ResearchArticle Host-TailoredSensorsforLeucomalachite ...recipp.ipp.pt/bitstream/10400.22/6734/1/ART_MoreiraFelismina_2013.pdf · JournalofChemistry 5 Wavenumbers (cm−1) Sulfonic

Hindawi Publishing CorporationJournal of ChemistryVolume 2013 Article ID 605403 13 pageshttpdxdoiorg1011552013605403

Research ArticleHost-Tailored Sensors for LeucomalachiteGreen Potentiometric Measurements

F T C Moreira R B Queiroacutes L A A Truta T I Silva R M CastroL R Amorim andM G Sales

BioMarkISEP and School of Engineering Polytechnic Institute of Porto Road Dr Antoacutenio Bernardino de Almeida431 4200-072 Porto Portugal

Correspondence should be addressed to M G Sales goretisalesgmailcom

Received 19 June 2012 Revised 8 August 2012 Accepted 8 August 2012

Academic Editor Jose Alberto Pereira

Copyright copy 2013 F T C Moreira et alis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

A new biomimetic sensor for leucomalachite green host-guest interactions and potentiometric transduction is presented earticial host was imprinted in methacrylic acid or acrylamido-2-methyl-1-propanesulfonic acid-based polymers Molecularlyimprinted particles were dispersed in 2-nitrophenyloctyl ether and trapped in poly(vinyl chloride) e potentiometric sensorsexhibited a near-Nernstian response in steady state evaluations with slopes and detection limits ranging from 458 to812mV decademinus1 and 028 to 101 120583120583gmLminus1 respectively ey were independent from the pH of test solutions within 3 to 5Good selectivity was observed towards drugs that may contaminate water near sh cultures such as oxycycline doxycyclineenrooxacin trimethoprim creatinine chloramphenicol and dopaminee sensors were successfully applied to eld monitoringof leucomalachite green in river samples e method offered the advantages of simplicity accuracy applicability to colored andturbid samples and automation feasibility

1 Introduction

Industrial aquaculture is a rapidly growing industry inmany developed and developing countries such as CanadaJapan Russia and China A signicant growth at food shproduction has been observed over the past decade due to theprevention or elimination of sh diseases e introductionof veterinary medicines such as antimicrobials at the foodproduction area has born the main responsibility for thisscenario However the wide use of antibiotics in aquacultureled to environmental and food spread of antimicrobials andmay result in the emergence of antibiotic-resistant bacteriain aquaculture environments [1 2] For food safety purposessh samplesmust be subect of rigorous and frequent controlsthat ensure that residues of antimicrobials are below themaximum legal levels [3]

Malachite green (MG) is among the drugs nding exten-sive use all over the world in the sh farming industrysince the 1930s [4] It is a dye and acts as a fungicideectoparasiticide and disinfectant Recent studies pointed outthat it is a suspected carcinogen showing highly cytotoxic

effects to mammalian cells and acting as a liver tumor-enhancing agent ese observations lead to its restricted orprohibited use in aquaculture farming activities worldwidence absorbed by sh tissue MG is metabolically reducedto leucomalachite green (LMG) its reduction product andmaor metabolite that is found in waters and sh tissuesus an efficient and low-cost method for monitoring LMGwould be highly appreciated [5] in order to prevent furtherdissemination of MG in the environment by means ofaquaculture activities

e analytical procedures suggested in the literaturefor LMG include microbiological methods [6ndash8] or liquid-chromatographic techniques [7ndash17] with uorescence [1819] andor in combination with UV DAD or MS detection[20ndash27] e rst one is unsuitable for routine controlprocedures as each trial may take several days laboratoriesrequire also proper facilities to handle biological compoundssafely Chromatographic techniques are accurate precise androbust but contribute to dispose highly toxic compoundsand are not sufficiently quick for routine control purposese same comments may apply to the electrophoretic-based

2 Journal of Chemistry

procedures reported in literature [28] Other reported meth-ods are based on immunoassays [29] although they providespecic responses the overall proceduremay take a long timeand turns out expensive

Alternative and advantageous methods should rely onexpeditious and ecient procedures providing highly specicand sensitive measurements e specic recognition of acertain analyte may be achieved by molecularly imprintedpolymers (MIPs) ese synthetic polymers are preparedby letting functional and cross-linking monomers organizearound a template (to be imprinted) and copolymerize themto form a highly cross-linked polymeric structure [30 31]e template is removed aer creating vacant places wherethe functional groups of the monomeric forms are strictlyoriented to bind this specic compound e producedpolymer is thus capable of rebinding the analyte with a highspecicity In many occasions it shows binding anitiesapproaching those presented by antigen-antibody systems[32 33] while offering higher stability at extremes of pHand temperature high mechanical strength and lower costthan their natural counterparts More recently several paperson the synthesis of MIP materials for malachite green havebeen published [34ndash37] ese materials are always obtainedby bulk imprinting being methacrylic acid (MAA) the onlyfunctional monomer tested and applied (or intended for)chromatographic applications

e great potential of MIP materials has led to theirinclusion as recognition elements in sensors Transduction ofdifferent nature has been employed for this purpose [38 39]with great emphasis on electrochemisty [40] Potentiometryis included here and offers the advantages of not requiringthe template to be extracted from the membrane and thespecies to be diffused through the membrane [40] Despitethese advantages most research work within this led hasbeen conducted only most recently [41ndash50]

erefore the present work tries to (i) develop MIPmaterials designed for LMG insteadMG because LMG is themajor sh metabolite that may be found in waters (ii) searchfor an alternative functional monomer by replacing thetraditionalMAAby acrylamido-2-methyl-1-propanesulfonicacid (AMPSA) (iii) and open new horizons on the use ofthesematerials by applying these in biosensing developmentsomething never done before for LMG nor MG e sen-sory materials were thus synthesized with methacrylic acid(MAA) and acrylamido-2-methyl-1-propanesulfonic acid(AMPSA) functional monomers cross-linked by ethyleneglycol dimethacrylic acid (EGDMA) within the templatemolecule e selective membranes were obtained by dis-persing these materials in a PVC matrix plasticized with o-nitrophenyl octyl ether (oNPOE)epotentiometric sensorswere evaluated in steady-state and owingmedia and appliedto the analysis of contaminated water

2 Experimental

21 Apparatus All potential measurements were made by aCrison 120583120583pH 2002 decimilivoltammeter (plusmn01mV sensitivity)at room temperature and under constant stirring by means

of a Crison micro ST 2038 e output signal in steady stateevaluations was redirected to a commutation unit linked tosix ways out enabling the simultaneous reading of six ISEse assembly of the potentiometric cell was as follows con-ductive graphite | LMG selectivemembrane | buffered samplesolution (phosphate 5 times 10minus2mol Lminus1 pH 45) || electrolytesolution KCl | AgCl(s) | Ag e reference electrode wasan Orion AgAgCl double junction (Orion 90-02-00) eselective electrode was prepared in conventional or tubularcongurations [51] for batch and ow mode evaluationsrespectively Both devices had no internal reference solutionand epoxy graphite was used as solid contact

e Flow Injection Analysis (FIA) assembly had a GilsonMinipuls 2 peristaltic pump tted with PVC tubing (080160 andor 200mm id) and a four-way Rheodyne 5020injection valve holding a loop of variable volume eseveral components were gathered by PTFE tubing (OmnitTeon 08mm id) Gilson end-ttings and connectorsesupport devices for tubular and reference electrodes andthe conuence point accessory were constructed in PerspexAer reaching a stable baseline the emf was recordedcontinuously by means of a homemade high-impedance dataacquisition eight-channel box connected to a PC through theinterface ADC 16 (Pico Tech UK) and PicoLog for windows(version 507) soware

When necessary the pH was measured by a CrisonCWLS7 combined glass electrode connected to a decim-ilivoltammeter Crison pH meter GLP 22 A double-beamUV-vis spectrophotometer (Shimadzumodel 1601) equippedwith 10-mm quartz cells was used for absorbance measure-ments in binding studies Infrared spectra were collected bya Nicolet 6700 FTIR spectrometer surface measurementswere carried out in ATR (attenuated total reectance) modewith the Nicolet ATR sampling accessory of diamond contactcrystal

22 Reagents All chemicals were of analytical grade anddeionized water (conductivity lt01120583120583S cmminus1) was employedLMG potassium tetrakis (4-chlorophenyl)borate (TpClPB)o-nitrophenyloctyl ether (oNPOE) poly (vinyl chloride)(PVC) of high molecular weight EGDMA AMPSA andMAA were purchased from Fluka Benzoyl peroxide (BPO)methanol (MeOH) chloroform and tetrahydrofuran (THF)were obtained from Riedel-deHaumlen

23 Synthesis of Host-Tailored Polymers For molecularlyimprinted polymers the template (LMG 10mmol) wasplaced in a glass tube (140mm id) with the functionalmonomer (40mmol MAA) the cross-linker (EGDMA200mmol) and the radical initiator (BPO 024mmol) alldissolved in 8mL chloroform A polymer of AMPSA wasprepared similarly e mixture was sonicated degassedwith nitrogen for 5min and cured at 70∘C for 30minNonimprinted polymers (NIP) were prepared in a similarway by excluding the template from the procedure

e resulting polymerswere ground and sieved to particlesizes ranging from 50 to 150 120583120583m Extraction of the templatemolecule and washout of nonreacted species was carried

Journal of Chemistry 3

T 1 Membrane composition of LMG sensors cast in 200mg of PVC

ISE Active ingredient Plasticizer Additive Weight (mg)I MIPMAA oNPOE mdash 15 350II NIPMAA oNPOE mdash 15 350III MIPAMPSA oNPOE mdash 15 350IV NIPAMPSA oNPOE mdash 15 350V MIPMAA oNPOE TpClPB 15 350 7VI MIPAMPSA oNPOE TpClPB 15 350 7VII MIPMAA washed oNPOE mdash 15 350VIII MIPAMPSA washed oNPOE mdash 15 350VIII mdash oNPOE mdash mdash 350

out with methanolacetic acid (9 1 vv) All polymers(MIPMAA NIPMAA MIPAMPSA and NIPAMPSA)were let dry at ambient temperature before use

