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Contents lists available at SciVerse ScienceDirect

Analytica Chimica Acta

j ourna l ho mepage: www.elsev ier .com/ locate /aca

se of potentiometric detection in (ultra) high performance liquidhromatography and modelling with adsorption/desorptioninding kinetics

. Daemsa,b, G. Van Campb, M. Fernandeza, Y. Guisezc, E. Prinsenc, L.J. Nagelsa,∗

University of Antwerp, Department of Chemistry, Groenenborgerlaan 171, B-2020 Antwerp, BelgiumUniversity of Antwerp, Centre of Medical Genetics, Universiteitsplein 1, B-2610 Antwerp, BelgiumUniversity of Antwerp, Department of Biology, Groenenborgerlaan 171, B-2020 Antwerp, Belgium

i g h l i g h t s

The first use of a potentiometricdetector in UPLC is described.The potentiometric detector is alsoused in HPLC.Alkaloids were sensitively deter-mined.A model is given that describeshow adsorption/desorption kineticsdetermines the detector characteris-tics.Binding kinetics can be performedwith the detector, in Flow injectionanalysis.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

rticle history:eceived 29 December 2012eceived in revised form 5 March 2013ccepted 12 March 2013vailable online xxx

eywords:

a b s t r a c t

Observation of a potentiometric sensor’s response behaviour after injection in flow injection analysisat different concentrations allowed studying “on” and “off” kinetics of the analyte’s adsorption/diffusionbehaviour. The alkaloid metergoline was mostly used as an example. kon and koff rate constant values weremeasured, and the association constant Kass, and �G values of the analyte–surface interaction were cal-culated with an adsorption-based model which proved to be fully applicable. kon increased by decreasingthe sensor dimensions, while koff was unaffected by miniaturization. Increasing acetonitrile concentra-

otentiometryensorgramIAinetics

U)HPLC, Alkaloids

tions in the running buffer increased koff, while kon was unaffected. The experimentally determined �Gvalues of the analyte–surface interaction showed a linear relation to the response of the sensor, in mV.This knowledge was applied to optimize the potentiometric detection of plant alkaloids in (U)HPLC. Sub-micromolar detection limits were obtained with the potentiometric detector/(U)HPLC combination. Thisis the first time that the response rates and the response itself can be modelled accurately for coated wirepotentiometric sensors, and it is the first application of a potentiometric detector in UPLC.

Please cite this article in press as: D. Daems, et al., Use of potentiometrimodelling with adsorption/desorption binding kinetics, Anal. Chim. Acta (2

∗ Corresponding author at: Chromatographic Organic Trace Analysis, Chemistryepartment, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp,elgium. Tel.: +32 3 2653385; fax: +32 3 2653233.

E-mail address: luc.nagels@ua.ac.be (L.J. Nagels).

003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.aca.2013.03.031

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Potentiometric sensors are used as well in conjunction withsmall benchtop laboratory apparatus, as well as with automated

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high throughput equipment. They appear in different formats forthe determination of inorganic and organic ionic substances. Theworking principles and developments of potentiometric sensorswere recently reviewed [1–3]. In the last decade, potentiometric

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ensors attracted the attention of the biotechnological world, as aossible basis for biosensors [4,5]. Although a high impedance volt-eter is mostly used as a readout system, other technologies are

xplored for miniaturized and array-type sensors in high through-ut assays [5,6]. The target analytes include large biomolecules7–10] and even bioparticles such as viruses and bacteria [11,12].

Different kinds of organic ionic analytes have already beenroven to be potentiometrically detectable in high performance liq-id chromatography (HPLC) and in capillary electrophoresis (CE).wo reviews by Trojanowicz [13,14] were devoted to the use oflectrochemical principles for detection in analytical systems basedn a hydrodynamic flow. While amperometric, coulometric andonductivity detectors are well established in HPLC and conductiv-ty detectors for miniaturized analytical systems, this is not the caseor potentiometry. After some very early publications on the usef potentiometric detectors for Liquid chromatography mentionedy Trojanowicz, pioneering work on their applications to this fieldtarted in the 1980s with work from Alexander [15] and Haddad16]. Later, our group considered the applications of potentiomet-ic detectors to HPLC [17,18] and to CE [19]. Although it was clearhat these detectors had development potential, the behaviour ofhese adsorption/diffusion dependent detectors in hydrodynamicystems remained open to discussion.

The reason for the lack of market penetration of potentiom-try in (ultra) high performance liquid chromatography (U)HPLCay be its limited rate of response [14]. Characteristics such as the

esponse rates of potentiometric sensors have seldom been studiedystematically, and are poorly understood. A reason for this may behe fact that these sensors are mainly used in batch measurements,here response times of several seconds are seldom a disadvan-

age. Another reason may be the fact that the voltage output ofhese sensors has a complex relation to concentration if the sub

icromolar concentration range (important in (U)HPLC) has to bencluded, which complicates a study of response rates. A main goalf the present study will therefore be to evaluate the factors thatffect the rate of response of these sensors, and to evaluate theirompatibility to (U)HPLC.

