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Talanta 75 (2008) 841–845

Short communication

The simultaneous determination of chloride, nitrate and sulphateby isotachophoresis using bromide as a leading ion

Jeff E. Prest ∗, Peter R. FieldenSchool of Chemical Engineering and Analytical Science, The University of Manchester, PO Box 88, Manchester M60 1QD, UK

Received 27 July 2007; received in revised form 27 November 2007; accepted 7 December 2007Available online 23 December 2007

bstract

A new method has been devised to allow the determination of small inorganic anions using isotachophoresis. This method makes use ofndium(III) as a counter ion to manipulate the effective mobilities of inorganic anion species by means of complexation reactions. This new

rocedure successfully allowed the simultaneous determination of nitrate, chloride and sulphate to be realised on a capillary scale instrument andn a chip-based separation device. The electrolyte system developed to allow the separation to be achieved employed a 10 mM bromide-basedeading electrolyte containing 1.25 mM indium(III) at pH 3.15 and a terminating electrolyte of cyanoacetic acid.

2007 Elsevier B.V. All rights reserved.

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eywords: Isotachophoresis; Inorganic anions; Chloride; Nitrate; Sulphate

. Introduction

Isotachophoresis is a powerful analytical technique that hasroven to be highly suited to the analysis of small ionic species1,2]. The technique offers a number of features such as the abil-ty to preconcentrate dilute samples and the capacity to handle aide variety of concentrations, which can be of benefit when per-

orming practical analytical measurements. However, to performn isotachophoretic separation it is necessary to use a discontin-ous electrolyte system comprising of a leading electrolyte andterminating electrolyte. The leading electrolyte must contain

n ion with a higher mobility than the sample species and theerminating electrolyte an ion with a lower mobility than theample species. The latter requirement can usually be realisedasily but the former can cause problems if there is a requiremento analyse ions which exhibit a high electrophoretic mobility.

The ability to analyse nitrate, chloride and sulphate simulta-eously is of great importance to many applications particularlyhose involving the analysis of waters. However, such a separa-

ion is difficult to realise using isotachophoresis because all threef these anions possess high electrophoretic mobilities. The mostroblematic of the species to analyse is chloride because in most

∗ Corresponding author. Tel.: +44 161 3068900; fax: +44 161 3064896.E-mail address: j.prest@manchester.ac.uk (J.E. Prest).

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ractical situations this ion has a higher effective mobility thanitrate or sulphate. Indeed, in most electrolyte systems devel-ped for anionic separations using isotachophoresis, chloride ismployed as the leading ion. Previously several methods haveeen proposed for allowing the simultaneous determination ofitrate, chloride and sulphate to be made using isotachophore-is. The most successful of these saw the use of dithionate asleading electrolyte, which has a higher mobility than chlo-

ide, together with magnesium as a complexing agent to retardhe effective mobility of sulphate behind that of nitrate [3].owever, whilst this method works well and has been sub-

equently used for chip-based separations [4] it requires these of dithionic acid, a substance which is difficult to source.nly the sodium salt of dithionate is commonly available so

hat an ion exchange process is needed to obtain the acid.ther solutions to the problem have seen the use of hydrox-

de as a leading ion together with calcium as a complexinggent [5] and simply using chloride as the leading ion with 1,3-is-[tris(hydroxymethyl)methylamino]propane as a complexinggent [6]. Problems with these approaches are that the former isn unbuffered system and thus likely to suffer irreproducibilityroblems [7] and the latter only allows quantification of chlo-

ide via an indirect method based on the change in length of theeading ion zone.

This paper describes the findings of a preliminary study intohe development of a new method to enable the simultaneous

8 / Talanta 75 (2008) 841–845

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42 J.E. Prest, P.R. Fielden

etermination of nitrate, chloride and sulphate to be made usingsotachophoresis. Through the employment of indium(III) as aomplexing counter ion it was possible to identify a set of con-itions where the required separation could be achieved usingromide as a leading ion.