24 FTIR Analysis FTIR spectra were collected under roomtemperaturehumidity control aer background correctione number of scans was 32 for both sample and background119909119909-axis was wavenumber ranging from 525 to 4000 cmminus1 and119910119910-axis was transmittance e resolution was 4000

25 Binding Experiments e binding studies were car-ried out aer suitable washing of the synthesized particlesis was conrmed by measuring the absorbance of thewashout solution against blank at 253 nm the particleswere repeatedly washed with methanol until LMG was nolonger detected meaning that its concentration was belowthe limit of detection of the spectrophotometric readings(lt021120583120583gmLminus1)e polymer was let dry at ambient temper-ature before use

Binding experiments were carried out by placing 200mgof MIP particles (washed and dry) in contact with 100mL ofLMG standard solutions of varying concentrations rangingfrom 012 to 40mmol Lminus1 e mixtures were oscillated for12 h at room temperature and the solid phase was separatedby centrifugation (3000 rpm 10min) e concentration offree LMG in the supernatant was calculated by UV spec-trophotometry at 120582120582max = 253 nme increase in absorbancewas directly proportional to the concentration of LMG in thesupernatant obeying Beerrsquos law from 25 to 530 120583120583gmLminus1

e amount of LMGbound to the polymerwas calculatedby subtracting the concentration of LMG in solution (freeLMG) from the initial concentration the free LMG concen-tration was obtained by means of the previously indicatedcalibration data e obtained data was used for Scatchardanalysis

26 Sensory Membranes e sensing membranes were pre-pared by mixing 200mg of PVC 350mg of plasticizeroNPOE and 15mg of the sensing polymer (Table 1) cor-responding to a mass composition of 354 619 and 27respectively An amount of 7mg of TpClPB acting as anionicadditive was added to some of these membranes in thiscase the corresponding mass composition was 350 612

26 and 12 respectively e mixture was stirred untilthe PVC was well moistened and dispersed in 30mL THFese membranes were placed in conductive supports ofconventional or tubular shapes [51] Membranes were let dryfor 24 h and conditioned aerwards in a 1 times 10minus3mol Lminus1LMG solution e electrodes were also kept in this solutionwhen not in use e dry membranes were about 10 (plusmn01)mm thick and got thicker aer aqueous conditioning

27 Potentiometric Procedures All potentiometric measure-ments were carried out at room temperature e emf valueswere always obtained in solutions of xed pHionic strengthIncreasing concentration levels of LMG was obtained bytransferring 00200ndash100mL aliquots of 10 times 10minus3mol Lminus1LMG aqueous solutions to a 100mL beaker containing500mL of 50 times 10minus2mol Lminus1 of suitable buffer Potentialreadingswere recorded aer stabilization toplusmn02mVe cal-ibration graphs plotted emf as a function of logarithm LMGconcentration and were used for subsequent determinationof unknown LMG concentrations

28 Determination of LMG inRiverWater Samples ewatersamples were collected from different points along DouroRiver e collected samples had no antibiotics and weresubsequently spiked with two solutions of different concen-trations in LMG in order to produce a nal concentrationin LMG of 50 and 100 120583120583gmLminus1 A volume of 50mL ofthese solutions was further diluted in 25mL of 005mol Lminus1phosphate solution of pH 45 e direct potential methodwas applied to determine LMG in these samples

3 Results and Discussions

e ionophore or the ion carrier is the most vital compo-nent in a polymeric membrane sensor for potentiometrictransduction in terms of selectivity and sensitivity [52] Ionexchangers and neutral macrocyclic compounds have beenemployed for this purpose over the past decades Until nowsome reports found in the literature describe the use of MIPas potentiometric sensing materials [41ndash50]

Noncovalent interactions between the MIP active sitesand the template have been considered for allowing fastand reversible binding [52] AMPSA and MAA were used


100

150

200

250

300

350

400

0

50

100

150

200

250

(A1) AMPSA (B1) AMPSA

(A2) MAA (B2) MAA

0 1 2 3 4 5

0 1 2 3 4 5

0

02

04

06

08

10

12

0

01

02

03

04

0 100 200 300 400 500

0 50 100 150 200 250

F 1 Binding isotherm for LMGMAA and LMGAAMPSO imprinted polymer 119876119876 is the amount of LMG bound to 200mg of thecorresponding polymer 119905119905 119905 119905119905∘C 119881119881 = 100mL binding time 20 h (A2) Scatchard plot to estimate the binding characteristic of LMGimprinted polymer

as monomers as they allow electrostatic interactions mostlyby hydrogen bonds with the template compound Ion-pairinteractions may also be achieved with AMPSA

31 Binding Characteristic of the MIP In liquid phase appli-cations ofMIPs amolecule in solution interacts with bindingsites in a solid adsorbente free ligand concentration in theliquid phase aer equilibrium and reached is constant and isused to plot the corresponding adsorption isotherm

Adsorption isotherms plot the equilibrium concentra-tions of bound versus free ligand e bound ligand isexpressed in terms of binding capacity and was calculatedaccording to the following equation

119876119876 119905120583120583mol (LMG bound)119892119892 (MIP)

11990510076501007650119862119862119894119894 minus 11986211986211989111989110076661007666119881119881119904119904 times 1000119872119872MIP

(1)

where 119876119876 is the binding capacity of MIPs (120583120583mol gminus1) 119862119862119894119894the initial concentration of LMG (120583120583molmLminus1) 119862119862119891119891 the nalconcentration of LMG (120583120583molmLminus1)119881119881119904119904 the volume of the testsolution (mL) and MIP the mass of dry polymer (mg)

us the synthesized particles were let stand with a widerange of LMG concentrations for several hours and undercontinuous stirring When equilibrium was reached thefree LMG concentration was calculated by UV spectropho-tometry e corresponding binding capacities were thencalculated and plotted against the initial LMG concentration(Figure 1(a)) In general the adsorption data showed that thebinding capacity of the imprinted polymers increased withthe increasing of the initial concentration of LMG and had atendency to saturation for higher analyte concentrations

e above experimental data was used to carry out theScatchard analysis and estimate the binding parameters ofMIP particles e Scatchard equation

119876119876119862119862free11990510076491007649119876119876max minus 11987611987610076651007665119870119870119889119889

(2)

was applied for this purpose where 119876119876 was the bindingcapacity 119862119862free the free analyte concentration in equilibrium(120583120583mol Lminus1)119876119876max was the maximum apparent binding capac-ity 119870119870119889119889 was the dissociation constant of the binding sitePlotting 119876119876119876119862119862free versus 119876119876 a liner function was expected


Wavenumbers (cmminus1)

Sulfonic acid

stretch

stretch

stretch

C

C

O

C O

C O

C O

Bendamide

MAA MIPS

AMPSA MIPs

EsterAmide

stretchcarboxylic acidester

stretch ester

N H

H

stretch amide

3500 3000 2500 2000 1500 1000

80

70

60

50

40

30

20

10

80

70

60

50

40

30

20

10

0

Tra

nsm

itta

nce

(

)T

ran

smit

tan

ce (

)

2953

8 1636

1

1716

1

1450

2

1295

912

472

1138

4

943

2

3286

7

2982

9

1664

416

1115

762

1457

7

1352

513

298

1307

612

447

1219 11

753

1130

6 1115

510

379

991

8

F 2 FITR spectra of the MAA- and AMPSA-based polymers ATR accessory number of scans 32 scans resolution 4000

where 119870119870119889119889 was the slope and the y-intercept the apparentmaximum number of binding sites

e Scatchard plot for MIP particles made with AMPSA(Figure 1(B1)) showed a concave curve that is indicative ofthe presence of multiple classes of binding sites with different119870119870 values Its x-asymptote behavior may also indicate thepresence of nonspecic binding [53] Overall this concavecurve suggests that the interaction of the ligand is governedby a positive cooperativity phenomenon between strong andweak binding sites [54ndash56] On the contrary the Scatchardplot of MAA MIPs displayed a linear behavior meaning thatthe binding sites were uniform (Figure 1(B2)) and mostprobably based on hydrogen-bonding interactions e 119870119870119889119889and119876119876max of the binding sites were equal to 1283120583120583mol Lminus1 and268 120583120583mol gminus1 for dry polymer

32 FTIR Data FTIR spectra were taken by direct surfaceanalysis of the materials placing the MIP particles over adiamond crystal support of the ATR accessory e resultingspectra are indicated in Figure 2 Similar spectra wereobserved forNIPmaterials because they bear the same chem-ical functionse two or one peaks at about 3000ndash2950 cmminus1

were assignable to absorption bands from sp3 CndashH stretchingin ndashCH3 and ndashCH2 groups of atoms e absence of similarbands close to these and just above 3000 cmminus1 suggested thatmost monomers were suitably polymerized because no sp2

CndashH stretching was found Absorption peaks near 1450 cmminus1

were also attributed to sp3 CndashH bonds but resulted frombending the ndashCH3 or ndashCH2 groups

e amide function in AMPSAMIPs the carboxylic acidfunction in MAA MIPs and the ester function in the cross-linker have in common a carbonyl group making it difficultto distinguish the nature of the carbonyl involved in thisstrong and typical adsorption band In general its stretchand bend may be identied in several points of the spectralying within 1610ndash1720 cmminus1 (Figure 2) Regarding AMPSApolymers the amide function is clearly identied by the broadand strong adsorption band centered in 3287 cmminus1 corre-sponding to the stretch of the NndashH bond e sulfonic acidgroup has a typical strong adsorption at about 1350 cmminus1also present in the AMPSA MIP spectrum Regarding MAApolymers the carboxylic acid is identiable because of thestrong and sharp adsorption band of the carbonyl group

