, labelled 2 in Fig. 2, does not undergo

ignificant hydration, and as such exhibits smaller and less nega-ive �K values compared to other compounds of similar boilingoint. Conversely acetonitrile and propionitrile, labelled 3 inig. 2, are highly polar and will interact strongly with water lead-

ng to disproportionately large and negative �K values relativeo species of comparable volatility.

The general shift to lower compensation fields with increas-ng molecular ion size/boiling point, also has the effect ofeducing the resolution of the differential mobility spectrom-ter’s responses to the ion clusters. Fig. 3 amplifies the generalrends apparent in Fig. 2 with the effect of ion mass on thebserved compensation field for a series of protonated monomernd proton-bound dimer ketones [1]. The reduction in the analyt-cal space and differentiation between different ions is marked,eaching a limit with a boiling point of about 410 K, or aolecular mass of 200. Above these limits significant disper-

ion no longer occurred in the experimental configuration used.

olecules larger and less volatile than this exhibit a different

ehaviour. In such compounds the temperature effects outweighny declustering, or there is a slight increase in collisional cross-ection at high field as they unfold. (The exact mechanism has

132 M. Basanta et al. / J. Chromatog

Fig. 3. Previously published data reveals the relationship between the compen-sation field value of a peak and the molecular mass of the ion for a series ofprotonated ketones and proton-bound ketones dimers, identified in the tablebelow [1]. The reduction in the differentiation between different species ismarked and above a molecular mass of 175 differentiation between differentspecies becomes problematic under the operating conditions used.

Species Molecular mass Species Molecular mass

Acetone monomer 59.0 Acetone dimer 117.0Butan-2-one monomer 73.0 Butan-2-one dimer 147.0Pentan-2-0ne monomer 87.0 Pentan-2-one dimer 175.0HH

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f[eotit8lti[nmiiuide

dddacsaro

ctc

2

2

bwt

TM

A

ρ

m[

N

exan-2-one monomer 101.0 Hexan-2-one dimer 203.0eptan-2-one 115.0 Heptan-2-one dimer 231.0

et to be fully described, but whatever its cause the result is ahift to positive �K values.) The reduction in resolution withncreasing molecular size is an important feature of differential

obility spectrometry, as unaddressed it results in a decrease inhe “analytical space” available to the experiment with increas-ng ion mass [9]. Such an attribute has to be improved for theffective use of chemometric processing of the complex datarofiles obtained from clinical samples.

The effect of modification of the drift gas in ion mobilitypectrometry on the collisional cross-sections of ion clustersnd the importance of polarisability and stereochemical factorsave been demonstrated in an elegant series of studies where theelectivity and separation of ion-clusters in ion mobility haveeen optimised through control of the composition of the driftas. Blends of carbon dioxide, helium, argon and nitrogen werehown to affect the resolution of the separation of glycine pep-ides, cocaine, amphetamines and benzodiazepines [10,11]. The

xtension of these concepts was advanced yet further in a studyhat used (S)-(+)-2-butanol to affect the enantiometric separa-ion of l- and d-tryptopan among others [12,13]. In differentialobility, modification of the drift gas has been used success-

gfci

able 1asses and average eluent concentrations of the compounds used in this study

nalyte 1,3-Butandiol Butanone Ethylbe

(g cm−3) 1.01 0.81 0.87

c (ng) 9.85 7.89 8.50i](mg m−3) 3.64 2.91 3.14

ote: ρ, relative density; mc, on-column mass of analyte; [i], average eluent concentr

