Sergio MoralesRafael Cela

Departamento de QuímicaAnalítica, Nutrición yBromatología, Facultad deQuímica, Universidad deSantiago de Compostela,Santiago, Spain

Field-amplified sample stacking and nonaqueouscapillary electrophoresis determination of complexmixtures of polar aromatic sulfonates

Nonaqueous CE and field-amplified sample stacking have been used in the determina-tion of complex mixtures of polar aromatic sulfonates (AS; mainly benzene- andnaphthalenesulfonates) of environmental concern. The analytical procedure consistsof an on-column aqueous sample enrichment, followed by the nonaqueous electro-phoretic determination of stacked aromatic sulfonates. Various organic solvents wereused as separation medium, acetonitrile and N-methylformamide gave the best results.Optimum capillary electrophoresis separation is obtained with ammonium acetate(25 mM) dissolved in N-methylformamide-methanol (90:10) as background electrolyte.This combined method was applied to the analysis of surface water samples spikedwith selected aromatic sulfonates derivatives.

Keywords: Nonaqueous capillary electrophoresis / Polar aromatic sulfonates / Sample stacking /Surface waters EL 4770

1 Introduction

A wide variety of polar aromatic sulfonates – mainly linearalkylbenzene sulfonates (LAS) and substituted benzeneand naphthalenesulfonates – are frequently used in indus-trial and domestic processes [1, 2] and, subsequently,released to aquatic environment. Although the aromaticsulfonates (AS) have been produced and applied for along time, little information on their toxicology, ecotoxicol-ogy and environmental behavior is available. In fact, lim-ited attention has been paid to AS other than LAS withinaquatic environments, probably due to their apparentlycomforting ecotoxicological potential: its toxicity and therisk of bioaccumulation appears to be small. However, incontrast to LAS, which have been found to be readily bio-degradable [3], other AS without long alkyl side chains areconsidered as biologically persistent compounds [4, 5].Moreover, polar AS possess a high mobility within theaquatic system and, in consequence, they can cause pol-lution of surface waters and pass the water treatment pro-cess. AS are frequently found in natural waters [6–9], andthey have even been detected in tap and drinking watersat concentrations of about 1 �g/L [10]. In industrial wastewater and water treatment plants the concentrations ofthese sulfonates may reach the mg/L range [11–13].

The low biodegradability of polar AS makes them potentiallyhazardous, however, the most dangerous effect of thesecompounds on the environment probably comes from theirsurface activity, which enables the mobilization and subse-quent release into groundwater of other toxic substances,commonly adsorbed in soils and sediments. Because oftheir polarity, nonvolatilityand thermal lability, gaschromato-graphy (GC) is only of limited value for AS determinations.Derivatizationprior to GC analysis is useful forLAS and othermonosulfonates. However, derivatives of di- and polysulfo-nates lack volatility. Furthermore, hydroxy- and aminosulfo-nates are only incompletely derivatized with most reagents[14]. Thus, liquid chromatography (LC) and capillary electro-phoresis (CE) methods are preferred in environmental deter-minations of AS. In liquid chromatography, published proce-dures include reversed-phase, ion exchange and ion-pair,but most of currently available methods for AS determina-tion are based on ion-pair reversed phase-high-perfor-mance liquid chromatography [2, 15–17].

Since aromatic sulfonates are permanently charged, CE,with its high efficiency and peak capacity can be consid-ered the technique of choice for their determination. Themajor advantages of CE over HPLC are its simple, inex-pensive and routine operation, higher efficiency andshorter analysis time. In addition, the use of nonaqueousmedia for CE separations [18–28] of small molecules hasproven to be very beneficial for reaching high selectivity,even with simple electrolytes. In this way, the introductionof organic solvents has extended the range of applicationand the effectiveness of this technique. Increasingly,reports are being published on the advantages of devel-oping CE separation in organic media: depending onthe solvent, improved selectivity and efficiency can be

Correspondence: Dr. R. Cela, Departamento de Química Analí-tica, Nutrición y Bromatología, Facultad de Química, Universidadde Santiago de Compostela, E-15706 Santiago, SpainE-mail: qnrctd@usc.esFax: +34-81-54-71-41