In general the observed spectra conrmed the expectedchemical functions suggesting a high degree of polymer-ization and conrming the adequate removal of nonreactedmonomers as well as template e washout from the tem-plate was also conrmed by analyzing theMIPmaterials aeralong the several washing steps

33 Sensor Performance LMG sensors contained eitherMIP or NIP particles as electroactive materials dispersedin plasticized PVC eir main analytical features wereobtained under batch conditions and followed the IUPACrecommendations [57] e results obtained are shown inTable 2 LMG sensors based in MIP particles displayeddifferent behavior in terms of sensitivity and detection limite sensors prepared with MAA and AMPSA showedrespectively linear responses starting at 50 times 10minus5 and


T 2 Main analytical features of the LMG potentiometric sensors in 50 times 10minus2mol Lminus1 phosphate buffer pH 45

ISE Slope (mV decademinus1) 1198771198772 (119899119899 119899 5) LOD (mol Lminus1) LLLR (mol Lminus1) 120590120590119907119907 (mV) C 119907119907119908119908 ()I 507 plusmn 05 0994 30 times 10minus5 50 times 10minus5 142 11II 56 plusmn 5 0996 32 times 10minus5 89 times 10minus5 354 32III 46 plusmn 3 0933 12 times 10minus5 16 times 10minus5 608 53IV 68 plusmn 2 0997 15 times 10minus5 30 times 10minus5 233 16V 78 plusmn 8 0998 43 times 10minus5 89 times 10minus5 177 095VI 68 plusmn 4 0993 13 times 10minus5 16 times 10minus5 816 60VII 81 plusmn 3 0992 36 times 10minus5 50 times 10minus5 mdash mdashVIII 78 plusmn 3 0993 39 times 10minus5 50 times 10minus5 523 60VIII 7 plusmn 3 0972 53 times 10minus4 42 times 10minus4 523 18C119907119907119908119908 coefficient variation if weekly calibration for 272 times 10minus4M 1198771198772 correlation coefficient LOD limit of detection LLLR lower limit of linear range 120590120590standard deviation of the analytical measure for a concentration in the middle of the calibration curve considering 3 consecutive calibrations

log[LMG M]

MIPMAA washed

MIPMAA NIPMAAMIPAMPSA washed

MIPAMPSA NIPAMPSA

minus6 minus55 minus5 minus45 minus4 minus35 minus3 minus25

MIPMAA minus additive MIPAMPSA minus additive

60m

Vd

ecad

e

log[LMG M]


F 3 Potentiometric response of LMG and PVC membrane selective electrodes Buffer phosphate stock solution 5 times 10minus3mol Lminus1under stirring and ambient temperature

16 times 10minus5mol Lminus1 LMG cationic slopes of 507 and 458mVdecademinus1 and detection limits of 99 and 40 120583120583gmLminus1 ecorresponding NIP particles displayed a linear response aer89 times 10minus5mol Lminus1 and 30 times 10minus5 LMG cationic slopesof 557 and 677mV decademinus1 and detection limits of 51and 50 120583120583gmLminus1 respectively In general MAA MIP sensorswere the only ones presenting a near-Nernst behavior whileNIP AMPSA displayed a clear super-Nernstian performance(Figure 3) Overall the MIP sensors displayed wider linearranges than the NIP-based sensors but this feature wascoupled to the lowest slopes is behavior may be attributedto the presence of template and unreacted monomers on theMIP materials hindering the performance of the electrodes

e sensors based in MIP particles were subsequentlywashed with methanolacetic acid Aer this procedure theyshowed linear responses starting at 50 times 10minus5mol Lminus1 LMGfor MAA and AMPSA respectively cationic slopes of 812and 778mV decademinus1 and detection limits of 119 and129 120583120583gmLminus1 In general terms super-Nernstian behaviorwasobserved for bothMIP sensorsmade withMAA andAMPSAmonomers When compared to the corresponding sensorswithout washing a signicant improve in terms of slope wasobserved for both sensors It seems that the existence oftemplate inside the membrane hinders the potential changeswithin the membranesolution interfaceis behavior couldnot be attributed to the mediating solvent because a blank


1 2 3 4 5 6 7 8

pH

MIPMAA washed

NIPMAA MIPMAA

1 2 3 4 5 6 7 8

pH

MIPAMPSA washed

NIPAMPSA

MIPAMPSA + additiveMIPMAA + additive

50

mV

50

mV

F 4 Reilley diagram for LMG

membrane (without ionophore) did not respond to LMG (seeTable 2)

To improve the operating features of the previous mem-branes the MIP-based sensors were added of an anioniclipophilic compound Typically the addition of ionic com-pounds of lipophilic nature to potentiometric sensors reducesthe anionic interference and lowers the electrical resistanceof the membranes [58 59] In this work TpClPB was selectedfor this purpose (Table 1) Sensors based in MIPMAA andMIPAMPSA showed linear response ranges within 89 times10minus5 and 16 times 10minus5mol Lminus1 142 and 43 120583120583gmLminus1 detectionlimits and near-Nernstian responses of 783 and 679mVdecademinus1 respectivelyWhen compared to the correspondingsensors without additive a signicant improve in terms ofslope was observed for both sensors (see Figure 3)

34 Response Time and Lifetime e time required to achievea steady potential response (plusmn3mV) using the proposedsensors in 10minus6 to 10minus4mol Lminus1 LMG solutions with arapid 10-fold increase in concentration was lt15 s Replicatecalibrations for each sensor indicated low potential drilong-term stability and negligible change in the responseof the sensors e sensors were stored and conditioned in1 times 10minus3mol Lminus1 LMG solution of pH 45 With all sensorsexamined the detection limits response times linear rangesand calibration slopes were kept within plusmn3 of their originalvalues over a period of at least 7 weeks

35 Potential Stability and LifetimeReusability e LMGsensors were stable for 1 month and were reused almostin a daily basis e slope dried less than 5mV decademinus1over this period and the linear range was unalteredAlong this period (excluding weekends) all electrodes were

conditioned independently and washed with water beforecalibration

e intermediate precision of the sensor was assessed bycomparing three independent potentiometric measures of aspecic concentration within calibrations made in differentdays For a concentration of 14 times 10minus4mol Lminus1 in LMGsolution the average potential dri was of plusmn35mV thusconrming the good precision of the device e intra-assay precision was accessed similarly by checking threeconsecutive calibrations and showed potential variations ofplusmn6mV Overall these results suggested a high stability of thepotentiometric device

36 Effect of pH LMG has two ionizable amine groupsas may be seen in Figure 4 with a pK1 of 69 [60] isfeature points out the need to evaluate and control the effectof pH over the potentiometric response is was studiedfor a test solution of 50 times 10minus4mol Lminus1 of LMG in whichpH was altered by the addition of small aliquots of con-centrated hydrochloric acid or saturated sodium hydroxidesolution

eReilley diagrams were obtained by plotting the emf ofthis solutions against its pH (Figure 4) e results indicatedthat the electrode did not respond to the pH change from20 to 50 Lower pHs rendered an increase of the analyticalsignal Since the LMG selective electrode responds to acationic species this increase in potential was correlatedto an interference of H+ Above pH 50 potentials starteddecreasing is behavior was attributed to the formationof the free LMG base in the solution leading to a decreasein the concentration of LMG ion is was conrmed bya perceptible precipitation occurring at higher pH valuesA pH of 45 was selected for further studies because the


H3CH3C

CH3

CH3

NH2

NH2

NH2

NH2

NH2

H2N

MIP

AM

PS

A+

ad

dit

ive

MIP

MA

A+

ad

dit

ive

minus05

minus1

minus15

minus2

minus25

minus3

0

MIP

AM

PS

A

wash

ed

MIP

AM

PS

A

NIP

AM

PS

A

DOXY

OXY

ENR

TMP

CREA

CHML

DPM

LMG

LMG

DOXY

TMP

ENRO

OXY

CREA

CHML

DPMM

IPM

AA

wash

ed

MIP

MA

A

NIP

MA

A

OO

O

O

O

O

O

O O O

O

O

O

OOH OHOH

OH

OH

OH

OH OH

OH

OH

OH

OH N

N

N

N

N

N

N

N

N N

N

HH

HH

FHO

HO

HO

HO

HN

Cl

ClN+

Ominus

F 5 Potentiometric selectivity coefficients (log KPOT) of LMG membrane-based sensors SSM solutions in buffer concentrationstested 138 times 10minus4 512 times 10minus4 and 102 times 10minus3mol Lminus1 under stirring and ambient temperature

solubility and ionization of LMG were both promoted underthis condition

37 Sensor Selectivity e selectivity of the chemical sen-sors was evaluated by calculating potentiometric selectivitycoefficients (119870119870Pot) by the separate solution method (SSM)[61] e obtained values of log119870119870Pot were plotted in Fig-ure 5 and indicated the degree of preferential interactionfor LMG over different organic and inorganic species thatare common in biological and food samples e formergroup includes several antibiotics such as oxycycline (Oxy+)doxycycline (Doxy+) enrooxacin (ENR) and trimethoprim(TMP) and creatinine (Crea+) chloramphenicol (CHML)and dopamide (DPM)

e sensors with AMPSA materials showed a similarselectivity pattern e log 119870119870Pot relative order for MIPAMPSAwas LMG≫DOXYgtOXYgtENRgtTMPgtCREAgtDPM gt CHML e corresponding washed and NIP sensorsdisplayed similar patterns excluding the higher interferencefrom DPM and CHML and the lower one from DOXYe additive on the selective membrane did not alter thegeneral relative order of selectivity but the absolute values oflog 119870119870Pot were much higher us the additive deterioratedthe selectivity of the MIP AMPSA-based sensor