r. A 1173 (2007) 129–138

ully in the development of an explosives detection methodology9]. Dichloromethane, propanol, water and acetone have beenvaluated as drift gas modifiers for the enhanced separationf the vapours from ten explosives. Dichloromethane addedo the drift gas at a concentration of 3.5 g m−3 was shown toncrease the “analytical space” by three to six times enablinghe development of a highly sensitive [limit of detection (LOD)0–3 ppb (v), with no preconcentration], and fast (response timeess than 1 s) explosives detector to be demonstrated. An addi-ional consequence was to change the negative mode reactantonisation chemistry from [O2(H2O)n]− and [CO4(H2O)n]− toCl(H2O)n]−. Such approaches rely on the successful combi-ation of promoting ionisation and enhancing the differentialobility analytical space. The adoption of such a methodology

s well suited in optimising the response towards target analytesn specific applications. However, in the production of data forse in chemometric processing the modification of the reactanton chemistry may reduce the sensitivity of responses to candi-ate target analytes in the sample, and hence interfere with theffectiveness of post-analysis data processing.

Analytical space may also be enhanced by increasing theispersion field, see Eqs. (10) and (11). In a gas chromatography-ifferential mobility spectrometry experiment programming theispersion field against retention time would appear to offer anpproach to addressing the problem of the reduction of analyti-al space with increasing molecular size in differential mobilitypectrometry. Such an approach however will necessarily beccompanied by a reduction in sensitivity through the decrease ineactant ion concentrations due to wall losses at elevated valuesf the dispersion field.

This study, based on six candidate breath markers, sought toharacterise the effect of programming the dispersion field onhe analytical space and the sensitivity of the responses in gashromatography-differential mobility spectrometry analysis.

. Experimental

.1. Reagents

Methanolic standards of 2-butanone, 1,3-butanediol, ethyl-enzene, 2-heptan-2-one, o-xylene, nonanal (0.05%, v/v, Sigma)ere used in the characterisation studies, Table 1 summarises

he masses and concentrations used in these experiments. Nitro-

en was obtained from a gas generator (Dominick Hunter) andurther purified by passing the gas through a gas clean systemomprising a charcoal (Chrompack) and a moisture filter (Var-an) with an indicator. Helium was obtained from a cylinder

nzene Heptan-2-one Nonanal o-Xylene

0.82 0.82 0.888.04 8.07 8.632.97 2.98 3.18

ation of analyte in the differential mobility spectrometer.

romatogr. A 1173 (2007) 129–138 133

s(

2

Dtcc(c3eofi5hTtZnV

iivT(a(usp

2

s12vsdfii

pmatTartto

Table 2Summary of TPD/GC/DMS instrumental parameters

Optic thermal desorption unitStart temperature (◦C) 50Ramp rate (◦C s−1) 8Holding temperature (◦C) 100Hold time (min) 1Final temperature (◦C) 200Desorption time (min) 10

Gas chromatographCarrier gas and flow rate He, 1 cm3 min−1

Column temperature programme 45 ◦C (1 min),12 ◦C min−1, 150 ◦C(2 min)

Transfer line temperature (◦C) 60

Differential mobility spectrometerTransport gas and flow rate Nitrogen,

270 cm3 min−1

DMX chip temperature (◦C) 100Dispersion field strengths (kV cm−1) 14–28Scan range (V cm−1) −860 to 300Scan duration (s) 2.45Scan rate (V s−1) 473.5Scan step size 100 stepsVc step (V cm−1) 11.6Dispersion field programme (kV cm−1) 18–25

Fig. 4. Surface plots comparing the total ion response surface (top) to the

M. Basanta et al. / J. Ch

upply and purified using water, oxygen and hydrocarbon filtersThames Restek).

