ce frontal analysis employing contactless conductivity detection for determination of cmcs of non-uv...

Henrik JensenJesper ØstergaardSteen H. Hansen

Department of Pharmaceuticsand Analytical Chemistry,Faculty of PharmaceuticalSciences,University of Copenhagen,Copenhagen, Denmark

Received March 30, 2007Revised May 3, 2007Accepted May 3, 2007

Research Article

CE frontal analysis employing contactlessconductivity detection for determination ofCMCs of non-UV absorbing chargedsurfactants

Micellar systems composed of surfactants are used extensively in academia and industry formany different applications. In this work a highly versatile CE method for determination ofCMCs of charged surfactants has been developed. In the case of positively charged surfac-tants a coating procedure of the fused-silica capillary was used, whereas negatively chargedsurfactants were analyzed using uncoated capillaries. The CE method is based on frontalanalysis (FA) employing use of contactless conductivity and UV detection. The mainadvantages of the method are that it can be used for non-UV absorbing surfactants withoutintroducing marker compounds which previously has been found to affect CMCs, requiresvery limited sample volume and is easily implemented and automated using standard CEequipment. The fact that counterions and different aggregated states are separated allows adetailed characterization of the micelle systems using the developed method. In the case ofUV absorbing surfactants similar results were obtained employing contactless conductivityand UV detection. Finally, CMCs obtained using conductometry gave similar results ascompared to the developed CE-FA procedure.

Keywords:

ACE / Contactless conductivity detection / CMC / Frontal Analysis / SurfactantDOI 10.1002/elps.200700236

Electrophoresis 2007, 28, 2975–2980 2975

1 Introduction

Micellar systems are used in a wide variety of applications indifferent scientific disciplines, for instance due to their abil-ity to enhance solubility of compounds that are intrinsicallypoorly soluble in water [1–3]. A number of methods based onfor example, conductometry [4–6], surface tension [1, 3],potentiometry [7], and CE [8] are available for determinationof CMC values. Important advantages of CE-based methodsare rapid analysis (ease of automation), high efficiency andvery low sample volume requirements. As CMC values arehighly dependent on factors such as ionic strength, solvent

composition, temperature, cosurfactants, and counterions [3]it is practical to have convenient and fast methods for CMCdeterminations, e.g., in relation to drug development andformulation. Furthermore, some pharmaceutically relevantsurfactants or surface active drugs may only be available insmall amounts for analysis. The three most common CEmethods for CMC determinations are based on the variationof the retention factor of a model solute surfactant con-centration [9], variation of electric mobility of a marker com-pound as a function of surfactant concentration [10–15], or isbased on measuring the total capillary conductivity as afunction of surfactant concentration [16, 17]. However, thetwo former methods are indirect in the sense that they relyon marker compounds which may not be generally applica-ble to any surfactant system. Furthermore, rather dramaticdifferences in reported CMC values have appeared in the lit-erature suggesting that in some cases the CMC values aredirectly influenced by the presence of marker compounds[13, 18]. Another drawback is related to the fact that thesemethods require fast kinetics of the interaction between themicelle and the marker compound. Finally, a given markercompound is not optimal for any surfactant/micelle systemand should thus be optimized for new systems. The methodbased on measuring the capillary conductivity as a function

Correspondence: Dr. Henrik Jensen, Department of Pharmaceu-tics and Analytical Chemistry, Faculty of PharmaceuticalSciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, DenmarkE-mail: [email protected]: 145-35306010

Abbreviations: DTAB, dodecyltrimethylammonium bromide; FA,

frontal analysis; PDMAC, poly(diallyldimethylammonium chlo-ride); SDeS, sodium decylhydrogensulfate; SDS, sodium dode-cylhydrogensulfate; SOBS, sodium 4-octylbenzoylsulfonate;TTAB, tetradecyltrimetylammonium bromide;

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

2976 H. Jensen et al. Electrophoresis 2007, 28, 2975–2980

of surfactant concentration (which is essentially similar toordinary conductometry) is of limited use as the inflectionpoint of the conductivity versus surfactant concentration plotis often not clearly defined.