The potential generated by a potentiometric sensor is dependentn the strength of the chemical interactions between the chargednalyte and the surface of the sensor as well as its interior com-onents [20–22]. Potentiometry can be used to measure thesehemical interactions [22]. It can even become a new importantool in this respect, complementing an established but more expen-ive optical technique such as surface plasmon resonance (SPR)23] and more recent approaches such as the surface acoustic wave

ethod developed by Przybylski’s group [24]. The method reliesn the measurement of adsorption/diffusion rates of analytes athe surface of the potentiometric sensor, at different concentra-ions. The rate constants kon and koff (also called kassociation anddissociation) as used in binding kinetics of biomolecules and indsorption phenomena, are the important parameters. For use as

chromatographic detector, analyte/sensor adsorption/diffusionhenomena should be fast. We will show in the present articlehat these rate constants are determining whether this adsorp-ion/diffusion dependent detector can be used in analytical systemsased on a hydrodynamic flow.

. Materials and methods

.1. Chemicals

Please cite this article in press as: D. Daems, et al., Use of potentiometrimodelling with adsorption/desorption binding kinetics, Anal. Chim. Acta (2

All chemicals were of analytical reagent grade. The eluentsere prepared by dilution of a phosphoric acid solution obtained

rom Acros Organics and by dilution of a formic acid solu-ion obtained from Fluka. The acetonitrile (CH3CN) was obtained

PRESSa Acta xxx (2013) xxx– xxx

from Acros Organics. High-molecular mass PVC was obtainedfrom Janssen Chimica. The other membrane components wereof the highest quality grade available from Fluka. These includepotassium tetrakis(p-chlorophenyl)borate (TCPB), the plasticizerdioctylsebacate (DOS) and the solvent tetrahydrofuran (THF). Thedrugs, biogenic amines and organic acids were obtained fromSigma–Aldrich.

2.2. Instrumentation

The Flow injection analysis (FIA) and HPLC analyses wereperformed using a LC-10ADvp pump (Shimadzu Liquid Chromatog-raphy), SPD-10Avp UV detector (Shimadzu UV-VIS detector) anda Rheodyne 7125 six port external sample injector (VICI, US). A1 mL sample loop was used to generate square concentration pulsesfor recording in FIA conditions. The polyether ether ketone tub-ing (Alltech, USA) of the injection loop and the injector–detectorconnections had an internal diameter of 0.18 mm. Poiseuille peakbroadening effects were kept to a minimum using short injector-detector connections (15 cm). To avoid such effects at the end of thesquare concentration pulse, the injector was switched from injectto load well before the sample loop volume was totally emptied.This resulted in a sharp pulse with negligible broadening as well atthe start as at the end of the pulse.

The electrolyte solution used in the FIA experiments was5 mM H3PO4 (pH 2.4), unless otherwise stated. The flow rate was1 mL min−1. The injector outlet (in an FIA setup), or the columnoutlet (in an HPLC setup) was directed perpendicularly towards thesensitive membrane of the coated wire electrode in a “wall-jet” flowcell (see [25] for details). The distance from the LC tubing outlet tothe electrode was 100 �m unless otherwise stated. The membranepotential was measured against an Orion 800500Ross® referenceelectrode (Ag/AgCl) using a high impedance (1013 �) homemadeamplifier. The detection signals were recorded on a data stationcomposed of a PC equipped with a 6013 NI DA converter and Lab-VIEW 7 (National Instruments, US) based software. The overall timeconstant of the high impedance amplifier plus data station was setto 200 ms.

For the HPLC analyses, an injector sample loop of 20 �L was usedin a Rheodyne 7125 HPLC injector. The eluent buffer was a mixtureof solvent A (5 mM H3PO4) and solvent B (30% (v/v) CH3CN/5 mMH3PO4) with gradient elution from 25% solvent B to 100% solvent Bin 30 min. The flow rate was 1 mL min−1. A CC 125/4 Nucleodur 100-5 CN-RP column (Macherey–Nagel, 125 mm × 5 mm, 5 �m) wasused.

For the UPLC analyses, an injection volume of 6 �L was usedin a Waters Acquity (U)HPLC set-up. The eluent buffer was amixture of solvent A (0.1% (v/v) formic acid) and solvent B(CH3CN + 0.1% (v/v) formic acid) with gradient elution from 15%solvent B to 50% solvent B in 4.40 min, unless otherwise stated. Theflow rate was 0.7 mL min−1. An Acquity (U)HPLC BEH C18 1.7 �m2.1 mm × 100 mm Column (Waters, Ireland) with an Acquity(U)HPLC VanGuardTM Pre-Column 3/Pk 2.1 mm × 5 mm (Waters,Ireland) or an Acquity (U)HPLC T3 1.8 �m 2.1 mm × 100 mmwith an Acquity (U)HPLC T3 1.8 �m VanGuardTM pre-column2.1 mm × 5 mm was used. The same set-up was used with a WatersAcquity Triple Quadrupole Detector to obtain the MS results. Detec-tion limits were obtained with tandem MS.