.2. Instrumentation

A temperature-programmed thermal desorption (TD-GC-MS system was assembled by fitting a programmable

emperature injector (Optic 600, AI Cambridge Optic) to a gashromatograph (800 series Fison Instruments). The system wasompleted by interfacing a differential mobility spectrometerDMx Sionex) fitted with a 63Ni ionisation source to the gashromatograph using an annular heat pipe (CRS Engineering),0 cm long with an outer diameter of 15 mm and an inner diam-ter of 9 mm (CRS Engineering), maintained at a temperaturef 180 ± 4 ◦C with a 1/4 in. cartridge heater, 50 W, 240 V (RS)tted to the aluminium mounting block. The transfer line was a0 cm long BPX1-Sim deactivated aluminium clad capillary thatad a 0.53 mm inner diameter and a 0.1 �m thick film (Restek).he analytical column was 30 m long with an internal diame-

er of 0.25 mm. The stationary phase was 1 �m thick and wasB-5 low bleed 5% phenyl and 95% polydimethylsiloxane (Phe-omenex). The column and transfer line were connected with au-2 Union Connector (Restek).

Two types of injections were used in this work: liquidnjections and thermal desorption of adsorbent traps. Liquidnjections were into a Pyrex liner (Anatune) packed with deacti-ated borosilicate glass wool (Restek), maintained at 200 ◦C.hermal desorption studies used Optic Pyrex injector liners

Anatune UK) packed with 70 mg Tenax TA 60/80 mesh andpproximately 70 mg Carbotrap 20/40 mesh adsorbent materialsSupelco). These adsorbent traps were prepared in the laboratorysing deactivated borosilicate glass wool (Thames Restek) toeparate the adsorbents and hold them in place. The experimentalarameters are summarised in Table 2.

.3. Procedures

Four dispersion field amplitude experiments were used in thistudy with a differential mobility spectrometer temperature of00 ◦C, the dispersion field amplitudes were 14, 18, 20, and,4 kV cm−1. Four replicates were run for each with an injectionolume of 1 �l and 50:1 split ratio. The differential mobilitypectrometer was operated in a scanning mode with a fixedispersion field amplitude, or with a programmed dispersioneld where the dispersion field amplitude was incrementally

ncreased at the end of each scan (Table 2).A total ion response surface response was recorded and

rocessed to provide background corrected GC-differentialobility surfaces for each of the six standards used. Fig. 4 is

n example of a total ion response surface for the responseo ethyl benzene at a dispersion field strength of 18 kV cm−1.he ammonia and water-based reactant ion peaks are evidents is the presence of some column bleed. The ethyl benzene

esponse may be seen at approximately 491 s emerging fromhe column bleed artefact. Note also the simultaneous reduc-ions in the ammonia and water product ion signals, indicativef charge transfer ionisation processes. The subtraction of the

background corrected response (bottom) for ethylbenzene for a dispersion fieldstrength of 18 kV cm−1. The ammonia-based and water-based reactant ion peaksare resolved from the ethyl benzene response, which is superimposed on the col-umn bleed artefact. A background subtraction of the column bleed signal yieldsa resolved response to ethylbenzene.

134 M. Basanta et al. / J. Chromatog

Fig. 5. Effect of dispersion field amplitude on the sensitivity of the responsesto six VOC found in exhaled. Heptanone (open triangles) and 1,3-butandiol(filled triangles) were observed to yield an initial rise in the sensitivity of theresponse with increasing dispersion field amplitude followed by a reduction insensitivity in line with that observed for nonanal (open diamonds), butanone(bi

cm

3

3

rararhfi

2imabdfiaabirtiidtt

w

oprrbtr

3

tpeih(nwut

ypafiw3ta−i

ihhwb

pam(ottmm

filled diamonds) ethyl benzene (open circles) and o-xylene (filled circles). Theehaviour of heptanone and 1,3-butandiol is thought to be due to dissociativeonisation initiated by increasing dispersion field amplitudes.

olumn bleed component of the signal results in a defined chro-atographic response for ethyl benzene.

. Results

.1. The effect of dispersion field on sensitivity

With a dispersion field amplitude of 14 kV cm−1 theesponses for nonanal, butanone, 1,3-butanediol, ethylbenzenend o-xylene could not be resolved from the hydrated protoneactant ion peak response, these species were first observedt dispersion field strengths of 18 kV cm−1. The most sensitiveesponse observed in this study was nonanal at 18 kV cm−1, andeptan-2-one alone yielded a resolvable response at a dispersioneld strength of 14 kV cm−1.