For the potentiometric sensors with MIP MAA-basedparticles the relative order of log 119870119870Pot was LMG ≫ ENR gtDOXY gtTMP gtOXY gtDPM gtCHML gtTMP gtCREAecorresponding washed andNIP sensors displayed completely


different patterns when compared to the previous sequencee MIP washed sensor showed the relative sequenceLMG≫ ENR asymp TMP asympgtOXY gtDOXY≫ CREA gtDPM≫CHML e NIP sensor had a relative order of selectivityequal to LMG ≫ ENR gt DOXY ≫ OXY gt TMP gt DPM gtCHML ≫ CREA Unlike in AMPSA the additive on theMAA-based sensors improved the selectivity properties withthe exception of CHML

In general all sensors displayed a good selectivity forLMG with DOXY OXY ENR and TMP species interferinga bit more than DPM CHML and CREA e additive onAMPSA sensors decreased the selectivity properties of thedetector for which its use in routine applications is notrecommended MG was not tested as an interfering speciesbut it is expected to contribute signicantly to the totalresponse of the device So the analytical response under realconditions may be assumed as a measure of a global amountfrom LMG andor MG

38 Optimization of Flow Injection System Routine analysisprocedures require an expedite method In this case acontinuousmode of operation is of regular selection andmaybe achieved by means FIA systems ese are particularlyattractive in view of their versatility simplicity and suitabilityfor large-scale analyses

e ow assembly had a double-channel allowing theon-line adjustment of pH and ionic strength A ow cellhad a tubular conguration as described by amel andcoauthors [51] It had the same graphiteepoxy conductivematerial as the conventional electrodes is material lledthe inner gap of a cylindrical tube (1 cm length made ofPerspex) and was machined aer dry to have a 10mmdiameter central hole is inner hole was coated later bythe selective membranee resulting working electrode wasof simple fabrication and allowed full membranesamplecontact maintaining the general features of conventionalconguration ISEs in terms of homogeneity thickness andxed area e reference electrode was placed aer theworking electrode while the auxiliary (required to elimi-nate the noise created by the owing stream) was placedbefore

To take full advantages of this FIA system ow rateand injection volume were optimized in terms of sampledilution sensitivity sampling rate reagent consumption andwastewater generation e dispersion is amongst the mostimportant FIA parameters to estimate the degree of sampledilution because it depends on the capability of the electrodeto reachmaximum response (which relates the response timeand the ow rate of the carrier) ere are several waysto calculate dispersion one of which compares the signalproduced by introducing the sample as carrier (steady-statesignal 1198671198670) or by injecting it through the loop (119867119867) eresulting dispersion (119863119863) for the potentiometric response isgiven by

log119863119863 119863100764910076491198671198670 minus 11986711986710076651007665119878119878 (3)

where 119878119878 is the slope of the ow-potentiometric systemevaluated exactly under the same experimental conditionse percentage of steady state is another way to infer aboutthe dilution of the sample with the carrier inside the owtubes It corresponds to the percentage value of1198671198671198671198671198670 Unlike119863119863 it is however independent from the slope of the electrodeunder the underlying conditions

e sample loopwas variedwithin 100 and 500120583120583L (Figure6) For each injection volume a set of LMG standardsranging 10 times 10minus4 to 10 times 10minus2mol Lminus1 and preparedin water was injected into the buffer carrier stream esensitivity of the response increased markedly with theinjection volume up to 250 120583120583L For higher volumes the slopeof the potentiometric response remained almost constant(less than 5 increase) is observation was coupled todecreased sampling rates sample consumption and wastegeneration

e effect of ow rate was examined from 40 to120mLminminus1 recording for each condition of the aboveindicated calibration No signicant changes were observedin terms of slope and dispersion but the peak width and peakheight decreased with increasing ow ratesis good featurewas however coupled to the production of higher amountsof wastewaters On the other hand for lower ow rates thesensor required long time to recover to baseline lowering thenumber of sample outputs and broadening the peaks usas a compromise a ow rate of 9mLminminus1 was selected forfurther studies

e sensor ofwashedMIPMAAwas calibrated under theoptimum conditionsis electrode was selected for showingthe best compromise between selectivity and sensitivity issensor gave slopes of 450mV decademinus1 with detection limitsof 107 120583120583gmL and lower limits of linear range of 10 times10minus5mol Lminus1 e sampling-rate was approximately 170 runsper hour

39 Analytical Application Considering the detection capa-bilities of the proposed device one of its applications couldbe analytical sample handling prior the chromatographicanalysis Alternatively it could be used to monitor MGapplications in local farming units where its restricted useis allowed So the potentiometric analysis was conductedin steady state over river water e collected samples hadno antibiotics and were subsequently doped with LMG toproduce contamination levels of 50 and 100120583120583gmLminus1 (Table3) ese solutions were subsequently diluted 5 times withbuffer to carry out the potentiometric analysis e obtainedrecoveries were of 973 (plusmn66) or 1065 (plusmn01) respec-tively suggesting that the analytical results were accuratee relative errors were minus26 and +65 respectively alsoaccounting for the good accuracy of the methode relativestandard deviation was also small conrming the goodprecision of the analytical data e t-student test conrmedthat there were no signicant differences between the meansof the added amount and the potentiometric set of resultse calculated t-student was 063 below the theoretical one(127)


Sam

ple

Water

Buer

Waste

Pump

WasteReference

electrodeGround

electrode

Injection

valve

LMG sensor

(a)

Stea

dy

stat

e (

)

08

09

1

11

12

13

14

15

Dis

per

sio

n

70

80

90

100

110

120

Sam

pli

ng

rate

(h

)

80

85

90

95

100

105

0 100 200 300 400 500

(b)St

ead

y st

ate

()

Dis

per

sio

n

Sam

pli

ng

rate

(h

)0

1

2

3

4

90

130

170

210

250

50

70

90

110

130

150

4 6 8 10 12 14

Flow rate (mLmin)

(c)

F 6 Double-channel FIA assembly with phosphate buffer carrier (a) and the effect of loop (b) and ow rate (c) in the dispersionpercentage steady state and sampling rate of the reported signals Samplesstandards are prepared in water

T 3 Potentiometric determination of LMG in river water using MAAMIP-based membrane sensor

Sample Added (120583120583g LMGmLminus1) Found(a) (120583120583g LMGmLminus1) Recovery () Relative standard deviation () Relative error ()River water 1 50 49 plusmn 2 97 plusmn 7 50 minus26River water 2 100 107 plusmn 1 107 plusmn 1 01 +65(a)Found = mean plusmn standard deviation

4 Conclusions

Molecular imprinting technique was employed to produceLMG host-tailored sensors for potentiometric transductionMAA andor AMPSA were used as monomers to producedifferent MIP materials Both MAA- and AMPSA-basedsensors offered good analytical features ey were capableof discriminating LMG from other amine drugs in aqueousmediae advantages of these sensors include the simplicityin designing short measurement time good precision highaccuracy high analytical throughput low limit of detectionand good selectivity

e MIPMAA sensors were successfully applied to theanalysis of river water samples both in steady state and inowing media e proposed method is simple of low costprecise accurate and inexpensive regarding reagent con-sumption and equipment involved Wastewaters dischargedare of small concern to environment regarding its volume andcomposition

e tubular devices are particularly suitable for theroutine screening control of LMG in sh food ey produce

quicker responses for LMG than those provided by micro-biological methods and are much less expensive than thechromatographicmethods that are used for routine purposes

Acknowledgment

e authors acknowledge the nancial support from Fun-daccedilatildeo para a Ciecircncia e Tecnologia (FCT) by means of projectPTDCAGR-AAM683592006

References

[1] F C Cabello ldquoHeavy use of prophylactic antibiotics in aquacul-ture a growing problem for human and animal health and forthe environmentrdquo Environmental Microbiology vol 8 no 7 pp1137ndash1144 2006

[2] T Maki I Hirono H Kondo and T Aoki ldquoDrug resis-tance mechanism of the sh-pathogenic bacterium Lactococcusgarvieaerdquo Journal of Fish Diseases vol 31 no 6 pp 461ndash4682008


[3] Council Regulation (EEC) 237790 of 26 June 1990 ldquoConsoli-dated with previous amendmentsrdquo

[4] S J Culp F A Beland R H Heich et al ldquoMutagenicity andcarcinogenicity in relation to DNA adduct formation in ratsfed leucomalachite greenrdquoMutation Research vol 506-507 pp55ndash63 2002

[5] A Swarbrick E J Murby and P Hume ldquoPost-column elec-trochemical oxidation of leuco malachite green for the analysisof rainbow trout esh using HPLC with absorbance detectionrdquoJournal of Liquid Chromatography and Related Technologies vol20 no 14 pp 2269ndash2280 1997

[6] A L Henderson T C Schmitt T M Heinze and C ECerniglia ldquoReduction of malachite green to leucomalachitegreen by intestinal bacteriardquo Applied and Environmental Micro-biology vol 63 no 10 pp 4099ndash4101 1997

[7] J J Jones and J O Falkinham ldquoDecolorization of malachitegreen and crystal violet by waterborne pathogenic mycobacte-riardquo Antimicrobial Agents and Chemotherapy vol 47 no 7 pp2323ndash2326 2003

[8] M C Yang J M Fang T F Kuo et al ldquoProduction ofantibodies for selective detection of malachite green and therelated triphenylmethane dyes in sh and shpond waterrdquoJournal of Agricultural and Food Chemistry vol 55 no 22 pp8851ndash8856 2007

[9] H YiW Qu andWHuang ldquoElectrochemical determination ofmalachite green using a multi-wall carbon nanotube modiedglassy carbon electroderdquo Microchimica Acta vol 160 no 1-2pp 291ndash296 2008