2.3. Procedures

The indicator electrode is made of a PVC cylinder. It contains

c detection in (ultra) high performance liquid chromatography and013), http://dx.doi.org/10.1016/j.aca.2013.03.031

a cylindrical substrate electrode (3 mm diameter × 1 mm length or1 mm diameter × 1 mm length), which is an electronically conduct-ing graphite/PVC composite material [26]. The composite substrateelectrode was polished with Carbimet grid 600 (Buehler Ltd, USA).

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Fig. 1. Potential output (in mV) of different concentrations of metergoline. The 3 mmcoated wire electrode contains PVC, DOS and TCPB. The eluent was 5 mM H3PO4.T −4 −4.5 −5

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he indicator electrodes had a rubber-phase membrane coated onhe composite substrate electrode. Electrodes with a compositeubstrate have the important property that the membrane coatingnd the composite are tightly mixed at the interface, and sensorsonstructed in this way show very good impedance characteristics26].

The coating used for the detection of positively charged analytesontains 2% tetra(p-chlorophenyl)borate (TCPB), 33% PVC and 65%ioctylsebacate (DOS), unless otherwise stated. In our work, 300 mgf this mixture was dissolved in 3 mL tetrahydrofuran (THF), and

× 40 �L of this solution was applied on the 3 mm (2 × 3.6 �l onhe 1 mm) substrate electrode material by drop-coating with anutomatic pipette. After each application of the solution, the sol-ent was allowed to evaporate for 30 min at room temperature. Thelectrodes were allowed to dry for at least 3 h. After evaporation,he resulting ionically conductive rubber-phase polymer coatingas a thickness of 100 �m.

Coated electrodes were kept in running buffer (5 mM H3PO4,nless otherwise stated) for at least 3 h until a stable baselineas obtained in the FIA measurements. The sensorgrams of each

nalyte were measured on three electrodes, and used when thenter-electrode reproducibility was better than 10%. At least threeensorgrams (injections) were performed on each electrode afteronditioning and stabilization in the running buffer. As the wordsensorgram” refers to a signal recording for a non-specified sensor,e will use the word “potentiogram” for a potentiometric detector

n the rest of the text.

. Results and discussion

.1. The potentiometer detector output signal, and potentiogramso measure “on” and “off” kinetics

The type of potentiometric sensor used in this study has anonically conducting coating (sensing) material deposited on anlectronically conducting “substrate” electrode material. This sub-trate electrode is connected electronically to a reference electrode,nd the potential (mV) between both is measured. In (U)HPLC appli-ations, we are mostly concerned about changes in potential ratherhan in absolute values. Changes in potential occur if an analyte ion

Please cite this article in press as: D. Daems, et al., Use of potentiometrimodelling with adsorption/desorption binding kinetics, Anal. Chim. Acta (2

s interacting with the sensing layer (see the insert in Fig. 1). Thisnteraction (adsorption, diffusion, ion-exchange, etc.) changes theurface potential. This change in potential is our analytical signal20]. In applications where there is a short contact time between

PRESSa Acta xxx (2013) xxx– xxx 3

the analytes and the sensor, adsorption phenomena will be preva-lent over diffusion and ion-exchange. The relative importance ofthese phenomena depends on the analyte and on the sensor coat-ing material used. The situation is summarized by Wojciechowski[27], but remains a matter of discussion.

The potential (mV) output of a potentiometric detector inresponse to an injection of a square concentration pulse of an ana-lyte in an FIA setup is shown in Fig. 1. When the potential observedat the plateau values is plot against log(canalyte), the well-knownNicolskii–Eisenmann (NE) equation is followed: see Eq. (1), and[19,22].

E(mV, plateau) = E0 + S × log�canalyte + Cst� (1)

In Eq. (1), E(mV, plateau) is the mV response at the plateau val-ues reached by the potentiograms shown in Fig. 1. It is also thesignal (peak height) which can be obtained in U(HPLC) conditions,in ideal circumstances (fast responding analytes). E0 is the valueof the potential, obtained by extrapolating the analyte concentra-tion, canalyte, to a 1 M concentration. S (slope) is the usual 2.303RT/nF term, wherein R is the gas constant, T is the kelvin tem-perature, n is the charge of the analyte ion, and F is the faradayconstant. The empirically obtained NE equation is commonly usedin potentiometry. With the analyte concentrations canalyte, used inU(HPLC), which are mostly below 10−3 M (injected concentration),it is unproblematic to use the molar concentration of the analyte,canalyte, and not the analyte activity. The “Cst” term in this equationtakes into account interferences. In U(HPLC) conditions, these aredue to eluent buffer components, and they are usually very small.This term becomes significant if injected concentrations canalyte arebelow micromolar. With the method which is proposed here, molarconcentration ranges from 10−3 M down to the detection limit ofthe sensor/analyte combination (which can be many decades belowmicromolar) can be dealt with.