Two types of behaviour were observed. Responses to heptan--one and butanediol were observed to increase initially withncreasing dispersion field strength before reaching a maxi-

um and then reducing in sensitivity. Such behaviour may bettributed to dissociative ionisation processes that were inducedy the energy imparted to the parent ion from the high fieldispersion voltage. Increasing the field-strength of the high-eld segment of the dispersion waveform would enhance suchprocess increasing the yield of product ions and boosting the

nalytes’ responses. The abundance of the fragment ions woulde directly dependant on the levels of the parent ion, and its helpful to note that the parent ion concentration necessarilyeduces with increasing dispersion field amplitude (caused byhe removal of the reactant ion from enhanced wall losses) giv-ng rise to two competing mechanisms. The yield of fragmentons increases with dispersion field amplitude while the abun-ance of starting material (the parent ion) is reducing at the same

ime. In the limit the effect of wall loses of ions dominated andhe sensitivity of the response reduced (Fig. 5).

The other four molecules exhibited a range of sensitivities,ith the reduction in sensitivity observed when the amplitude

acp

r. A 1173 (2007) 129–138

f the dispersion field was increased that was inversely pro-ortional to the observed low-field sensitivity. Note the 70%eduction in the responses of nonanal and butanone over theange 18–24 kV cm−1. In contrast the effect on the hydrocar-ons ethylbenzene and o-xylene was not as pronounced withhe sensitivity reducing between 25 and 30% over the sameange.

.2. The effect on resolution of dispersion field amplitude

Fig. 6 compares GC-DMS responses to the six standards athree dispersion field amplitudes, with the data shown as contourlots. These data show how the nature of the responses is influ-nced by the dispersion field amplitude. The two hydrocarbonsn the study ethylbenzene (D in Fig. 6) and o-xylene (F in Fig. 6)ad almost identical responses. Ethyl benzene and o-xyleneretention times of 492 and 528 s, respectively) were not sig-ificantly affected by increasing the dispersion field amplitudeith constant compensation fields of −39.8 and −28 V cm−1

ntil at 24 kV cm−1 both compounds compensation field shiftedo −16.8 V cm−1.

1,3-Butanediol (B in Fig. 6 with a retention time of 470 s)ielded a complex assembly of product ions with two distincteaks −86.6 and 7 V cm−1 at 18 kV cm−1, which then splitnd resolved into three distinct product ions with compensationelds of −157, −110.2 and −18.8 V cm−1 at 20 kV cm−1. Thereere other minor and unresolved features also present at −86.6,9.8 and −4.6 V cm−1. Increasing the dispersion field ampli-ude to 24 kV cm−1 resulted in a complex and poorly resolvedssembly of peaks with partially resolved features at −157 and110 V cm−1, and as has already been noted above and shown

n Fig. 6, a significant reduction in sensitivity.The low-field products of the atmospheric pressure chemical

onisation of carbonyl compounds (oxygen containing bases)ave been described previously [7]. Mass spectrometric studiesave shown that as the analyte concentration increases the chargeithin the system is distributed in an equilibria between proton-ound monomers and dimmers.

In this study the maximum concentrations of these com-ounds entering the differential mobility spectrometer waspproximately 0.15 ppm (v). This would have occurred at theaximum of the chromatographic elution profile and 0.15 ppm

v) was a high enough concentration to result in the productionf protonated dimer ion species. Consequently it was anticipatedhat differential mobility responses from carbonyls would con-ain at least two components associated with the formation of

onomer and dimer ion clusters. In this study nominal assign-ents were made on this data model.