[10] G Chen and S Miao ldquoHPLC determination andMS conrma-tion of malachite green gentian violet and their leuco metabo-lite residues in channel catsh musclerdquo Journal of AgricultureFood Chemistry vol 58 no 12 pp 7109ndash7114 2010

[11] K Halme E Lindfors and K Peltonen ldquoA conrmatoryanalysis of malachite green residues in rainbow trout with liq-uid chromatography-electrospray tandem mass spectrometryrdquoJournal of Chromatography B vol 845 no 1 pp 74ndash79 2007

[12] W C Andersen J E Roybal and S B Turnipseed ldquoLiquidchromatographic determination of malachite green and leuco-malachite green (LMG) residues in salmon with in situ LMGoxidationrdquo Journal of AOAC International vol 88 no 5 pp1292ndash1298 2005

[13] C Long Z Mai Y Yang et al ldquoDetermination of multi-residuefor malachite green gentian violet and their metabolites inaquatic products by high-performance liquid chromatographycoupled with molecularly imprinted solid-phase extractionrdquoJournal of Chromatography A vol 1216 no 12 pp 2275ndash22812009

[14] W C Andersen S B Turnipseed and J E Roybal ldquoQuan-titative and conrmatory analyses of malachite green andleucomalachite green residues in sh and shrimprdquo Journal ofAgricultural and Food Chemistry vol 54 no 13 pp 4517ndash45232006

[15] M J M Bueno S Herrera A Ucleacutes et al ldquoDetermination ofmalachite green residues in sh using molecularly imprintedsolid-phase extraction followed by liquid chromatography-linear ion trapmass spectrometryrdquoAnalytica Chimica Acta vol665 no 1 pp 47ndash54 2010

[16] K Mitrowska A Posyniak and J Zmudzki ldquoDetermination ofmalachite green and leucomalachite green in carp muscle byliquid chromatographywith visible and uorescence detectionrdquoJournal of Chromatography A vol 1089 no 1-2 pp 187ndash1922005

[17] K Halme E Lindfors and K Peltonen ldquoDetermination ofmalachite green residues in rainbow trout muscle with liquidchromatography and liquid chromatography coupled with tan-demmass spectrometryrdquo FoodAdditives and Contaminants vol21 no 7 pp 641ndash648 2004

[18] K Mitrowska A Posyniak and J Zmudzki ldquoDeterminationof malachite green and leucomalachite green residues in waterusing liquid chromatography with visible and uorescencedetection and conrmation by tandem mass spectrometryrdquoJournal of Chromatography A vol 1207 no 1-2 pp 94ndash1002008

[19] L G Rushing and E B Hansen ldquoConrmation of malachitegreen gentian violet and their leuco analogs in catsh and trouttissue by high-performance liquid chromatography utilizingelectrochemistry with ultraviolet-visible diode array detectionand uorescence detectionrdquo Journal of Chromatography B vol700 no 1-2 pp 223ndash231 1997

[20] H Sun L Wang X Qin and X Ge ldquoSimultaneous determi-nation of malachite green enrooxacin and ciprooxacin insh farming water and sh feed by liquid chromatography withsolid-phase extractionrdquo Journal of Environmental Monitoringvol 179 pp 421ndash429 2011

[21] X Wu G Zhang Y Wu X Hou and Z Yuan ldquoSimultaneousdetermination of malachite green gentian violet and theirleuco-metabolites in aquatic products by high-performanceliquid chromatography-linear ion trap mass spectrometryrdquoJournal of Chromatography A vol 1172 no 2 pp 121ndash1262007

[22] A Khodabakhshi and M M Amin ldquoDetermination of mala-chite green in trout tissue and euent water from sh farmsrdquoInternational Journal of Environmental Health Engineering vol1 pp 51ndash56 2012

[23] K C Lee J L Wu and Z Cai ldquoDetermination of malachitegreen and leucomalachite green in edible goldsh muscle byliquid chromatography-ion trap mass spectrometryrdquo Journal ofChromatography B vol 843 no 2 pp 247ndash251 2006

[24] X Hu K Jiao W Sun and J Y You ldquoElectrochemical andspectroscopic studies on the interaction ofmalachite greenwithDNA and its applicationrdquo Electroanalysis vol 18 no 6 pp613ndash620 2006

[25] Z Hall C Hopley and G OrsquoConnor ldquoHigh accuracy determi-nation of malachite green and leucomalachite green in salmontissue by exact matching isotope dilution mass spectrometryrdquoJournal of Chromatography B vol 874 no 1-2 pp 95ndash1002008

[26] J A Tarbin K A Barnes J Bygrave and W H H FarringtonldquoScreening and conrmation of triphenylmethane dyes andtheir leuco metabolites in trout muscle using HPLC-vis andESP-LC-MSrdquo Analyst vol 123 no 12 pp 2567ndash2571 1998

[27] S B Turnipseed W C Andersen and J E Roybal ldquoDetermi-nation and conrmation ofmalachite green and leucomalachitegreen residues in salmon using liquid chromatographymassspectrometrywith no-discharge atmospheric pressure chemicalionizationrdquo Journal of AOAC International vol 88 no 5 pp1312ndash1317 2005

[28] C H Tsai J D Lin and C H Lin ldquoOptimization of theseparation of malachite green in water by capillary elec-trophoresis Raman spectroscopy (CE-RS) based on the stackingand sweepingmodesrdquo Talanta vol 72 no 2 pp 368ndash372 2007

[29] US Patent Application 20070072242 ldquoImmunoassay methodand kit to leucomalachite green and malachite greenrdquo


httpwwwpatentstormusapplications20070072242descrip-tionhtml

[30] P Speacutegel L Schweitz and S Nilsson ldquoMolecularly imprintedpolymersrdquo Analytical and Bioanalytical Chemistry vol 372 no1 pp 37ndash38 2002

[31] L I Andersson ldquoMolecular imprinting for drug bioanalysis areview on the application of imprinted polymers to solid-phaseextraction and binding assayrdquo Journal of Chromatography B vol739 no 1 pp 163ndash173 2000

[32] L Ye and K Haupt ldquoMolecularly imprinted polymers asantibody and receptor mimics for assays sensors and drugdiscoveryrdquo Analytical and Bioanalytical Chemistry vol 378 no8 pp 1887ndash1897 2004

[33] G Vlatakis L I Andersson R Muller and K Mosbach ldquoDrugassay using antibody mimics made by molecular imprintingrdquoNature vol 361 no 6413 pp 645ndash647 1993

[34] Y H Li T Yang X L Qi YW Qiao and A P Deng ldquoDevelop-ment of a group selectivemolecularly imprinted polymers basedsolid phase extraction of malachite green from sh water andsh feed samplesrdquo Analytica Chimica Acta vol 624 no 2 pp317ndash325 2008

[35] H Cao F Xu D X Li X-G Zhang and J-S Yu ldquoPrepara-tion and performance valuation of high selective molecularlyimprinted polymers for malachite greenrdquo Research on ChemicalIntermediates In press

[36] S Yan Z Gao Y Fang Y Cheng H Zhou and H WangldquoCharacterization and quality assessment of binding propertiesof malachite green molecularly imprinted polymers preparedby precipitation polymerization in acetonitrilerdquo Dyes and Pig-ments vol 74 no 3 pp 572ndash577 2007

[37] C M Lok and R Son ldquoApplication of molecularly imprintedpolymers in food sample analysismdasha perspectiverdquo InternationalFood Research Journal vol 16 pp 127ndash140 2009

[38] K Haupt and K Mosbach ldquoMolecularly imprinted polymersand their use in biomimetic sensorsrdquo Chemical Reviews vol100 no 7 pp 2495ndash2504 2000

[39] F T C Moreira V A P Freitas andM G F Sales ldquoBiomimeticnoroxacin sensors made of molecularly-imprinted materialsfor potentiometric transductionrdquo Microchimica Acta vol 172no 1 pp 15ndash23 2011

[40] M C Blanco-Loacutepez M J Lobo-Castantildeoacuten A J Miranda-Ordieres and P Tuntildeoacuten-Blanco ldquoElectrochemical sensors basedon molecularly imprinted polymersrdquo Trends in AnalyticalChemistry vol 23 no 1 pp 36ndash48 2004

[41] M Javanbakht S E Fard A Mohammadi et al ldquoMolecu-larly imprinted polymer based potentiometric sensor for thedetermination of hydroxyzine in tablets and biological uidsrdquoAnalytica Chimica Acta vol 612 no 1 pp 65ndash74 2008

[42] L Codognoto E Winter K M Doretto G B Monteiroand S Rath ldquoElectroanalytical performance of self-assembledmonolayer gold electrode for chloramphenicol determinationrdquoMicrochimica Acta vol 169 no 3 pp 345ndash351 2010

[43] R Shoji T Takeuchi and I Kubo ldquoAtrazine sensor basedon molecularly imprinted polymer-modied gold electroderdquoAnalytical Chemistry vol 75 no 18 pp 4882ndash4886 2003

[44] T Kitade K Kitamura T Konishi et al ldquoPotentiometricimmunosensor using articial antibody based on molecularlyimprinted polymersrdquo Analytical Chemistry vol 76 no 22 pp6802ndash6807 2004

[45] G DrsquoAgostino G Alberti R Biesuz and M Pesavento ldquoPoten-tiometric sensor for atrazine based on a molecular imprintedmembranerdquo Biosensors and Bioelectronics vol 22 no 1 pp145ndash152 2006

[46] A Warsinke and B Nagel ldquoTowards separation-free electro-chemical affinity sensors by using antibodies aptamers andmolecularly imprinted polymersmdasha reviewrdquo Analytical Lettersvol 39 no 13 pp 2507ndash2556 2006