Frontal analysis (FA) is frequently used in CE for asses-sing noncovalent molecular interactions [19–23]. In frontalanalysis a relatively large pre-equilibrated sample is intro-duced in the capillary. Upon application of an electrical fielda separation of free and complexed ligand is obtained. Be-cause of the large sample volume plateaus or plateau peaksare obtained. If the experimental conditions are chosen cor-rectly, the height of the plateau or plateau peak correspond-ing to the ligand is directly proportional to the concentrationof free ligand in the original sample. Recently, a new methodfor determining CMC values of UV-absorbing anionic sur-factants based on FA was introduced [24]. This method isattractive as it does not rely on marker compounds andappears to be quite accurate and easily implemented. It is,however, only applicable to anionic UV-absorbing surfactantswhich somewhat limits the usefulness as most of the cur-rently used surfactants do not have suitable chromophores tobe detected. Furthermore, it is not directly applicable to cati-onic surfactants as they are known to adsorb strongly ontouncoated silica capillaries. It has been attempted to extendthe FA procedure to non-UV-absorbing surfactants by intro-ducing UV-absorbing markers [25], however, this proceduremay be expected to suffer from the same limitations as otherCE methods involving marker compounds.

Contactless conductivity detection has been employed asa detector in CE in a number of cases [26–31]. An advantageof this technique is that it does not rely on particular chro-mophores or fluorophores, which is a requirement of spec-troscopically based detectors.

In this work we have developed an FA methodology basedon in-line contactless conductivity and UV detection for de-termining CMC values of non-UV- and UV-absorbing anionicand cationic surfactants. The method is demonstrated usingthree negatively charged (sodium dodecylhydrogensulfate(SDS), sodium 4-octylbenzoylsulfonate (SOBS), and sodiumdecylhydrogensulfate (SDeS)) and two positively charged(dodecyltrimethylammonium bromide (DTAB) and tetra-decyltrimetylammonium bromide (TTAB)) surfactants,respectively. In the case of the cationic surfactants the methodinvolves a coating procedure of the fused-silica capillary.

2 Materials and methods

2.1 Chemicals

SOBS, �97% from Aldrich; sodium decylhydrogensulfate(SDeS), �99%; SDS, �99%, from Merck; dodecyltrimethyl-ammonium bromide (DTAB), �99%; TTAB, �99%;tetraethylammonium bromide �99%, from Sigma.Poly(diallyldimethylammonium chloride) (PDMAC), highmolecular weight, 20% in water, Mr = 4–500 000 Da was

obtained from Aldrich. All other chemicals were at least ofanalytical grade and used as received. All solutions wereprepared using purified water from a Milli-Q system (Milli-pore, Bedford, MA, USA).

2.2 Instrumentation

The CE experiments were performed using a Hewlett Pack-ard 3DCE instrument equipped with a diode array UV–Visdetector and an HP multichannel interface (model 35900E)for recording the signal from the conductivity detector. Theelectropherograms from the UV detector and multichannelinterface were recorded using Chemstation software.

Uncoated fused-silica capillaries (33 cm650 mm id and365 mm od; Polymicro Technologies) were used in all experi-ments. The UV window was positioned 8.5 cm from theshort end of the capillary.

For the contactless conductivity detection a lock-inamplifier from Stanford Research Systems (SR830) equip-ped with a preamplifier (SR550) was used. The signal fromthe internal oscillator of the SR830 was amplified using aTEGAM, model 2340 single channel high-voltage amplifier.Shielded BNC cables were used for all connections.

The electrodes for the conductivity detector were madefrom stainless-steel syringe needles (od = 0.4 mm). Theelectrode connections to the SR830 were enabled using aspecially designed detection cell made from poly-methylmethacrylate (PMMA). The gap (2 mm) between thetwo detector electrodes (length 1.5 cm) was positioned12.5 cm from the short end of the capillary. The frequency ofthe sinusoidal waveform used was 100 kHz. Further detailson the experimental setup and detection cell have beenreported elsewhere [31].

Conductometry was performed using a Metrohm 712conductometer equipped with a parallel plate electrode(Metrohm, model 6.0912.110). The sample solutions wereplaced in a water bath termostated to 257C (Lauda E100). Theelectrode was calibrated using a standard 0.100 M KCl solu-tion (Metrohm, no: 6.2301.060) immediately prior to use.

2.3 Sample and buffer preparation

For all experiments sodium phosphate-buffered sample andelectrophoresis run buffer solutions were used (10 mM,pH 7.0).

2.4 CE experiments

New capillaries were conditioned by flushing the capillariesfor 60 min with 1 M NaOH and 30 min using the run buffer.