In former publications from our group [19,22], we derived andtested a transpose of the NE equation (Eq. (1)), expressing the ana-lyte concentration as a function of the measured potential over theentire concentration interval: see Eq. (2).

canalyte =(

10mV/S − 1)

× Cst (2)

We experience a complete fitting of Eq. (2) type graphs in theconcentration range from 10−4.5 M down to 10−8 M and lower.Above 10−4.5 M concentrations, deviations start to occur which areattributed to surface saturation. The validity of Eqs. (1) and (2) wasverified for an extensive set of compounds in our (U)HPLC and FIAcalibration measurements. Plotting 10mV/S − 1 against canalyte yieldsR2 values close to one.

The response rate of the sensors was measured with differentanalyte concentrations in a flow injection analysis setup: see Fig. 1.Metergoline was used as an example, and injected as square con-centration pulses of different concentrations in the FIA setup. It isa mycotoxin which can occur in Ergot contaminated food samples.The electrode material was made as described under Section 2.3.The upgoing part of the obtained potentiograms is often referredin this text as the “on” part, and the downgoing part is referredas the “off” part. The data were used to estimate the kinetics andthe Gibbs free energy of interaction between the sensor coatingmaterial and the analyte. The obtained results perfectly fit themodel published by our group [22]. We were able to calculate kon

(3.36 × 106 min−1 M−1), koff (3.26 min−1), Kass (1.03 × 106 M−1) and�G values (6.72 kcal mol−1) for the interaction of metergoline tothe PVC-based sensor coating, using the data shown in Fig. 1, asexplained in the next paragraph.

c detection in (ultra) high performance liquid chromatography and013), http://dx.doi.org/10.1016/j.aca.2013.03.031

Kass is the association constant for the formation of an ana-lyte/surface complex (adsorption of the analyte on the surface:see insert drawing in Fig. 1). Especially in the molecular biologyand pharmacy literature, its inverse, the dissociation constant Kdiss

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s mostly used to describe the complex formation between twoolecular species. The reaction rate constant of the complex for-ation is kon, and the reaction rate constant of complex breakdown

s koff. An extensive description of how these adsorption/desorptionate constants are calculated from potentiograms as shown inig. 1 is described in [22]. The potential determining process in theotentiometric sensors studied here is the formation of a complexetween the analyte ion and the sensor surface:

nalyte + Surface � Analyte × Surface

Complex formation rates are described by the rate constant kon,nd complex dissociation rates by the rate constant koff. The ratiof both is equal to the equilibrium constant of the Analyte × Surfaceomplex association, Kass: Kass = kon/koff (in the study of associ-tion/dissociation phenomena, the inverse of Kass, Kdiss, is oftensed).

The potentiogram data of Fig. 1 are first converted from a mV y-xis to the 10mV/S − 1 y-axis, which is linearly related to the injectedoncentration of the analyte, canalyte [22]. In this conversion of theeasured potential (in mV) to a concentration related signal, “S”

s the slope of the Nicolskii–Eisenmann calibration graph for thenalyte: see Eq. (1). Potentiograms plotted in this way show expo-ential upgoing (“on”) and downgoing (“off”) curves of the typeiven in Eqs. (3) and (4) respectively [22]:

= Rplateau × (1 − e−kobscanalyte·t) (3)

= Rplateau × e−koff·t (4)

In these equations, koff has the meaning explained above. Theate constant kobs (“obs” stands for “observed”) in the upgoing oron” phase of the potentiograms, is a function of kon and koff, as inhis phase both adsorption and desorption are active: see Eq. (5)

obs = kon × canalyte − koff (5)

The most accurate way to calculate kon and koff is now to plot kobsalues versus canalyte. This should give a straight line with slope andntercept respectively equal to kon and −koff. The method is calledhe “linearization method” in the literature. For confirmation, welso calculated koff values from the “off” phase curves, using Eq. (4),nd checked whether they matched the koff value calculated usingq. (5).