[47] F T C Moreira A H Kamel J R L Guerreiro and MG F Sales ldquoMan-tailored biomimetic sensor of molecularlyimprinted materials for the potentiometric measurement ofoxytetracyclinerdquo Biosensors and Bioelectronics vol 26 no 2 pp566ndash574 2010

[48] K P Prathish K Prasad T P Rao and M V S SuryanarayanaldquoMolecularly imprinted polymer-based potentiometric sensorfor degradation product of chemical warfare agents Part IMethylphosphonic acidrdquo Talanta vol 71 no 5 pp 1976ndash19802007

[49] J R L Guerreiro V Freitas and M G F Sales ldquoNew sensingmaterials of molecularly-imprinted polymers for the selectiverecognition of Chlortetracyclinerdquo Microchemical Journal vol97 no 2 pp 173ndash181 2011

[50] A H Kamel F T C Moreira S A A Almeida and M GF Sales ldquoNovel potentiometric sensors of molecular imprintedpolymers for specic binding of chlormequatrdquo Electroanalysisvol 20 no 2 pp 194ndash202 2008

[51] A H Kamel S A A Almeida M G F Sales and F TC Moreira ldquoSulfadiazine-potentiometric sensors for ow andbatch determinations of sulfadiazine in drugs and biologicaluidsrdquo Analytical Sciences vol 25 no 3 pp 365ndash371 2009

[52] F Faridbod M R Ganjali R Dinarvand and P NorouzildquoDevelopments in the eld of conducting and non-conductingpolymer based potentiometric membrane sensors for ions overthe past decaderdquo Sensors vol 8 no 4 pp 2331ndash2412 2008

[53] J A Garciacutea-Calzoacuten andM E Diacuteaz-Garciacutea ldquoCharacterization ofbinding sites in molecularly imprinted polymersrdquo Sensors andActuators B vol 123 no 2 pp 1180ndash1194 2007

[54] F Gritti and G Guiochon ldquoHeterogeneity of the surfaceenergy on unused C18-Chromolith adsorbents in reversed-phase liquid chromatographyrdquo Journal of Chromatography Avol 1028 no 1 pp 105ndash119 2004

[55] S Sharma and G P Agarwal ldquoInteractions of proteins withimmobilized metal ions role of ionic strength and pHrdquo Journalof Colloid and Interface Science vol 243 no 1 pp 61ndash72 2001

[56] C Hidayat M Nakajima M Takagi and T Yoshida ldquoMultiva-lent binding interaction of alcohol dehydrogenase on dye-metalaffinitymatrixrdquo Journal of Bioscience andBioengineering vol 96no 2 pp 168ndash173 2003

[57] Y Umezawa P Buumlhlmann K Umezawa K Tohda and SAmemiya ldquoPotentiometric selectivity coefficients of ion-selective electrodes part I Inorganic cationsmdash(technicalreport)rdquo Pure and Applied Chemistry vol 72 no 10 pp1851ndash2082 2000

[58] M Telting-Diaz and E Bakker ldquoEffect of lipophilic ion-exchanger leaching on the detection limit of carrier-based ion-selective electrodesrdquo Analytical Chemistry vol 73 no 22 pp5582ndash5589 2001

[59] M Lizondo M Pons M Gallardo and J Estelrich ldquoPhysico-chemical properties of enrooxacinrdquo Journal of Pharmaceuticaland Biomedical Analysis vol 15 no 12 pp 1845ndash1849 1997


[60] F L Dickert andOHayden ldquoMolecular imprinting in chemicalsensingrdquo Trends in Analytical Chemistry vol 18 no 3 pp192ndash199 1999

[61] Y Umezawa K Umezawa and H Sato ldquoSelectivity coeffi-cients for ion-selective electrodesmdashrecommended methods forreporting 119870119870pot119860119860119860119860119860 valuesmdash(technical report)rdquo Pure and AppliedChemistry vol 67 no 3 pp 507ndash518 1995

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International


CatalystsJournal of

ElectrochemistryInternational Journal of



procedures reported in literature [28] Other reported meth-ods are based on immunoassays [29] although they providespecic responses the overall proceduremay take a long timeand turns out expensive

Alternative and advantageous methods should rely onexpeditious and ecient procedures providing highly specicand sensitive measurements e specic recognition of acertain analyte may be achieved by molecularly imprintedpolymers (MIPs) ese synthetic polymers are preparedby letting functional and cross-linking monomers organizearound a template (to be imprinted) and copolymerize themto form a highly cross-linked polymeric structure [30 31]e template is removed aer creating vacant places wherethe functional groups of the monomeric forms are strictlyoriented to bind this specic compound e producedpolymer is thus capable of rebinding the analyte with a highspecicity In many occasions it shows binding anitiesapproaching those presented by antigen-antibody systems[32 33] while offering higher stability at extremes of pHand temperature high mechanical strength and lower costthan their natural counterparts More recently several paperson the synthesis of MIP materials for malachite green havebeen published [34ndash37] ese materials are always obtainedby bulk imprinting being methacrylic acid (MAA) the onlyfunctional monomer tested and applied (or intended for)chromatographic applications

e great potential of MIP materials has led to theirinclusion as recognition elements in sensors Transduction ofdifferent nature has been employed for this purpose [38 39]with great emphasis on electrochemisty [40] Potentiometryis included here and offers the advantages of not requiringthe template to be extracted from the membrane and thespecies to be diffused through the membrane [40] Despitethese advantages most research work within this led hasbeen conducted only most recently [41ndash50]

erefore the present work tries to (i) develop MIPmaterials designed for LMG insteadMG because LMG is themajor sh metabolite that may be found in waters (ii) searchfor an alternative functional monomer by replacing thetraditionalMAAby acrylamido-2-methyl-1-propanesulfonicacid (AMPSA) (iii) and open new horizons on the use ofthesematerials by applying these in biosensing developmentsomething never done before for LMG nor MG e sen-sory materials were thus synthesized with methacrylic acid(MAA) and acrylamido-2-methyl-1-propanesulfonic acid(AMPSA) functional monomers cross-linked by ethyleneglycol dimethacrylic acid (EGDMA) within the templatemolecule e selective membranes were obtained by dis-persing these materials in a PVC matrix plasticized with o-nitrophenyl octyl ether (oNPOE)epotentiometric sensorswere evaluated in steady-state and owingmedia and appliedto the analysis of contaminated water

2 Experimental

21 Apparatus All potential measurements were made by aCrison 120583120583pH 2002 decimilivoltammeter (plusmn01mV sensitivity)at room temperature and under constant stirring by means

of a Crison micro ST 2038 e output signal in steady stateevaluations was redirected to a commutation unit linked tosix ways out enabling the simultaneous reading of six ISEse assembly of the potentiometric cell was as follows con-ductive graphite | LMG selectivemembrane | buffered samplesolution (phosphate 5 times 10minus2mol Lminus1 pH 45) || electrolytesolution KCl | AgCl(s) | Ag e reference electrode wasan Orion AgAgCl double junction (Orion 90-02-00) eselective electrode was prepared in conventional or tubularcongurations [51] for batch and ow mode evaluationsrespectively Both devices had no internal reference solutionand epoxy graphite was used as solid contact

e Flow Injection Analysis (FIA) assembly had a GilsonMinipuls 2 peristaltic pump tted with PVC tubing (080160 andor 200mm id) and a four-way Rheodyne 5020injection valve holding a loop of variable volume eseveral components were gathered by PTFE tubing (OmnitTeon 08mm id) Gilson end-ttings and connectorsesupport devices for tubular and reference electrodes andthe conuence point accessory were constructed in PerspexAer reaching a stable baseline the emf was recordedcontinuously by means of a homemade high-impedance dataacquisition eight-channel box connected to a PC through theinterface ADC 16 (Pico Tech UK) and PicoLog for windows(version 507) soware

When necessary the pH was measured by a CrisonCWLS7 combined glass electrode connected to a decim-ilivoltammeter Crison pH meter GLP 22 A double-beamUV-vis spectrophotometer (Shimadzumodel 1601) equippedwith 10-mm quartz cells was used for absorbance measure-ments in binding studies Infrared spectra were collected bya Nicolet 6700 FTIR spectrometer surface measurementswere carried out in ATR (attenuated total reectance) modewith the Nicolet ATR sampling accessory of diamond contactcrystal

22 Reagents All chemicals were of analytical grade anddeionized water (conductivity lt01120583120583S cmminus1) was employedLMG potassium tetrakis (4-chlorophenyl)borate (TpClPB)o-nitrophenyloctyl ether (oNPOE) poly (vinyl chloride)(PVC) of high molecular weight EGDMA AMPSA andMAA were purchased from Fluka Benzoyl peroxide (BPO)methanol (MeOH) chloroform and tetrahydrofuran (THF)were obtained from Riedel-deHaumlen

23 Synthesis of Host-Tailored Polymers For molecularlyimprinted polymers the template (LMG 10mmol) wasplaced in a glass tube (140mm id) with the functionalmonomer (40mmol MAA) the cross-linker (EGDMA200mmol) and the radical initiator (BPO 024mmol) alldissolved in 8mL chloroform A polymer of AMPSA wasprepared similarly e mixture was sonicated degassedwith nitrogen for 5min and cured at 70∘C for 30minNonimprinted polymers (NIP) were prepared in a similarway by excluding the template from the procedure

e resulting polymerswere ground and sieved to particlesizes ranging from 50 to 150 120583120583m Extraction of the templatemolecule and washout of nonreacted species was carried

















100

150

200

250

300

350

400

0

50

100

150

200

250


(A2) MAA (B2) MAA

0 1 2 3 4 5

0 1 2 3 4 5

0

02

04

06

08

10

12

0

01

02

03

04

0 100 200 300 400 500

0 50 100 150 200 250





119876119876 119905120583120583mol (LMG bound)119892119892 (MIP)

11990510076501007650119862119862119894119894 minus 11986211986211989111989110076661007666119881119881119904119904 times 1000119872119872MIP