Abbreviations: AS, aromatic sulfonates; FASS, field-amplifiedsample stacking; NACE, nonaqueous capillary electrophoresis;NMF, N-methylformamide

408 Electrophoresis 2002, 23, 408–413

ª WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002 0173-0835/02/0302–408 $17.50+.50/0

reached by changing acid-base properties and hydrody-namic volume of solutes. Other advantages of nonaque-ous electrolytes include reduced adsorption of hydro-phobic compounds to capillary wall and lower Joule heat-ing, allowing higher field strengths compared to typicalaqueous buffers. However, for environmental analysis,CE using common UV absorption detection has a well-known limitation. Usually, detection limits are well abovethe ones needed in environmental studies. For this rea-son, an appropriate sample pretreatment procedure, on-column sample stacking or a high sensitive detectionmethod should be frequently applied.

Classical solid-phase extraction (SPE), the preferredtechnique for LAS, is less straightforward for other sulfo-nates lacking a hydrophobic alkyl chain. Ion-pairing SPE,with cationic reagents and hydrophobic sorbents is suita-ble for the extraction of these highly water-soluble sulfo-nates. Good recovery values for many AS have beenreported [15–17]. Nevertheless, very hydrophilic amino-and hydroxysubstituted benzenesulfonates, as well asnaphthalenesulfonates with two or three hydrophilicgroups cannot be extracted quantitatively, even by ion-pair SPE. Furthermore, as the nonvolatile ion-pairingreagent interferes with electrophoretic separation, ionpair SPE is incompatible with CE.

In our previous work [29, 30], we reported the significanteffect of nonaqueous electrolytes for reaching better elec-trophoretic separations of complex mixtures of chargeablecompounds. This paper reports the development of meth-ods for the determination of a wide variety of AS derivativesby CE using nonaqueous buffers. Acetonitrile, methanol,N-methylformamide and its combinations were assayed asbuffer solvents. Better separations were obtained withN-methylformamide-based electrolytes. These methodswere applied to the determination of sulfonates in riverwater samples that were spiked with known concentrationsof selected compounds in the range levels found in surfacewaters. Field-amplified sample stacking (FASS) in nonaqu-eous media was successfully applied as sample enrich-ment methodology for the analysis of these samples.

2 Materials and methods

2.1 Chemicals

Acetonitrile (HPLC grade), toluene-4-sulfonic acid (sodiumsalt), 4-aminonaphthalene-1-sulfonicacid (sodium salt) and4-amino-5-hydroxinaphthalene-2,7-disulfonic acid (mono-sodium salt) were obtained from Merck (Darmstadt, Ger-many). N-Methylformamide, ammonium acetate, aniline-2-sulfonic acid, sulfanilic acid (sodium salt hydrate), 4-amino-toluene-3-sulfonic acid and 1-naphthol-3,6-disulfonic acid

(disodium salt hydrate) were obtained from Aldrich (Madrid,Spain). Benzenesulfonic acid (sodium salt), 3-aminobenze-nesulfonic acid, 3-nitrobenzenesulfonic acid (sodium salt),phenol-4-sulfonic acid (sodium salt dihydrate), benzene-1,3-disulfonic acid (disodium salt), naphthalene-1-sulfonicacid (sodium salt), naphthalene-2-sulfonic acid (sodiumsalt), 1-naphthylamine-5-sulfonic acid, 1-naphthylamine-7-sulfonic acid, 2-naphthylamine-1-sulfonic, 6-amino-1-naphthol-3-sulfonicacid, 6-amino-4-hydroxy-2-naphthale-nesulfonic acid, 1-naphthol-4-sulfonic acid (sodium salt),naphthalene-1,5-disulfonic acid (disodium salt), 2,6-naphthalenedisulfonic acid (disodium salt), 2-naphthyla-mine-1,5-disulfonic acid (disodium salt), 2-naphthol-3,6-disulfonic acid (disodium salt), nafthalene-1,3,6-trisulfonicacid (trisodium salt hydrate) and anthraquinone-2-sulfonicacid (sodium salt) were from Fluka (Buchs, Switzerland).Sodium hydroxide was supplied from BDH (Poole, UK).Ultrapure water was obtained from a Milli-Q system (Milli-pore Corp., Bedford, MA, USA). Methanol was purchasedfrom Scharlau (Barcelona, Spain) and potassium hydroxidefrom Carlo Erba. Stock solutions of each sulfonate deriva-tive were prepared at about 500 �g/g. Chemical standardswere dissolved in the solvent or solvent mixture corre-sponding to the electrophoresis medium. All solutionswere refrigerated at 4�C and protected against daylight.These solutions were used to make working standardssolutions by appropriate dilution.