In Eqs. (3) and (4), “Rplateau” represents the response value,btained at the plateau of the potentiogram. R is the response atny other place in the potentiogram. These R values are the concen-ration related responses, R = 10mV/S − 1. In electronic applications,he “time constant” is generally expressed as the time requiredor the signal to drop down to Rplateau/e (“e” being the base of theatural logarithm, equal to 2.718), which means to 37% of Rplateau.or the “off” exponentials, which have concentration independentime constants, this is calculated to be 18.3 s for the 3 mm diame-er electrode used in the conditions of Fig. 1. This is far too high forPLC, and certainly for UPLC. Time constants in the “on” parts of theotentiograms are concentration (canalyte) dependent. Higher con-entrations show a more rapid “on” response. We can calculate theime, required to go from a zero response to 63% of Rplateau (which

eans we are 37% below the final value, in this case Rplateau). Atanalyte values of 10−4, 10−5 and 10−6 M, they are respectively 0.18,.8 and 18 s. So, when dealing with 10−6 M concentrations or lower,hese time constants are also to be reduced drastically in order to

ake the sensors compatible with (U)HPLC where time constantsower than 1 s are usually expected. In the next paragraphs, we willhow how our adsorption/desorption model [22] allows to study

Please cite this article in press as: D. Daems, et al., Use of potentiometrimodelling with adsorption/desorption binding kinetics, Anal. Chim. Acta (2

ime constant optimization. It is quite obvious that the choice ofensor coating material will be crucial in the future developmentf rapid sensors. In the present study, we will first focus on theffects of sensor dimensions and of organic modifier.

coated wire electrode contains PVC, DOS and TCPB. The eluent was 5 mM H3PO4.The potentiograms are in descending order 10−4 M (upper graph), 10−4.5 M, 10−5 M,10−5.5 M, 10−6 M, 10−6.5 M and 10−7 M (lower graph) injections of metergoline.

3.2. Effects of sensor dimensions and organic modifier

A 3D drawing of the large volume wall-jet flow-cell is given in[22]. All of the above results were obtained with 3 mm diameterelectrodes, as these could be constructed with good reproducibil-ity in high quantities. Changing to 1 mm diameter sensors hassome clear effects, which are already obvious when inspectingthe potentiograms in Fig. 2. The kon value, calculated using allthe potentiograms in Fig. 2 with our adsorption model (describedin [22] and briefly explained under Section 3.1) increases from3.4 × 106 min−1 M−1 for a 3 mm diameter electrode (see Section3.1) up to 1.1 × 107 min−1 M−1. The time constant was estimatedto be 0.056 s, 0.56 s and 5.6 s for 10−4, 10−5 and 10−6 M concentra-tions respectively. This is 3 times faster than the 3 mm diameterelectrode (see data under Section 3.1) in the upgoing part of theblock pulse. This means that, at least for the upgoing part of the sig-nal, miniaturization is very beneficial. The reason for this is as yetunclear. Reduction of the sensor surface with a factor 9 reduced the“on” time constant with a factor 3.2. Further reduction of the sensordimension would still be required however for (U)HPLC purposesbecause this could provide smaller time constants, especially in thelow concentration ranges. Desorption rates on the other hand areindependent on the surface area. The koff value measured from thedata shown in Fig. 2 was 2.28 min−1. This is a rate constant whichis comparable to the one mentioned above for a 3 mm diameterelectrode (see Section 3.1).

If the Rplateau value of a 10−5 M metergoline injection on a 3 mmelectrode (see Fig. 1, 118 mV) is compared with the one on a 1 mmelectrode (see Fig. 2, 198 mV), an increase of the maximum responseis noticed at a 1 mm electrode. This effect was not systematicallyinvestigated however.

Further miniaturization to sub-mm electrodes will most prob-ably speed up the “on” kinetics, but will leave the “off” kineticsunchanged. We successfully used 100 �m electrodes in capillaryelectrophoresis [19]. Decreasing the electrode diameter below1 mm still poses practical problems to obtain enough quantitiesof such electrodes with good impedance characteristics, in reason-able time periods. Therefore, we choose 1 mm diameter electrodesfor our (U)HPLC experiments (see Sections 3.3 and 3.4). Theirbehaviour was first tested in FIA, with 30% CH3CN in the runningbuffer: see Fig. 3.

c detection in (ultra) high performance liquid chromatography and013), http://dx.doi.org/10.1016/j.aca.2013.03.031

Adding an organic modifier such as CH3CN to the eluent doesnot affect the rate of association (kon), but accelerates the rateof dissociation (koff): see Table 1, and Fig. 3. kon is constant inthe four eluents with varying CH3CN concentrations (average:

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Fig. 3. Potentiograms (in mV) of 10−4 M (upper graph), 10−4.5 M, 10−5 M, 10−5.5 M,10−6 M, 10−6.5 M and 10−7 M (lower graph) of metergoline in a running buffer con-tc

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Fig. 4. Separation of a mixture of alkaloids and plant phenolics, detection withUV (red) and potentiometry (blue). MN-Nucleodur 100-5 CN-RP 125/4 mm col-umn. Linear gradient elution from 7.5% CH3CN in 5 mM H3PO4 to 30% CH3CN in30 min. Flow-rate is 1 mL min−1. 1 mm diameter sensor containing PVC, DOS andTCPB. Cocaine (A) 1.5 × 10−4 M, p-coumaric acid (B) 1.5 × 10−4 M, p-coumaric acid

Cst.