(1)




119876119876119862119862free11990510076491007649119876119876max minus 11987611987610076651007665119870119870119889119889

(2)




Sulfonic acid

stretch

stretch

stretch

C

C

O

C O

C O

C O

Bendamide

MAA MIPS

AMPSA MIPs

EsterAmide


stretch ester

N H

H

stretch amide

3500 3000 2500 2000 1500 1000

80

70

60

50

40

30

20

10

80

70

60

50

40

30

20

10

0

Tra

nsm

itta

nce

(

)T

ran

smit

tan

ce (

)

2953

8 1636

1

1716

1

1450

2

1295

912

472

1138

4

943

2

3286

7

2982

9

1664

416

1115

762

1457

7

1352

513

298

1307

612

447

1219 11

753

1130

6 1115

510

379

991

8














log[LMG M]

MIPMAA washed


MIPAMPSA NIPAMPSA



60m

Vd

ecad

e

log[LMG M]






1 2 3 4 5 6 7 8

pH

MIPMAA washed

NIPMAA MIPMAA

1 2 3 4 5 6 7 8

pH

MIPAMPSA washed

NIPAMPSA


50

mV

50

mV











H3CH3C

CH3

CH3

NH2

NH2

NH2

NH2

NH2

H2N

MIP

AM

PS

A+

ad

dit

ive

MIP

MA

A+

ad

dit

ive

minus05

minus1

minus15

minus2

minus25

minus3

0

MIP

AM

PS

A

wash

ed

MIP

AM

PS

A

NIP

AM

PS

A

DOXY

OXY

ENR

TMP

CREA

CHML

DPM

LMG

LMG

DOXY

TMP

ENRO

OXY

CREA

CHML

DPMM

IPM

AA

wash

ed

MIP

MA

A

NIP

MA

A

OO

O

O

O

O

O

O O O

O

O

O

OOH OHOH

OH

OH

OH

OH OH

OH

OH

OH

OH N

N

N

N

N

N

N

N

N N

N

HH

HH

FHO

HO

HO

HO

HN

Cl

ClN+

Ominus












log119863119863 119863100764910076491198671198670 minus 11986711986710076651007665119878119878 (3)







Sam

ple

Water

Buer

Waste

Pump

WasteReference

electrodeGround

electrode

Injection

valve

LMG sensor

(a)

Stea

dy

stat

e (

)

08

09

1

11

12

13

14

15

Dis

per

sio

n

70

80

90

100

110

120

Sam

pli

ng

rate

(h

)

80

85

90

95

100

105

0 100 200 300 400 500

(b)St

ead

y st

ate

()

Dis

per

sio

n

Sam

pli

ng

rate

(h

)0

1

2

3

4

90

130

170

210

250

50

70

90

110

130

150

4 6 8 10 12 14

Flow rate (mLmin)

(c)




4 Conclusions





Acknowledgment


References











































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of



















100

150

200

250

300

350

400

0

50

100

150

200

250


(A2) MAA (B2) MAA

0 1 2 3 4 5

0 1 2 3 4 5

0

02

04

06

08

10

12

0

01

02

03

04

0 100 200 300 400 500

0 50 100 150 200 250





119876119876 119905120583120583mol (LMG bound)119892119892 (MIP)

11990510076501007650119862119862119894119894 minus 11986211986211989111989110076661007666119881119881119904119904 times 1000119872119872MIP

(1)




119876119876119862119862free11990510076491007649119876119876max minus 11987611987610076651007665119870119870119889119889

(2)




Sulfonic acid

stretch

stretch

stretch

C

C

O

C O

C O

C O

Bendamide

MAA MIPS

AMPSA MIPs

EsterAmide


stretch ester

N H

H

stretch amide

3500 3000 2500 2000 1500 1000

80

70

60

50

40

30

20

10

80

70

60

50

40

30

20

10

0

Tra

nsm

itta

nce

(

)T

ran

smit

tan

ce (

)

2953

8 1636

1

1716

1

1450

2

1295

912

472

1138

4

943

2

3286

7

2982

9

1664

416

1115

762

1457

7

1352

513

298

1307

612

447

1219 11

753

1130

6 1115

510

379

991

8














log[LMG M]

MIPMAA washed


MIPAMPSA NIPAMPSA



60m

Vd

ecad

e

log[LMG M]






1 2 3 4 5 6 7 8

pH

MIPMAA washed

NIPMAA MIPMAA

1 2 3 4 5 6 7 8

pH

MIPAMPSA washed

NIPAMPSA


50

mV

50

mV











H3CH3C

CH3

CH3

NH2

NH2

NH2

NH2

NH2

H2N

MIP

AM

PS

A+

ad

dit

ive

MIP

MA

A+

ad

dit

ive

minus05

minus1

minus15

minus2

minus25

minus3

0

MIP

AM

PS

A

wash

ed

MIP

AM

PS

A

NIP

AM

PS

A

DOXY

OXY

ENR

TMP

CREA

CHML

DPM

LMG

LMG

DOXY

TMP

ENRO

OXY

CREA

CHML

DPMM

IPM

AA

wash

ed

MIP

MA

A

NIP

MA

A

OO

O

O

O

O

O

O O O

O

O

O

OOH OHOH

OH

OH

OH

OH OH

OH

OH

OH

OH N

N

N

N

N

N

N

N

N N

N

HH

HH

FHO

HO

HO

HO

HN

Cl

ClN+

Ominus












log119863119863 119863100764910076491198671198670 minus 11986711986710076651007665119878119878 (3)







Sam

ple

Water

Buer

Waste

Pump

WasteReference

electrodeGround

electrode

Injection

valve

LMG sensor

(a)

Stea

dy

stat

e (

)

08

09

1

11

12

13

14

15

Dis

per

sio

n

70

80

90

100

110

120

Sam

pli

ng

rate

(h

)

80

85

90

95

100

105

0 100 200 300 400 500

(b)St

ead

y st

ate

()

Dis

per

sio

n

Sam

pli

ng

rate

(h

)0

1

2

3

4

90

130

170

210

250

50

70

90

110

130

150

4 6 8 10 12 14

Flow rate (mLmin)

(c)




4 Conclusions





Acknowledgment


References











































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of




100

150

200

250

300

350

400

0

50

100

150

200

250


(A2) MAA (B2) MAA

0 1 2 3 4 5

0 1 2 3 4 5

0

02

04

06

08

10

12

0

01

02

03

04

0 100 200 300 400 500

0 50 100 150 200 250





119876119876 119905120583120583mol (LMG bound)119892119892 (MIP)

11990510076501007650119862119862119894119894 minus 11986211986211989111989110076661007666119881119881119904119904 times 1000119872119872MIP

(1)




119876119876119862119862free11990510076491007649119876119876max minus 11987611987610076651007665119870119870119889119889

(2)




Sulfonic acid

stretch

stretch

stretch

C

C

O

C O

C O

C O

Bendamide

MAA MIPS

AMPSA MIPs

EsterAmide


stretch ester

N H

H

stretch amide

3500 3000 2500 2000 1500 1000

80

70

60

50

40

30

20

10

80

70

60

50

40

30

20

10

0

Tra

nsm

itta

nce

(

)T

ran

smit

tan

ce (

)

2953

8 1636

1

1716

1

1450

2

1295

912

472

1138

4

943

2

3286

7

2982

9

1664

416

1115

762

1457

7

1352

513

298

1307

612

447

1219 11

753

1130

6 1115

510

379

991

8














log[LMG M]

MIPMAA washed


MIPAMPSA NIPAMPSA



60m

Vd

ecad

e

log[LMG M]






1 2 3 4 5 6 7 8

pH

MIPMAA washed

NIPMAA MIPMAA

1 2 3 4 5 6 7 8

pH

MIPAMPSA washed

NIPAMPSA


50

mV

50

mV











H3CH3C

CH3

CH3

NH2

NH2

NH2

NH2

NH2

H2N

MIP

AM

PS

A+

ad

dit

ive

MIP

MA

A+

ad

dit

ive

minus05

minus1

minus15

minus2

minus25

minus3

0

MIP

AM

PS

A

wash

ed

MIP

AM

PS

A

NIP

AM

PS

A

DOXY

OXY

ENR

TMP

CREA

CHML

DPM

LMG

LMG

DOXY

TMP

ENRO

OXY

CREA

CHML

DPMM

IPM

AA

wash

ed

MIP

MA

A

NIP

MA

A

OO

O

O

O

O

O

O O O

O

O

O

OOH OHOH

OH

OH

OH

OH OH

OH

OH

OH

OH N

N

N

N

N

N

N

N

N N

N

HH

HH

FHO

HO

HO

HO

HN

Cl

ClN+

Ominus












log119863119863 119863100764910076491198671198670 minus 11986711986710076651007665119878119878 (3)







Sam

ple

Water

Buer

Waste

Pump

WasteReference

electrodeGround

electrode

Injection

valve

LMG sensor

(a)

Stea

dy

stat

e (

)

08

09

1

11

12

13

14

15

Dis

per

sio

n

70

80

90

100

110

120

Sam

pli

ng

rate

(h

)

80

85

90

95

100

105

0 100 200 300 400 500

(b)St

ead

y st

ate

()

Dis

per

sio

n

Sam

pli

ng

rate

(h

)0

1

2

3

4

90

130

170

210

250

50

70

90

110

130

150

4 6 8 10 12 14

Flow rate (mLmin)

(c)




4 Conclusions





Acknowledgment


References











































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of





Sulfonic acid

stretch

stretch

stretch

C

C

O

C O

C O

C O

Bendamide

MAA MIPS

AMPSA MIPs

EsterAmide


stretch ester

N H

H

stretch amide

3500 3000 2500 2000 1500 1000

80

70

60

50

40

30

20

10

80

70

60

50

40

30

20

10

0

Tra

nsm

itta

nce

(

)T

ran

smit

tan

ce (

)