. Experimental

.1. Instrumentation

Separations were performed using either an ItaChromA202M electrophoretic analyser (JH-Analytik, Aalen, Ger-any) or using a miniaturised poly(methyl methacrylate)

PMMA) separation device fabricated in-house. The ItaChromnstrument was fitted with two columns: a 100 mm long, 0.8 mmnternal diameter (ID) fluorinated ethylene–propylene copoly-

er preseparation column and a 140 mm long, 0.3 mm ID silicanalytical column. Both columns were fitted with contactlessonductivity detectors. The analytical column also contains ann-column UV detector. Control of the analyser and data pro-essing was carried out on a personal computer using ITPPro32oftware version 1.0 (Kas Comp, Bratislava, Slovakia). In thistudy the separations were carried out using only the presepara-ion column. To perform runs, samples were injected using thenternal 30 �l sample loop and then analysed using a two stepontrol program. In this program the first step involved apply-ng a current of 400 �A for 260 s. Detection was undertaken inhe second step, during which a lower current of 200 �A waspplied.

The miniaturised separation device comprised a PMMA chipith an integrated on-column conductivity detector, full detailsf which have been previously described [8]. Briefly, the deviceontained two channels; a 200 �m wide, 300 �m deep separa-ion channel with an effective length of 44 mm and a 57 mm longnjection channel with a width of 300 �m and depth of 300 �m.his arrangement results in the system having an injection vol-me of 5.1 �l. A schematic diagram of the layout of the chip ishown in Fig. 1. Control of the separations, data capture and datanalysis was done using LabVIEW software (National Instru-ents, Austin, TX, USA). Sample injection was made using a

ravity feed hydrodynamic system. Separations were performedy applying a current of 40 �A for 260 s and then reducing thisurrent to 20 �A. Detection was undertaken whilst the lowerurrent was applied.

.2. Chemicals

The electrolyte system developed incorporates a leading elec-rolyte composed of hydrobromic acid (48% analytical grade,iedel-de Haen, Gillingham, Dorset, UK) and indium bromide

99.99%, Alfa Aesar, Heysham, Lancashire, UK). The termi-ating electrolyte was 20 mM cyanoacetic acid (99%, Aldrich,illingham, Dorset, UK). To suppress electro-osmotic flow

g l−1 of hydroxyethyl cellulose (HEC) (molecular weight ca.50000, Aldrich) was added to the leading electrolyte. TheH of the leading electrolyte was adjusted using glycylglycine99+%, Acros, Loughborough, UK). Chloride samples were pre-

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ig. 1. Schematic diagram of the channel network in a miniaturised PMMAeparation device. LE = leading electrolyte and TE = terminating electrolyte.imensions shown are in mm.

ared using an ion standard solution (1000 mg l−1, BDH, Poole,K). Nitrite and sulphate samples were prepared using sodium

alts (>99%, Aldrich). Iodide (99.5%, BDH) and nitrate (99+%,ldrich) samples were produce using potassium salts. All sam-les were prepared using >18 M� water (Elga Maxima Ultraure, Vivendi Water Systems, High Wycombe, UK).

. Results and discussion

Previously the authors had discovered that by usingndium(III) as a complexing agent, the halide ions, chloride,romide and iodide could be readily separated using an elec-rolyte system based on using nitrate as the leading ion [9]. Thisroup of ions is notoriously difficult to separate using isota-hophoresis so this represented a useful finding. Unfortunately,he use of nitrate as the leading ion meant that the method wasot suitable for the analysis of this species. However, despitehis it was believed that the complexation that occurs betweenarious anions and indium(III) might offer a potential meansf enabling a separation of nitrate, chloride and sulphate toe achieved. The reasoning behind this belief was the fact thatndium(III) forms complexes of different stabilities with a rangef inorganic anions. This feature can be seen in the size of thetability constants (log K1) of the indium(III) complexes formedhich are 0.18, 2.01 and 2.41 with nitrate, bromide and chloride,

espectively [10] and 3.04 with sulphate [11].From the different stability constants of the indium(III)–

nion complexes it was thought that it may be possible to findset of conditions under which bromide could be employed asleading ion to allow the separation of nitrate, chloride and

ulphate to be achieved. Such an idea was thought possibleecause bromide possess a somewhat higher absolute mobil-ty of −80.9 × 10−9 m2 V−1 s−1 than that of nitrate which is

74.0 × 10−9 m2 V−1 s−1 [12]. Thus, in the absence of anyomplexation, bromide will be faster than nitrate and the twopecies can be readily separated. If indium(III) is added to theeading electrolyte the effective mobilities of these two ions

/ Talanta 75 (2008) 841–845 843

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Fig. 2. Effect of indium concentration on the relative step heights observed forciE