2.2 Capillary electrophoresis

CE was performed using an HP3D system (Hewlett-Pack-ard, Waldbronn, Germany) equipped with an on-columndiode-array detector (DAD). Detection wavelengths of210, 230 254 and 280 nm (direct UV detection) wereused for all samples, depending of species considered ineach particular mixture. 280 nm is a good compromise.Uncoated narrow-bore silica capillaries (supplied byComposite Metal Services, Hallow, UK) with an inner dia-meter of 75 �m, a total length of 70 cm and effectiveseparation length of 62.5 cm were used. The capillarywas thermostated to 20�C. A Techne RB-5A externalwater bath was used for thermostating the samples try to20�C. Samples were injected by applying a pressure of50 mbar for 5 s unless otherwise stated. The applied volt-age for separation was 30 kV (positive or negative polar-ity). New capillaries were rinsed with 1 M sodium hydrox-ide for 20 min. Before injections, capillaries were condi-tioned by washing them with 0.1 M sodium hydroxide for5 min, water for 5 min, organic solvent corresponding tothe electrophoresis medium for 10 min and 20 min withthe separation electrolyte. After each run the capillarywas flushed for 5 min with water. N-Methylformamide(NMF), acetonitrile (ACN), methanol (MeOH) and its mix-

Electrophoresis 2002, 23, 408–413 Sample stacking-NACE separation of polar aromatic sulfonates 409

CE

and

CE

C

tures were assayed as solvent for nonaqueous back-ground electrolyte preparation. Ammonium acetate,being readily soluble in all solvents investigated, wasused as electrolytic salt. All solutions were filtered beforeuse through a membrane of 0.22 �m pore size.

2.3 Sample preparation

A known volume of tap or surface water was taken andeventually spiked with sulfonate standards as specifiedbefore further processing. Surface water samples werepassed through a C-18 SPE cartridge in order to removenonpolar interferences prior to the enrichment step.

3 Results and discussion

3.1 Capillary electrophoresis

Some experimental parameters as capillary diameter andlength were chosen on the basis of previous works in non-aqueous CE (NACE) of small organic molecules [29, 30]. A75 �m inner diameter capillary was used for best UVdetection sensitivity whereas, owing to the number ofsample components in mixtures (26 sulfonate deriva-tives), the capillary length was set at 70 cm (62.5 cm tothe detector). The sequence described for capillary con-ditioning in 2.2 was designed by taking special care of thecompatibility between aqueous and organic solutions, inorder to avoid capillary blockade. About applied voltage,electric field strength does not affect significantly theseparation selectivity but migration times decrease asthe potential is increased, then, highest field strength willbring about the shortest analysis time. On the other hand,heat generation limits the application of high fieldstrengths. According to Ohm‘s law, for a given buffer, theplot of applied field strength versus the resulting currentshould be linear. At higher potential differences, increasedheat generation may cause deviations from this linearbehavior. However, as one of the most advantageousproperties of nonaqueous buffers is the lower Joule heat-ing, it was proven that, for every assayed electrolyte, thehighest allowable voltage can be applied without any signof Ohm‘s law deviation. Then, to speedup separations,30 kV (positive or negative) was applied in all cases. Bestseparations were obtained using normal polarity.