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aining 70% HCOOH (0.1%, v/v) and 30% CH3CN. The 1 mm coated wire electrodeontains PVC, DOS and TCPB.

on = 2.15 ± 0.16 × 107 min−1 M−1). We also determined Kass valuessing the new model published recently by our group [22], and dis-ussed briefly under Section 3.1. Each time, measurements wereone in the same manner and with the same series of concentra-ions as for the above-described experiments (Figs. 1 and 2). Kass inn eluent with 0% CH3CN is somewhat higher for the 1 mm sensorTable 1) than found in the experiment with the 3 mm sensor (seebove) where we used another buffer counter ion. The Kass valuesand correspondingly, �G values) decrease when the percentagef CH3CN increases: see Table 1. When plotting the �G values as aunction of Rplateau (mV), the number of mV generated by the sensorsee Table 1), a linear relationship is obtained (R2 = 0.9997; N = 4).

hen we project the Rplateau (mV) values to 1 M concentrations,he straight line (R2 = 0.9964) passes through the origin. These datare a nice proof of the approach that chemical energy of interac-ion (�G of association) is converted to electrical (capacitor) energy20]: �G = −nFE.

This is very convenient for use of the detector in (U)HPLC, wheren organic component is practically always present in the eluent.he progression from 0% CH3CN to 30% CH3CN increases koff with aactor of 5. This reduces the “off” time constant to 3.7 s. This is stilloo high for (U)HPLC systems. An additional advantage is experi-nced in gradient (U)HPLC analysis however, where the increase ofrganic modifier with time creates “regeneration” buffer (a buffersed to desorb components from surfaces) conditions giving anxtra decrease of the time constant.

CH3CN concentrations in the eluent, exceeding 50% (v/v), willecrease the performance of the detector in time, due to slow dis-olution of plasticizer components. At concentrations below 30%,he sensor is robust for long time periods.

Effects of the diameter of the PEEK tubing spray nozzle wereot yet investigated. It is expected to affect the thickness of the

Please cite this article in press as: D. Daems, et al., Use of potentiometrimodelling with adsorption/desorption binding kinetics, Anal. Chim. Acta (2

iffusion layer, and therefore also the response time. We estimatedowever that adsorption kinetics is still by far the more important

actor at this moment.

able 1alues of kon, koff, Kass and �G for metergoline measured in HCOOH running buffer contaiesponses (in mV) for a 10−5 M metergoline injection are presented.

Percentage of ACN [%] kon [min−1 M−1] koff [min−1]

0 2.37 × 107 3.180

10 1.83 × 107 4.90

20 2.23 × 107 7.51

30 2.19 × 107 16.13

methylester (C) 1.5 × 10−4 M, p-coumaric acid ethylester 1.5 × 10−4 M (D), ergo-cornine (E) 4.2 × 10−4 M, ergotamine (F) 1.5 × 10−4 M, impurity ergocornine (G),solanidine (H) 6 × 10−5 M and metergoline (I) 2.5 × 10−4 M.

3.3. Application in HPLC conditions

The potential of the potentiometric detection system is demon-strated in Fig. 4.

Here, we injected a mixture of plant alkaloids (cocaine, ergo-cornine, ergotamine, solanidine and metergoline) and non-ionic(not potentiometrically active) secondary plant phenolics withgood UV absorption properties (p-coumaric acid and its methyland ethylester). The electrode diameter used was 1 mm. Cationicorganics with log P values (a lipophilicity indicator used mainlyin Pharmacy) above 2 will generally have very low detectionlimits [21]. The potentiometric detection limits (peak heightequal to 12�noise, 20 �L injection) in the HPLC conditions ofFig. 4 were: cocaine 8.71 × 10−7 M, ergocornine 3.58 × 10−5 M,ergotamine 2.44 × 10−5 M, solanidine 2.47 × 10−7 M, and metergo-line 3.58 × 10−6 M (injected concentrations). These potentiometricdetection limits are all in the low ng range (injected amounts). Weused the detection limit as generally defined in HPLC. In poten-tiometry, another definition of detection limit is used, based on thedeflection point occurring in the NE calibration graph (Eq. (1)). Inpotentiometry, this graph is only used in the part where the sig-nal (mV) versus log canalyte curve is linear. However, Eq. (2) allowsus to use calibration graphs which are totally linear, even in thelow concentrations region. The only requirement is to offset thesensor response to 0 mV for canalyte = 0 (a blank sample), and toassume that the NE equation (Eq. (1)) is valid. This can be easilyverified with the “solver” plugin in Excel, which will fit the exper-imental measurements (response in mV, versus log canalyte) to aNE curve (Eq. (1)), and calculate the best fit values for E0, S, and

c detection in (ultra) high performance liquid chromatography and013), http://dx.doi.org/10.1016/j.aca.2013.03.031