Heptan-2-one (C in Fig. 6 with a retention time of 506.5 s)

nd butanone (E in Fig. 7, with a retention time of 244 s)hromatographic responses showed protonated monomer androton-bound dimer relationships as well as dissociative ionisa-

M. Basanta et al. / J. Chromat

Fig. 6. GC-DMS background corrected contour plots for the six standards at 18,20 and 22 kV cm−1 (bottom middle and top respectively) for (A) nonanal, (B)1,3-butandiol, (C) heptanone, (D) ethylbenzene, (E) butanone, (F) o-xylene. SeeTd

tpana

ptopIt−2ttIp−i−paIsaoan2

wd1firafitcrpraaFuhp

cdprdeati

able 1 for the masses and concentrations involved and Table 2 for experimentaletails. Responses are discussed in detail in the text.

ion at the higher concentrations present at the chromatographic

eak maxima. At 18 kV cm−1 the heptan-2-one response hadn intense proton-bound dimer peak at 20 V cm−1 with proto-ated monomer responses at −16.4 V cm−1. Butanone also hadproton-bound dimer response at 7 V cm−1 and accompanying

a

R

ogr. A 1173 (2007) 129–138 135

rotonated monomers at 242 s and 245 s with a compensa-ion field of −86.6 V cm−1. In addition there was a third typef product ion that appears at the same retention time as theroton-bound dimer at a compensation field of −51.6 V cm−1.ncreasing the field strength resulted in a clearer differentia-ion between the monomer (−35 V cm−1 for heptan-2-one and

110 V for butanone) and dimer species (30 V cm−1 for heptan--one and 7 V for butanone). In the heptan-2-one responsehe two product ion responses merged into each other, indica-ive perhaps of the formation of a third dissociation product.n contrast, the butanone response showed the dissociationroduct ion clearly resolved with a compensation field of51.6 V cm−1. At 24 kV cm−1 heptan-2-one shows a dimer

on peak at 65.6 V cm−1 with protonated monomer species at39.8 V cm−1 and retention times of 504.5 and 508.5 s. A third

roduct ion species associated with the proton-bound dimer hadlso emerged at a compensation field of 7 V cm−1 and 506.5 s.n contrast the nature of product ions for butanone had changedignificantly at 24 kV cm−1 with a single major peak at 244 s andcompensation field of −145 V cm−1. No evidence of monomerr dimer relationships was discernable at this field strengthlthough there were minor features, just discernable fromoise, at −98.4 and −63.2 V cm−1. All at a retention time of44 s.

Nonanal responses (A in Fig. 6 with a retention time of 756 s)ere consistent with protonated monomer and proton-boundimer responses across all three dispersion field amplitudes. At8 kV cm−1 a single, asymmetric, peak with a compensationeld of 7 V developed to give evidence of a monomer–dimerelationship with a highly asymmetric peak at 20 kV cm−1. Heren intense proton-bound dimer response with a compensationeld maximum of 18.8 V cm−1 had two lobes at compensa-

ion fields of −4.6 V cm−1 sitting on the Gaussian tail of thehromatographic peak. At 24 kV cm−1 the monomer–dimerelationships were still evident with a proton-bound dimereak at 54 V cm−1 with protonated monomers as partiallyesolved shoulders on a complex feature at 7 V cm−1. Notelso the maximum at the centre of the chromatographic peak,lso with a compensation field of 7 V cm−1 (marked × inig. 6A), such a feature indicates the existence of a third prod-ct ion at this dispersion field amplitude associated with theigher concentrations at the centre of the Gaussian elutionrofile.

These six examples demonstrate the nature and generalharacteristics of how the GC-DMS responses change withispersion field amplitude. Integration of the chromatographiceaks with retention time, see Fig. 7, enables the effect on theesolution of the disparate features of the response surfaces to beiscerned. Analysis of the summed compensation field spectranables the resolution of the most mobile ion clusters (nominallyssigned as a protonated monomer or a protonated fragment ion)o be estimated; see Fig. 8 which plots the resolution expressedn terms of the compensation field and the full width of the peak

t half maximum height Eq. (12).