Waaration of nitrate, chloride and sulphate. An example of such aseparation can be seen in Fig. 3. From this result it can be seenthat the three sample species could be well resolved from one

J.E. Prest, P.R. Fielden

ill change due to complexation and eventually the migra-ion order is reversed. Chloride has an absolute mobility of

79.1 × 10−9 m2 V−1 s−1 [12], a value similar to that of bro-ide. Thus, without complexation, bromide and chloride cannot

e separated. However, as chloride complexes with indium(III)ore strongly than bromide the effective mobility of the two

pecies changes so that such a separation can be readilychieved. Sulphate has a higher absolute mobility than bromidef −82.9 10−9 m2 V−1 s−1 [12]. However, as this species is diva-ent, even in the absence of any complexation, it exhibits a lowerffective mobility than bromide due to ionic strength effects.he addition of indium(III) to the leading electrolyte would bexpected to slow this down to a greater extent than the otherpecies of interest due to forming stronger complexes.

To see whether the required separation could be achieved inractice a series of experiments were carried out using a rangef leading electrolytes containing 10 mM bromide at pH 3.0s the leading ion. The terminating ion used was cyanoacetate.his species was selected for this purpose as compared to moreommonly employed terminating ions in isotachophoresis, suchs acetate, it has a reasonably high effective mobility underhe conditions used due to having a pKa of 2.45 [12]. Using

higher mobility terminating ion lowers the voltages arisinguring the separations, thus reducing the possibility of adverseeating effects such as bubble formation occurring and will alsoeduce the range between the leading and terminating steps. Thisatter effect was thought likely to assist in the identification of themall steps expected from the analysis of the high mobility inor-anic anions of prime interest in this study. To the leading ionere added varying concentrations of indium(III) of betweenand 1.5 mM. With each of the different electrolyte systems,

eparations were performed with single component samples ofitrate, chloride and sulphate. The resulting relative step heightsRSH) of the different species were recorded. In this work theSH was calculated as the height of the sample step dividedy the height of the step for the terminating ion. The resultsbtained are shown in Fig. 2. From the RSHs observed it can beeen that the use of bromide as a leading ion allowed sulphateo be determined under all conditions. When indium(III) con-entrations of 0.25 mM and above were used it was possible toeparate chloride from bromide whereas nitrate could be seenith indium concentrations of 1.25 mM and below. The results

ndicated that chloride and nitrate were likely to co-migrate withndium(III) concentrations of between 0.5 and 1.0 mM. There-ore, the two potential systems which seemed likely to allowhe for the analysis of nitrate, chloride and sulphate were thoseith leading electrolytes containing either 0.25 mM indium(III)r 1.25 mM indium(III) as a complexing agent.

The system that was selected as being most useful for fur-her investigation was that containing 1.25 mM indium(III), ast gave the biggest difference in RSHs between the three speciesf interest. Thus, further experiments were carried out using suchsystem. For these further experiments the pH of the system was

aised to pH 3.15. This change was made as it was thought it mayllow the method to also be used for the determination of nitrite.t pH 3.0 nitrite possess too low an effective mobility to migrate

sotachophoretically ahead of the cyanoacetate terminating ion.

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hloride, nitrate and sulphate. The leading electrolyte is at pH 3.0, the leadingon is 10 mM bromide and the terminating electrolyte is 20 mM cyanoacetate.rror bars represent ±S.D. (n = 3).

hen separations were attempted using the leading electrolytet pH 3.15 it did prove possible to achieve a simultaneous sep-

ig. 3. Isotachopherogram of a separation of a sample containing 20 mg l−1

hloride, nitrate and sulphate performed on an ItaChrom EA202 electrophoreticnalyser. Leading electrolyte 6.25 mM HBr, 1.25 mM InBr3, 1 g l−1 HEC,H 3.15 (glycylglycine). Terminating electrolyte 20 mM cyanoacetic acid.= nitrate; 2 = chloride; 3 = sulphate; L = leading ion; T = terminating ion.

844 J.E. Prest, P.R. Fielden / Talanta 75 (2008) 841–845

Table 1Relative step heights (RSH) observed for nitrate, chloride and sulphate whenusing a an electrolyte system comprising a leading electrolyte of 6.25 mMHBr, 1.25 mM InBr3, 1 g l−1 HEC at pH 3.15 (glycylglycine) and a terminatingelectrolyte of 20 mM cyanoacetic acid

RSH ± S.D.