The full potential of the sensitivity of potentiometry can clearlybe better exploited in (U)HPLC than in batch methods, as we canwork in the low concentration, non-linear part of the NE curve

ning various percentages of acetonitrile (CH3CN). In the last column, the maximum

Ka [M−1] �G [kcal mol−1] Rplateau [mV]

7.44 × 106 7.87 1553.73 × 106 7.47 1192.97 × 106 7.34 1091.36 × 106 6.88 68

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6 D. Daems et al. / Analytica Chimic

Fig. 5. Chromatograms obtained from an UPLC measurement with potentiometricdetection (upper curve, black) and MS (lower curve, blue). Cocaine (A), papaverine(m

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B), metergoline (C), solanidine (D) and drophenine (E) are present in the injectedixture (6 �L) at 2 × 10−5 M concentrations.

n (U)HPLC. The linear dynamic range of the detector with a con-entration related output (10mV/S − 1 versus canalyte) easily spans

decades of concentration, which can be advantageous whenhecking compounds for impurities. Fig. 4 also demonstrates theelectivity of the detector, which detects the alkaloids only, beingotally non-responsive to the phenolic secondary metabolites. Aon-UV absorbing alkaloid such as solanidine is detected very sen-itively (0.621 ng detection limit), in comparison with UV (5.11 ngetection limit). Even highly UV absorbing alkaloids such as cocainend metergoline have similar detection limits for the potentiomet-ic detector as compared to the UV detector. Generally, the timeonstant is low enough for HPLC. At the longer contact times appli-able in HPLC, diffusion of the analyte into the ionically conductiveensor coating may occasionally decrease koff values and provokeome peak broadening.

.4. Application in UPLC conditions

The sensor was examined as a detector in UPLC (see Fig. 5). Thenalytes metergoline, papaverine, cocaine, solanidine and drophe-ine were injected at 10−5 mol L−1 (dissolved in 100% HPLC gradecetonitrile and injected in 6 �L).

The potentiometric sensor/UPLC combination gave around threeimes lower detection limits when compared to the potentiomet-ic sensor/HPLC combination, and some ten times higher detectionimits than the tandem mass spectrometer detector/UPLC combi-ation (see Table 2).

As it can be seen in Fig. 5, the potentiometric sensor performsell in this rapid separation system, but still needs faster time

Please cite this article in press as: D. Daems, et al., Use of potentiometrimodelling with adsorption/desorption binding kinetics, Anal. Chim. Acta (2

onstants to avoid loss of resolution.

able 2etection limits (DL) of the alkaloids used in UPLC, expressed as nanomolar (nM)oncentrations injected. Potentiometric (Pot) and tandem mass spectrometric (MS)alues are shown (peak height equal to 12 × �noise, 6 �L injection).

Alkaloid Pot DL (nM) MS DL (nM)

Cocaine 248 31.2Papaverine 274 84.2Metergoline 1061 50.5Solanidine 637 85.2Drophenine 988 119

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PRESSa Acta xxx (2013) xxx– xxx

4. Conclusions

Adsorption/desorption analysis via potentiogram recordings atdifferent analyte concentrations reveals the strengths, and thepossibilities for optimization of potentiometric sensors for use inhydrodynamic techniques. The potentiograms show simple expo-nential “on” and “off” behaviour when the mV output is convertedto a canalyte dependent signal. Langmuir adsorption is experienced,as well as some diffusion control. Diffusion control is neglige-able in UPLC, and only appears if the analyte molecule has longcontact times with the sensor such as in HPLC peaks with over20 s peak width. In the latter case, a minor peak broadening wasobserved experimentally due to diffusion of the analyte out ofthe sensor coating. Potentiometric detection for (U)HPLC usesthe concentration related sensor output signal. The detector isable to work linearly in four to five decades of concentration.Sub-micromolar detection limits (injected concentrations) can beobtained in UPLC. Major improvements of sensor “on” kinetics areto be expected from further miniaturization of the electrode diam-eter. Finding ionically conducting materials with high “on” and“off” rates are goals for future research. Given the typical Langmuiradsorption behaviour, future studies will certainly also concentrateon the promising use of potentiogram and linearization methodsin the study of analyte/surface interactions with potentiometricsensors.

Acknowledgements

Financial support for this work was provided by the Univer-sity of Antwerp by granting L.N. and G.V.C. a BOF interdisciplinaryresearch project, L.N. a BOF project, and M.F. and L.N. an ErasmusMundus–EADIC project. We want to thank J. Everaert, A. Babiczand T. Willems for their help in interpreting the results. We wantto thank CeProMa (University of Antwerp, Belgium) and PGO (Uni-versity of Antwerp, Belgium) for the use of the UPLC and the MSinstruments.