3.2 Separations

CZE separations of AS were performed with several non-aqueous buffers. In order to analyze the electrophoreticbehavior of these broad family of compounds in organicmedia, 26 benzene and naphthalenesulfonates of a wide

range of different substitution were chosen (see Section2.1). These substances are of environmental concernand, most of them, have been found in river and waste-water samples [2, 16, 17, 31, 32]. With the exception ofaminobenzenesulfonic acids, AS are strong acids, whichare almost entirely dissociated in aqueous solutions. Itmeans that in solution, except at extremely acidic pH,they behave as charged particles: each -SO3H group willrepresent a negative charge. This fact provides own mobi-lity to AS in a given electrophoretic medium. Under con-ventional conditions (normal polarity) the target electrodein capillary zone electrophoresis is the negative chargedcathode. Then, for reaching the detector, the own electro-phoretic movement of sulfonate anions toward the anodemust be superseded by the EOF in cathodic direction.

3.2.1 Separations in ACN-based electrolyte

First runs were performed in ACN-based electrolytes,however, the addition of MeOH and acetic acid (HAc) wasneeded to enhance the solubility of ammonium acetate.Starting buffers were prepared with salt concentrationsfrom 0 to 200 mM in a mixture of ACN-MeOH-HAc(74:25:1). In all cases, under suitable conditions, the elec-trophoretic mobility of AS is higher than EOF and reversepolarity (anodic detection) is needed to bring the analytesto detector. Figure 1 shows the electropherogram corre-sponding to the injection of the complete mixture of 26 aro-matic sulfonates derivatives. Compared to other injections(electropherograms not shown), the substitution of MeOHby a 0.1 M potassium hydroxide (KOH) methanolic solutionimproves the resolution of electrophoretic system. KOHcontributes to increment pH and ionic concentration ofelectrolyte solution. The increment in ionic concentrationof the buffer modifies both analyte effective charge andelectroosmotic flow. This combined effect leads to lowermigration times and higher system selectivity. As shownin the figure, the majority of studied compounds appear inthe electropherogram as single peaks. Only a group (com-pounds 6, 9, 11 and 15) and a couple (compounds 5 and25) comigrate. Note that compounds 1 and 20 can beresolved by proper selection of the detection wavelength.

Effective analyte charge is probably the most importantseparation parameter and it may help to understand theelectrophoretic behavior of sulfonates. Average electricalcharge of each compound will be determined by thecontribution of three types of chargeable substituents:-SO3H, -NH2 and -OH. As stated before, each -SO3Hgroup represents a negative charge. As regards to -NH2

group, in a neutral or basic medium this group will notcarry any electrical charge, then its presence only contri-butes to the hydrodynamic volume. On the other hand,the influence of -OH substituents on the electromigration

410 S. Morales and R. Cela Electrophoresis 2002, 23, 408–413

Figure 1. Injection of the complete mixture (approxi-mately 10 �g/mL each) of AS. Electrolyte, 150 mM ammo-nium acetate in ACN-MeOH (KOH 0.1)-CH3CO2H(74:25:1); applied voltage, 30 kV; temperature, 20�C.Detection wavelength, 210 and 230 nm. Compounds:(1) benzenesulfonic acid; (2) 2-aminobenzenesulfonicacid; (3) 3-aminobenzenesulfonic acid; (4) 4-amino-benzenesulfonic acid; (5) 2-amino-5-methylbenzenesul-fonic acid; (6) 4-toluensulfonic acid; (7) 3-nitrobenzene-sulfonic acid; (8) 4-hydroxybenzenesulfonic acid; (9) 1,3-benzenedisulfonic acid; (10) 1-naphthalenesulfonic acid;(11) 2-naphthalenesulfonic acid; (12) 4-aminonaphtha-lene-1-sulfonic acid; (13) 1-aminonaphthalene-5-sulfonicacid; (14) 1-aminonaphthalene-7-sulfonicacid; (15) 2-ami-nonaphthalene-1-sulfonic acid; (16) 2-amino-5-hydroxy-naphthalene-7-sulfonic acid; (17) 2-amino-8-hydroxy-naphthalene-6-sulfonic acid; (18) 1-hydroxynaphthalene-4-sulfonic acid; (19) 1,5-naphthalenedisulfonic acid;(20) 2,6-naphthalenedisulfonic acid; (21) 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid; (22) 2-amino-naphthalene-1,5-disulfonic acid; (23) 1-hydroxynaphtha-lene-3,6-disulfonic acid; (24) 2-hydroxynaphthalene-3,6-disulfonic acid; (26) 2-anthraquinonisulfonic acid.