Capillary scale Chip

NO3− 0.025 ± 0.005 0.043 ± 0.017

Cl− 0.065 ± 0.006 0.125 ± 0.011S 2−

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a ± S.D. (s) b ± S.D. (s l mg−1) r n Concentrationrange (mg l−1)

Cl− 1.68 ± 0.39 0.647 ± 0.014 0.996 7 2.5–60NO3

− 1.42 ± 0.53 0.628 ± 0.024 0.994 7 2.5–60SO42− 0.25 ± 0.16 0.460 ± 0.007 0.996 7 2.5–60

a = intercept; b = slope; n = number of data points (three replications performeda

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tandard deviations (S.D.s) were calculated over 12 runs.

nother. The observed RSHs for nitrate, chloride and sulphateogether with associated standard deviations (S.D.s) are shownn Table 1. The results show that similar levels of reproducibil-ty were noted in the step heights for all three species. Whenttempts were made to analyse nitrite with the system this alsoroved possible. Nitrite was found to produce a higher step thanhe other species with a RSH ± S.D. of 0.663 ± 0.005 (n = 4).

A possible problem with other methods that have been pro-osed for allowing the simultaneous determination of nitrate,hloride and sulphate using isotachophoresis is that they are sus-eptible to interference from bromide and iodide. This is becauseromide and iodide have very similar mobilities and chemicalroperties to chloride and thus tend to co-migrate with this ion.ith the method developed in this study, bromide is used as the

eading ion so the presence of this species in any samples will notause any interference. Iodide forms weaker indium(III) com-lexes than either chloride or bromide (log K1 = 1.64 [10]) so wasot expected to cause any problems either. This expectation wasonfirmed when experiments were made with samples contain-ng iodide. The results obtained with such separations showedo visible steps and extended leading ion zones indicating thathe species was co-migrating with the leading ion.

To test the potential of the method and to see whethereproducible separations could be achieved a series of sevenixtures were analysed. The mixtures, which were analysed

n three repeat determinations, contained equal concentrationsf nitrate, chloride and sulphate. With these various mixtures,eproducible separations were observed. The average relativetandard deviations for the zone lengths observed in these mix-ures were 4.8% for nitrate, 5.2% for sulphate and 5.4% forhloride. The results from these analyses were used to produceseries of calibration curves. The findings of these experiments

howed that the method behaved in a linear manner over the.5–60 mg l−1 concentration range used, with correlation coef-cients of 0.996, 0.994 and 0.996 being achieved for chloride,itrate and sulphate, respectively. Full details of the calibrationurves, produced using weighted linear regression, can be foundn Table 2.

Whilst the aim of this work was primarily to investigate theotential of the method for enabling the separation of the three

pecies to be achieved, the results used to produce the calibra-ion curves could be used to estimate limits of detection (LOD)or the three species. Calculation of a LOD in isotachophoresiss not a straightforward procedure due to the stepwise form of

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t each); r = correlation coefficient; S.D. = standard deviation.

he output. This is because in isotachophoresis a blank usuallyoes not produce a step, unless there is background contami-ation present. Thus, no meaningful standard deviation in thelank result can be obtained. Therefore, it is not possible to cal-ulate a LOD on the basis of such a result. In this work LODsere taken as being the intercept, which is an estimate of thelank, plus three times the standard deviation of this parame-er. Using this procedure the LODs were found to be 1.0 mg l−1

or sulphate, 1.8 mg l−1 for chloride and 2.5 mg l−1 for nitrate.hese values represent a reasonable performance, being similar

o those previously achieved with a similar type of instrument13], and are adequate for many applications as these anions areften present in significant concentration. However, these lim-ts could be significantly reduced, albeit with increased analysisimes, if separations were carried out using the analytical columnf the instrument. Under such conditions LODs of the order of0–100 �g l−1 could be possible making the method applicableo trace analysis [1].