2953

8 1636

1

1716

1

1450

2

1295

912

472

1138

4

943

2

3286

7

2982

9

1664

416

1115

762

1457

7

1352

513

298

1307

612

447

1219 11

753

1130

6 1115

510

379

991

8














log[LMG M]

MIPMAA washed


MIPAMPSA NIPAMPSA



60m

Vd

ecad

e

log[LMG M]






1 2 3 4 5 6 7 8

pH

MIPMAA washed

NIPMAA MIPMAA

1 2 3 4 5 6 7 8

pH

MIPAMPSA washed

NIPAMPSA


50

mV

50

mV











H3CH3C

CH3

CH3

NH2

NH2

NH2

NH2

NH2

H2N

MIP

AM

PS

A+

ad

dit

ive

MIP

MA

A+

ad

dit

ive

minus05

minus1

minus15

minus2

minus25

minus3

0

MIP

AM

PS

A

wash

ed

MIP

AM

PS

A

NIP

AM

PS

A

DOXY

OXY

ENR

TMP

CREA

CHML

DPM

LMG

LMG

DOXY

TMP

ENRO

OXY

CREA

CHML

DPMM

IPM

AA

wash

ed

MIP

MA

A

NIP

MA

A

OO

O

O

O

O

O

O O O

O

O

O

OOH OHOH

OH

OH

OH

OH OH

OH

OH

OH

OH N

N

N

N

N

N

N

N

N N

N

HH

HH

FHO

HO

HO

HO

HN

Cl

ClN+

Ominus












log119863119863 119863100764910076491198671198670 minus 11986711986710076651007665119878119878 (3)







Sam

ple

Water

Buer

Waste

Pump

WasteReference

electrodeGround

electrode

Injection

valve

LMG sensor

(a)

Stea

dy

stat

e (

)

08

09

1

11

12

13

14

15

Dis

per

sio

n

70

80

90

100

110

120

Sam

pli

ng

rate

(h

)

80

85

90

95

100

105

0 100 200 300 400 500

(b)St

ead

y st

ate

()

Dis

per

sio

n

Sam

pli

ng

rate

(h

)0

1

2

3

4

90

130

170

210

250

50

70

90

110

130

150

4 6 8 10 12 14

Flow rate (mLmin)

(c)




4 Conclusions





Acknowledgment


References











































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of






log[LMG M]

MIPMAA washed


MIPAMPSA NIPAMPSA



60m

Vd

ecad

e

log[LMG M]






1 2 3 4 5 6 7 8

pH

MIPMAA washed

NIPMAA MIPMAA

1 2 3 4 5 6 7 8

pH

MIPAMPSA washed

NIPAMPSA


50

mV

50

mV











H3CH3C

CH3

CH3

NH2

NH2

NH2

NH2

NH2

H2N

MIP

AM

PS

A+

ad

dit

ive

MIP

MA

A+

ad

dit

ive

minus05

minus1

minus15

minus2

minus25

minus3

0

MIP

AM

PS

A

wash

ed

MIP

AM

PS

A

NIP

AM

PS

A

DOXY

OXY

ENR

TMP

CREA

CHML

DPM

LMG

LMG

DOXY

TMP

ENRO

OXY

CREA

CHML

DPMM

IPM

AA

wash

ed

MIP

MA

A

NIP

MA

A

OO

O

O

O

O

O

O O O

O

O

O

OOH OHOH

OH

OH

OH

OH OH

OH

OH

OH

OH N

N

N

N

N

N

N

N

N N

N

HH

HH

FHO

HO

HO

HO

HN

Cl

ClN+

Ominus












log119863119863 119863100764910076491198671198670 minus 11986711986710076651007665119878119878 (3)







Sam

ple

Water

Buer

Waste

Pump

WasteReference

electrodeGround

electrode

Injection

valve

LMG sensor

(a)

Stea

dy

stat

e (

)

08

09

1

11

12

13

14

15

Dis

per

sio

n

70

80

90

100

110

120

Sam

pli

ng

rate

(h

)

80

85

90

95

100

105

0 100 200 300 400 500

(b)St

ead

y st

ate

()

Dis

per

sio

n

Sam

pli

ng

rate

(h

)0

1

2

3

4

90

130

170

210

250

50

70

90

110

130

150

4 6 8 10 12 14

Flow rate (mLmin)

(c)




4 Conclusions





Acknowledgment


References











































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of




1 2 3 4 5 6 7 8

pH

MIPMAA washed

NIPMAA MIPMAA

1 2 3 4 5 6 7 8

pH

MIPAMPSA washed

NIPAMPSA


50

mV

50

mV











H3CH3C

CH3

CH3

NH2

NH2

NH2

NH2

NH2

H2N

MIP

AM

PS

A+

ad

dit

ive

MIP

MA

A+

ad

dit

ive

minus05

minus1

minus15

minus2

minus25

minus3

0

MIP

AM

PS

A

wash

ed

MIP

AM

PS

A

NIP

AM

PS

A

DOXY

OXY

ENR

TMP

CREA

CHML

DPM

LMG

LMG

DOXY

TMP

ENRO

OXY

CREA

CHML

DPMM

IPM

AA

wash

ed

MIP

MA

A

NIP

MA

A

OO

O

O

O

O

O

O O O

O

O

O

OOH OHOH

OH

OH

OH

OH OH

OH

OH

OH

OH N

N

N

N

N

N

N

N

N N

N

HH

HH

FHO

HO

HO

HO

HN

Cl

ClN+

Ominus












log119863119863 119863100764910076491198671198670 minus 11986711986710076651007665119878119878 (3)







Sam

ple

Water

Buer

Waste

Pump

WasteReference

electrodeGround

electrode

Injection

valve

LMG sensor

(a)

Stea

dy

stat

e (

)

08

09

1

11

12

13

14

15

Dis

per

sio

n

70

80

90

100

110

120

Sam

pli

ng

rate

(h

)

80

85

90

95

100

105

0 100 200 300 400 500

(b)St

ead

y st

ate

()

Dis

per

sio

n

Sam

pli

ng

rate

(h

)0

1

2

3

4

90

130

170

210

250

50

70

90

110

130

150

4 6 8 10 12 14

Flow rate (mLmin)

(c)




4 Conclusions





Acknowledgment


References











































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of




H3CH3C

CH3

CH3

NH2

NH2

NH2

NH2

NH2

H2N

MIP

AM

PS

A+

ad

dit

ive

MIP

MA

A+

ad

dit

ive

minus05

minus1

minus15

minus2

minus25

minus3

0

MIP

AM

PS

A

wash

ed

MIP

AM

PS

A

NIP

AM

PS

A

DOXY

OXY

ENR

TMP

CREA

CHML

DPM

LMG

LMG

DOXY

TMP

ENRO

OXY

CREA

CHML

DPMM

IPM

AA

wash

ed

MIP

MA

A

NIP

MA

A

OO

O

O

O

O

O

O O O

O

O

O

OOH OHOH

OH

OH

OH

OH OH

OH

OH

OH

OH N

N

N

N

N

N

N

N

N N

N

HH

HH

FHO

HO

HO

HO

HN

Cl

ClN+

Ominus












log119863119863 119863100764910076491198671198670 minus 11986711986710076651007665119878119878 (3)







Sam

ple

Water

Buer

Waste

Pump

WasteReference

electrodeGround

electrode

Injection

valve

LMG sensor

(a)

Stea

dy

stat

e (

)

08

09

1

11

12

13

14

15

Dis

per

sio

n

70

80

90

100

110

120

Sam

pli

ng

rate

(h

)

80

85

90

95

100

105

0 100 200 300 400 500

(b)St

ead

y st

ate

()

Dis

per

sio

n

Sam

pli

ng

rate

(h

)0

1

2

3

4

90

130

170

210

250

50

70

90

110

130

150

4 6 8 10 12 14

Flow rate (mLmin)

(c)




4 Conclusions





Acknowledgment


References











































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of









log119863119863 119863100764910076491198671198670 minus 11986711986710076651007665119878119878 (3)







Sam

ple

Water

Buer

Waste

Pump

WasteReference

electrodeGround

electrode

Injection

valve

LMG sensor

(a)

Stea

dy

stat

e (

)

08

09

1

11

12

13

14

15

Dis

per

sio

n

70

80

90

100

110

120

Sam

pli

ng

rate

(h

)

80

85

90

95

100

105

0 100 200 300 400 500

(b)St

ead

y st

ate

()

Dis

per

sio

n

Sam

pli

ng

rate

(h

)0

1

2

3

4

90

130

170

210

250

50

70

90

110

130

150

4 6 8 10 12 14

Flow rate (mLmin)

(c)




4 Conclusions





Acknowledgment


References











































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of




Sam

ple

Water

Buer

Waste

Pump

WasteReference

electrodeGround

electrode

Injection

valve

LMG sensor

(a)

Stea

dy

stat

e (

)

08

09

1

11

12

13

14

15

Dis

per

sio

n

70

80

90

100

110

120

Sam

pli

ng

rate

(h

)

80

85

90

95

100

105

0 100 200 300 400 500

(b)St

ead

y st

ate

()

Dis

per

sio

n

Sam

pli

ng

rate

(h

)0

1

2

3

4

90

130

170

210

250

50

70

90

110

130

150

4 6 8 10 12 14

Flow rate (mLmin)

(c)




4 Conclusions





Acknowledgment


References











































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of











































































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




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Journal of

Chemistry


Advances in

Physical Chemistry



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Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of












Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry




CatalystsJournal of



Documents

ResearchArticle Host-TailoredSensorsforLeucomalachite ...recipp.ipp.pt/bitstream/10400.22/6734/1/ART_MoreiraFelismina_2013.pdf · JournalofChemistry 5 Wavenumbers (cm−1) Sulfonic