will be considerable at the basic pH range. This meansthat even small changes in pH will strongly affect the aver-age electrical charge of a given sulfonate.

Obviously, the relation between the average charge andthe hydrodynamic volume of every compound determinesthe migration order. As depicted in Fig. 1, highest migra-tion times correspond to naphthalenemonosulfonates(compounds 16 and 17) with hydroxyl (probably low disso-ciated) and amino substituents, in other words, com-pounds with high hydrodynamic volume and low negativeaverage charge. The lowest migration time corresponds to3-nitrobenzenesulfonic acid (peak 7), a compound withlow hydrodynamic volume, which has an electron with-drawing substituent (-NO2). As expected, polysulfonatesexhibit low migration times. Both 4-hydroxybenzenesulfo-nic acid (peak 8) and 1-hydroxy-4-naphthalenesulfonicacid (peak 18), having the same substituents and differenthydrodynamic volume, migrate at similar times. 3-Amino-benzenesulfonic acid (peak 3) and 1-amino-7-naphthale-

nesulfonic acid (peak 14) behave in the same way. It meansthat for these couples, more than hydrodynamic volume,the effective charge determines the migration behavior.

3.2.2 Separations in NMF

NMF has advantageous properties for NACE such as itshigh dielectric constant, solubilizing power, amphiproticcharacter and low conductivity. The pure solvent, withoutadded electrolyte salt, was assayed as electrophoreticmedium and, even at the highest attainable field strength,the system maintained a stable current level and no signsof electrical breakdown were observed. Nevertheless,poor resolution of sulfonates mixture was obtained (elec-tropherogram not shown). Binary combinations of NMFwith ACN or MeOH (NMF as main component) at differentconcentrations of ammonium acetate provided betterresults. Fig. 2 shows the electropherogram correspond-ing to the injection in normal polarity of the complete mix-ture of sulfonates at the specified conditions. This electro-phoretic medium provided the best resolution of sulfonatemixture: only compounds 13 and 14 (1-amino-5-naphtha-lenesulfonic acid and 1-amino-7-naphthalenesulfonicacid) were fully overlapping. Peaks 12 and 18 can beresolved at 280 nm. As separations take place in normalpolarity, electromigration behaves as expected, in a re-verse order to analyte mobility. 1,3,6-Naphthalenesulfonicacid, having three -SO3H substituents, exhibits the high-est migration time. Next, at high times too, all disulfonatesmigrate. Naphthalenemonosulfonates, compounds withhigh hydrodynamics volume and low average charge,exhibit the shortest migration times. Benzenemonosulfo-nates appear in the middle of the electropherogram.These compounds have a low hydrodynamic volume buta low average charge, too.

3.3 Sample stacking injections: analysis of realsamples

The majority of currently available methods for the analy-sis of AS involve a previous enrichment sample stepbased on ion-pairing SPE. However, this method exhibits

Figure 2. Injection of the whole mixture (approximately10 �g/mL each) of AS derivatives in NMF-based electro-lyte. For the numeric key of compounds see Fig. 1.

Electrophoresis 2002, 23, 408–413 Sample stacking-NACE separation of polar aromatic sulfonates 411

limited extraction efficiency for very polar sulfonates.Many sorbents and extracting procedures have beendeveloped but apparently a single method, useful for allsulfonate derivatives, does not exist. If all analytes arestacked completely, FASS may be a promising methodto concentrate liquid samples of environmental concern,containing AS. The stacking process involves the injec-tion of large low-conductivity sample volumes. Then, thenature of sample matrix and its compatibility with theseparation process should be carefully considered for aproper system performance.