= EC

FWHM(12)

136 M. Basanta et al. / J. Chromatogr. A 1173 (2007) 129–138

F tegraf sens( e mas

Tphil

Fi(c

accounted for when the shift to more positive compensation fieldvalues is taken into account.

ig. 7. Background corrected GC-DMS responses for 18, 20 and 24 kV cm−1 inor the six breath volatile standards. The effect of dispersion field amplitude onC) heptanone, (D) ethylbenzene, (E) butanone, (F) o-xylene. See Table 1 for th

hese data show a mixed set of responses to increasing dis-ersion field amplitude. Four of the molecules 1,3-butanediol,

eptan-2-one, ethylbenzene, and butanone all showed increasesn the resolution of the observed responses. Nonanal’s reso-ution increased slightly, and o-xylene resolution behaviour is

ig. 8. The effect on the resolution of the protonated monomer peak with increas-ng dispersion field strength. (A) Nonanal, (B) 1,3-butandiol, (C) heptanone,D) ethylbenzene, (E) butanone, (F) o-xylene. See Table 1 for the masses andoncentrations involved and Table 2 for experimental details.

r

Fmp1Td

ted with respect to retention time to give a summed compensation field spectraitivity and resolution is evident in these data. (A) Nonanal, (B) 1,3-butandiol,ses and concentrations involved and Table 2 for experimental details.

Fig. 9 illustrates the effect on the dimensionality of theesponse in terms of the peak resolution between the most mobile

ig. 9. The effect of dispersion field strength on the resolution between theost mobile of the product ions (nominally assigned as a protonated monomer or

rotonated fragment) and the hydrated proton reactant ion peak. (A) Nonanal, (B),3-butandiol, (C) heptanone, (D) ethylbenzene, (E) butanone, (F) o-xylene. Seeable 1 for the masses and concentrations involved and Table 2 for experimentaletails.

romatogr. A 1173 (2007) 129–138 137

pw

R

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3

idfipitatFtttapiow

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Fig. 10. A comparison of surface plot data obtained from replicate breath sam-ples taken from a subject with chronic obstructive pulmonary disease. The traceon the bottom shows a close up of part of the response surface obtained with apws

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M. Basanta et al. / J. Ch

roduct ion peak and the water reactant ion on the basis of theidely accepted chromatographic term,

= 2

(ECP − ECR

WP + WR

)(13)

onanal (A), 1,3-butanediol (B), butanone (E) and o-xylene (F)esponses were all differentiated more clearly with increasingispersion field strength. The behaviour of heptan-2-one (C)nd ethylbenzene (D) were not so straightforward. The emer-ence of a partially resolved dissociative ionisation producton in heptan-2-one served to broaden the protonated monomeresponse Fig. 7C. The broadening of the product ion peak inhe ethylbenzene response offset any gains from the enhancedeparation observed in compensation field values for the waternd product ion peaks, see Fig. 7D.

.3. Dispersion field amplitude programming

Figs. 5–8 show how the resolution and information contentn a GC-DMS response show a general increase with increasingispersion field amplitude. However, the use of high-dispersioneld amplitudes results in a significant loss of sensitivity with theotential loss of information from surveys of volatile metabolitesn humans using GC-DMS. At the beginning of a GC tempera-ure programme where the boiling points of the eluent species aret their lowest there is no need to use large dispersion field ampli-udes to resolve and differentiate the responses, see Figs. 2–5 andig. 6B and E. As the chromatographic separation progresses so

he boiling points of the eluents increase and the amplitude ofhe dispersion field needs to be increased to enable the responseso the lower volatility compounds to be better differentiated. Incomplex volatile metabolite sample, exhaled breath for exam-le, or a bacterial pyrolysate a single dispersion field amplitudes unlikely to yield a response of either sufficient sensitivityr dimensionality to capture all the information and variabilityithin the sample.Initiating a dispersion field programme that runs concur-