References

[1] E. Bakker, E. Pretsch, Trends Anal. Chem. 27 (2008) 612–618.[2] J. Bobacka, A. Ivaska, A. Lewenstam, Chem. Rev. 108 (2008) 329–351.[3] A. Bratov, N. Abramova, A. Ipatov, Anal. Chim. Acta 678 (2010) 149–159.[4] D.G. Pijanowska, W. Torbicz, Biocybern. Biomed. Eng. 28 (2008) 11–19.[5] K. Maehashi, K. Matsumoto, Sensors 9 (2009) 5368–5378.[6] M. Graf, D. Barrettino, H.P. Baltes, A. Hierlemann (Eds.), CMOS Hotplate Chem-

ical Microsensors, Springer Verlag, Berlin, 2010.[7] Y. Bao, J. Everaert, M. Pietraszkiewicz, O. Pietraszkiewicz, H. Bohets, H.J. Geise,

B.X. Peng, L.J. Nagels, Anal. Chim. Acta 550 (2005) 130–136.[8] Y. Wang, Y. Zhou, J. Sokoloa, B. Rigas, K. Levon, M. Rafailovich, Biosens. Bioelec-

tron. 24 (2008) 162–166.[9] W.B. Liang, W.J. Yi, S.H. Li, R. Yuan, A. Chen, S. Chen, G.M. Xiang, C.M. Hu, Clin.

Biochem. 42 (2009) 1524–1530.10] A.K. Bell, L. Hofler, M.E. Meyerhoff, Electroanalysis 24 (2012) 53–59.11] D.P. Tang, R. Yuan, Y.Q. Chai, Y.Z. Fu, J.Y. Dai, Y. Liu, X. Zhong, Biosens. Bioelec-

tron. 21 (2005) 539–548.12] D.P. Tang, R. Yuan, Y.Q. Chai, L.Y. Zhang, X. Zhong, Y. Liu, J.Y. Dai, Sensor. Actuat.

B – Chem. 104 (2005) 199–206.13] M. Trojanowicz, Anal. Chim. Acta 653 (2009) 36–58.14] M. Trojanowicz, Anal. Chim. Acta 688 (2011) 8–35.15] P.W. Alexander, P.R. Haddad, G.K.C. Low, C. Maitra, J. Chromatogr. 209 (1981)

29–39.16] P.R. Haddad, P.W. Alexander, M. Trojanowicz, J. Chromatogr. 315 (1984)

261–270.17] D. Zielinska, A. Gil, M. Pietraszkiewicz, O. Pietraszkiewicz, D. Van de Vijver, L.J.

Nagels, Anal. Chim. Acta. 523 (2004) 177–184.18] B. Vissers, J. Everaert, J. Sekula, A. Malak, H. Bohets, G. Bazylak, L.J. Nagels, J. Sep.

Sci. 32 (2009) 167–179.19] J. Sekula, J. Everaert, H. Bohets, B. Vissers, M. Pietraszkiewicz, O. Pietraszkiewicz,

F. Du Prez, K. Vanhoutte, P. Prus, L.J. Nagels, Anal. Chem. 78 (2006) 3772–3779.

c detection in (ultra) high performance liquid chromatography and013), http://dx.doi.org/10.1016/j.aca.2013.03.031

20] L.J. Nagels, Pure Appl. Chem. 76 (2004) 839–845.21] B. Vissers, H. Bohets, J. Everaert, P. Cool, E.F. Vansant, F. Du Prez, J.M. Kauffmann,

L.J. Nagels, Electrochim. Acta 51 (2006) 5062–5069.22] K. De Wael, D. Daems, G. Van Camp, L.J. Nagels, Anal. Chem. 84 (2012)

4921–4927.

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ARTICLECA-232460; No. of Pages 7

D. Daems et al. / Analytica

Please cite this article in press as: D. Daems, et al., Use of potentiometrimodelling with adsorption/desorption binding kinetics, Anal. Chim. Acta (2

23] R.B.M. Schasfoort, A.J. Tudos (Eds.), Handbook of Surface Plasmon Resonance,Royal Society of Chemistry, Cambridge, UK, 2008.

24] M. Dragusanu, B.A. Petre, S. Slamnoiu, C. Vlad, T. Tu, M. Przybylski, J. Am. Soc.Mass Spectrom. 21 (2010) 1643–1648.

25] G. Bazylak, L.J. Nagels, J. Pharm. Biomed. Anal. 32 (2003) 887–903.

[

[

PRESSa Acta xxx (2013) xxx– xxx 7

c detection in (ultra) high performance liquid chromatography and013), http://dx.doi.org/10.1016/j.aca.2013.03.031

26] L.J. Nagels, H. Bohets, M. Jimidar, Potentiometric Electrode, Gradient Poly-mer, Uses and Method of Preparation Therefor. Patent, U.S. 7,857,962B2.

27] K. Wojciechowski, M. Kucharek, W. Wróblewski, P. Warszyn’sky, J. Electroanal.Chem. 638 (2010) 204–211.