In our study tap and river water samples were analyzed.Because no positive results in the analysis of sulfonateswere obtained, some of these samples were spiked witha randomly selected mixture of 18 sulfonate derivativesat concentration levels similar to those found in pre-viously published surface water studies [10]. Sampleswere hydrodynamically injected at 50 mbar by 100, 200and 300 s. Prior to injection, river water samples werecleaned by passing the sample through a reversed-phase SPE cartridge. The lack of interaction betweenpolar AS and C-18 material allows using this cartridgefor the removal of less polar interferences [6, 16, 33,34]. Tap water samples were directly injected. Onceinjected a large sample volume, negative polarity isapplied to produce analyte focusing at the injection endof the capillary whereas EOF removes samples matrix. Inthis way, the analytes are “stacked” at the injection endof the capillary. Then, separation is performed in a non-aqueous electrolyte using the described procedure. Fig. 3shows the electropherogram corresponding to the sam-ple stacking injection of a spiked river water sample.As the results suggest, water samples containing AScan be directly stacked followed by analyte separationin a nonaqueous electrophoretic system. No signs ofunsuitability (like current drops) were observed. In thisway, FASS, a concentration procedure of general applic-ability, and NACE, with its advantages in terms of qualityof the separations, conform an excellent analytical pack-age for the analysis of environmental liquid samples (likesurface water) containing aromatic sulfonates.

4 Concluding remarks

The advantageous properties of nonaqueous electrolytesallow the selective and efficient separation of complexmixtures of closely structural related AS derivatives.Once again, NMF has proven to be an excellent solventfor NACE: with an NMF-based electrolyte, most of the 26components in the mixture of AS can be satisfactorilyresolved. Whereas SPE fails in the extraction of polysulfo-nates, FASS may be the best choice for the determination

Figure 3. FASS injection of a river water sample spik-ed with selected sulfonates. Injection, 50 mbar/200 s;electrolyte, 25 mM ammonium acetate in NMF-MeOH(90:10); applied voltage, – 30 kV, 30 kV; detection wave-length, 254 nm. For the numeric key of compounds seeFig. 1. Compounds: (1) benzenesulfonic acid (111 ng/mL);(2) 2-aminobenzenesulfonic acid (125 ng/L); (3) 3-amino-benzenesulfonic acid (67 ng/mL); (4) 4-aminobenzene-sulfonic acid (154 ng/mL); (7) 3-nitrobenzenesulfonicacid (132 ng/mL); (8) 4-hydroxybenzenesulfonic acid(207 ng/mL); (10) 1-naphthalenesulfonic acid (120 ng/mL);(11) 2-naphthalenesulfonic acid (108 ng/mL); (12) 4-amino-naphthalene-1-sulfonic acid (137 ng/L); (13) 1-amino-naphthalene-5-sulfonic acid (41 ng/mL); (15) 2-amino-naphthalene-1-sulfonic acid (113 ng/mL); (16) 2-amino-5-hydroxynaphthalene-7-sulfonic acid (63 ng/mL); (19) 1,5-naphthalenedisulfonic acid (109 ng/mL); (20) 2,6-naphtha-lenedisulfonic acid (95 ng/mL); (22) 2-aminonaphthalene-1,5-disulfonic acid (84 ng/mL); (23) 1-hydroxynaphthalene-3,6-disulfonic acid (95 ng/mL); (24) 2-hydroxynaphtha-lene-3,6-disulfonic acid (92 ng/mL); (26) 2-anthraquinoni-sulfonic acid (75 ng/mL).

of AS in environmental liquid samples, especially inscreening studies. Water samples could be directlystaked prior to NACE determination of AS derivatives. Asthe results suggest, the present analytical methodology isa promising alternative for routine determination of AS insurface waters.

S. M. acknowledges a long-term fellowship from SpanishAgency for International Cooperation (Ministerio de Asun-tos Exteriores).

Received July 24, 2001

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Electrophoresis 2002, 23, 408–413 Sample stacking-NACE separation of polar aromatic sulfonates 413