To further test the applicability of the new method, work wasone to see whether separations could be achieved using a dif-erent type of separation device. Therefore, attempts were madeo employ the developed electrolyte system in analyses using a

iniaturised PMMA chip device. These attempts proved suc-essful and separations of nitrate, chloride and sulphate werechieved on such a device. Details of the RSHs and associated.D.s observed on the miniaturised device are shown in Table 1.n a similar result to that seen on the capillary scale instrument,he reproducibility of the heights noted on the chip device wereimilar for all three species. However, the reproducibility wasower in the results from the miniaturised system. In the minia-urised separations it was found that all species exhibited higherteps than were seen in the capillary scale separations as cane seen from a comparison of the two sets of results shownn Table 1. This difference was thought to arise from differ-nces in the behaviour of the conductivity detectors in the twoevices. Examples of chip-based separations are shown in Fig. 4.ne example is of a separation of a model sample of 20 mg l−1

f chloride, nitrate and sulphate whereas the other is of Sil-er Spring a commercially available mineral water. It can beeen that isotachopherograms for both samples are similar. Thisesult is in agreement with the stated composition of mineral

ater which was that it contained 20 mg l−1 sulphate, 21 mg l−1

itrate, 26 mg l−1 chloride and 292 mg l−1 carbonate.

J.E. Prest, P.R. Fielden / Tala

Fig. 4. Isotachopherograms of separations performed on a PMMA chip. (a)Sample containing 20 mg l−1 chloride, nitrate and sulphate and (b) mineralw(2

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ater. Leading electrolyte 6.25 mM HBr, 1.25 mM InBr3, 1 g l−1 HEC, pH 3.15glycylglycine). Terminating electrolyte 20 mM cyanoacetic acid. 1 = nitrate;= chloride; 3 = sulphate; L = leading ion; T = terminating ion.

. Conclusions

The simultaneous determination of nitrate, chloride andulphate using isotachophoresis is hard to accomplish. A pre-iminary study has thus been carried out into the feasibility of

sing bromide as a leading ion to allow such a separation to bechieved. It was found that it was possible to achieve such sepa-ations by incorporating a concentration of 1.25 mM indium(III)s a complexing agent in the leading electrolyte. This complex-

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nta 75 (2008) 841–845 845

ng agent interacts with the different anions to a different degreeeaning that the species separate out in the order nitrate fol-

owed by chloride followed by sulphate. The devised method iseen as offering a relatively simple means, using readily avail-ble chemicals, of being able to allow what is regarded as aifficult analysis when using isotachophoresis to be achieved.he developed method uses a leading electrolyte at pH 3.15eaning that it can also be used for nitrite determination.

cknowledgements

The authors would like to thank the United Kingdom Homeffice for funding this research programme and the Forensicxplosives Laboratory, of the Defence Science and Technologyaboratory, Fort Halstead, for their support.

eferences

[1] T. Meissner, F. Eisenbeiss, B. Jastorff, Fresen. J. Anal. Chem. 361 (1998)459.

[2] V. Madajova, E. Turcelova, D. Kaniansky, J. Chromatogr. 589 (1992) 329.[3] T. Meissner, F. Eisenbeiss, B. Jastorff, J. Chromatogr. A 838 (1999) 81.[4] R. Bodor, V. Madajova, D. Kaniansky, M. Masar, M. Johnck, B. Stanis-

lawski, J. Chromatogr. A 916 (2001) 155.[5] J. Vacık, I. Muselasova, J. Chromatogr. 320 (1985) 199.[6] I. Zelensky, V. Zelenska, D. Kaniansky, P. Havasi, V. Lednarova, J. Chro-

matogr. 294 (1984) 317.[7] L. Krivankova, F. Foret, P. Gebauer, P. Bocek, J. Chromatogr. 390 (1987)

3.[8] J.E. Prest, S.J. Baldock, P.R. Fielden, N.J. Goddard, B.J. Treves Brown,

Anal. Bioanal. Chem. 376 (2003) 78.[9] J.E. Prest, S.J. Baldock, P.R. Fielden, N.J. Goddard, B.J. Treves Brown,

Analyst 130 (2005) 1375.10] D.G. Tuck, Pure Appl. Chem. 55 (1983) 1477.11] E. Hogfeldt (Ed.), Stability Constants of Metal–Ion Complexes. Part A.

12] D.R. Lide (Ed.), CRC Handbook of Chemistry and Physics, ChemicalRubber Publishing Co., Boca Raton, 1993.

13] P. Kosobucki, B. Buszewski, J. Liquid Chromatogr. Relat. Technol. 29(2006) 1951.