ently with the GC column temperature programme providesdispersion field amplitude that constantly changes to match

he decreasing volatility (increasing molecular weight) andhanging differential mobility behaviour of the analytes. Imple-entation of dispersion field programmes, see Table 2, has

een found to significantly enhance the information content ofomplex biological samples. Fig. 10 shows a comparison of GC-MS responses obtained from replicate breath samples obtained

rom a adult subject with chronic obstructive pulmonary disease.he effect of dispersion field programming is evident with sig-ificantly higher resolution and clearer peak definition in allarts of the response surface. Further the reduction in the RIPntensity also resulted in a reduction in the trisiloxane feature aompensation field value of approximately 0 V cm−1 that was atrongly interfering feature of the response surface from 300 snwards, due to column bleed into the detector. The reduction of

he background signal enabled responses not discernable underonstant compensation field conditions to be observed.

For dispersion field programming to have a wider applica-ility in metabolite mapping and screening applications it is

pmas

rogrammable dispersion field while the trace on the top shows data obtainedith a constant dispersion field strength over the same part of the response

urface (18 kV cm−1).

mportant that the responses and operation of the differentialobility spectrometer are reproducible over a timescale of up

o 12 months. (A typical length for a clinic based trial.) A dis-ersion test was developed to characterise of the signal to noiseharacteristics across all operating states of the instrument torovide information about the operational state of the DMS usedn these studies. This test enabled field strength stabilities to bevaluated, and the sensitivity of the instrument to be verified.inally it allowed the presence of potential contaminants to behecked for. The dispersion test consisted of scanning the com-ensation field continuously while dispersion field strength wasrogrammed. This test was run with the differential mobilitypectrometer in a nominally clean condition, and was applied at

eriodic intervals to evaluate the reproducibility of the instru-ent over an 8-month period. Evaluation of the responses to

mmonia and water reactant ions at different dispersion fieldtrengths showed that the instrument was stable and reproducible

1 atog

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38 M. Basanta et al. / J. Chrom

ver the time of the study. Importantly, some of these dispersionests revealed a build up of column artefacts within the system,long with column ageing effects. This test was subsequentlyncorporated into the experimental preparative protocols for allC-DMS activity.Dispersion field programming has been used with studies

f the volatiles in breath of volunteers with chronic obstruc-ive pulmonary disease and asthma as well as in the pyrolysisf bacterial cultures. In these programmes it has proven to beuseful enhancement to GC-DMS methodology with prelim-

nary indications that it was possible to differentiate betweenhe bacterial strains Bacillus Subtilis B0014, Bacillus Subtilis1382 and Bacillus Magneterium B0010, and provide high-imensionality data sets for COPD and asymptomatic smokersnd non-smoker profiles. The findings of these studies using dis-ersion field programming will be the subject of future researchapers. At this stage in these studies programming the disper-ion field amplitude has been found shown to generate highimensionality and well resolved responses for complex clin-cal samples and provide a general method of addressing theptimisation problem of maximising the resolution (maximis-ng the dispersion field amplitude) while maintaining sensitivityminimising the dispersion field amplitude).

cknowledgements

This research was carried out under the UK National Ini-iative in Ion Mobility Spectrometry, a consortium supporting

esearch into the development and application of ion mobilitypectrometry at Nottingham Trent University and the Universityf Manchester, supported by AstraZeneca, GlaxoSmithKline,nd Waters Limited.

[

[

r. A 1173 (2007) 129–138

The authors gratefully acknowledge the vital support pro-ided by the Sionex Corporation through the loan of an SVACeated differential mobility spectrometer, and additionally theoan of a Micro GC-DMS from Varian Incorporated.

Finally the authors express their gratitude to the volunteerubjects and the nursing and research staff at the North Westung Research Centre, for without their support this researchould not have been possible.

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