2.4.1 Negatively charged surfactants

Before a series of experiments the capillary was flushed20 min using 1 M NaOH and 20 min using the electropho-resis buffer. Before and between experiments the capillary


Electrophoresis 2007, 28, 2975–2980 CE and CEC 2977

was flushed for 1 min with 1 M NaOH, 1 min with 50 mMsodium phosphate buffer (pH 7.0) and 1 min using the CErun buffer.

2.4.2 Positively charged surfactants

Before a series of experiments the capillary was flushed20 min using a 0.1% solution of the positively charged poly-mer PDMAC in 10 mM phosphate buffer (pH 7.0) and2 min using the electrophoresis buffer. Before and betweenexperiments the capillary was flushed for 2 min with 0.1%PDMAC in 10 mM phosphate buffer (pH 7.0) and 1 minusing the CE run buffer (10 mM phosphate, pH 7). Thecoating procedure results in positively charged capillarywalls leading to a reversal of the EOF [32, 33].

The capillary cassette chamber temperature was set to257C. The fact that the conductivity detection cell is placedinside the capillary cassette ensures that it is kept at a con-stant temperature during the experiment. This point israther important as conductivity and CMC values are highlytemperature-dependent. The applied voltage was 610 kVdepending on whether negatively (210 kV) or positively(110 kV) charged surfactants were studied. For CE-FA,samples were introduced continuously from the short end ofthe capillary using the separation voltage. Sample introduc-tion from the short end resulted in sufficient separation ofthe monomer and aggregate forms of the micelles and gen-erally led to more well-defined plateaus as compared tosimilar experiments employing sample injection from thelong end. Further, by using sample introduction from theshort end of the capillary the analysis time is significantlyshorter.

2.5 Data analysis

The electropherograms having plateaus corresponding to thesurfactants and their aggregate forms are obtained by plot-ting detector output versus time. The height of the plateaucorresponding to the free surfactant form is directly propor-tional to the concentration of free surfactant molecules. Byplotting the plateau height against the total surfactant con-centration two straight lines are obtained corresponding tothe concentration regions below and above the CMC. Theintersection point of the two straight lines is taken as theCMC.

3 Results and discussion

A method based on CE-FA employing contactless con-ductivity detection has been developed for the determinationof CMCs of charged surfactants. In this study three nega-tively charged surfactants (SDS, SdeS, and SOBS) and twopositively charged surfactants (DTAB and TTAB) werechosen as model compounds in order to demonstrate thegenerality of the method.

3.1 CE-FA procedures for determining CMCs

For negatively charged surfactants, sample introduction wasperformed from the short end of the capillary employingcontinous injection applying an electrophoresis voltage of210 kV. In the case of the UV active SOBS both contactlessconductivity (Fig. 1a) and UV detection (Fig. 1b) wasemployed. Plateau P1a and P1b in Fig. 1 can be attributed tothe free surfactant monomer. At all investigated concentra-tions an additional front was observed after that of themonomer (P2a and P2b), which can be assigned to inter-mediate aggregate forms. The plateau corresponding to themicellar form appears in the UV trace (P3b) at concentra-tions higher than the CMC, it does on the other hand notappear as clearly in the conductivity trace (P3a). It should benoted that the measured signal is in fact an impedance,composed of a resistance (determined by the conductivity ofthe capillary solution) and a capacitance which is determinedby the dielectric properties of the polyimide-coated silica

Figure 1. Electropherograms corresponding to 0, 2.5, 5.0, 7.5,and 12.061023 M SOBS in 10 mM sodium phosphate buffer atpH 7.0 and 257C obtained using contacless conductivity (a) andUV detection (b). P1 correponds to the surfactant monomerwhereas P2 and P3 can be ascribed to intermediate aggregatesand micelles, respectively. The EOF is indicated.



capillary and the solution. It is well known that micelle for-mation can be associated with rather dramatic changes in therelative permittivity of the solution [5, 6, 34] which mayinfluence the signal and lead to the observed decrease insignal observed at the plateaus corresponding to themicelles. Furthermore, it seems reasonable to assume thatother factors contribute to P3a as this plateau is also presentat concentrations lower than the CMC. However, in thepresent method only the plateau corresponding to the freesurfactant form (P1a) is used in the analysis regardless ofwhether UV or contactless conductivity detection wasemployed. Consequently, no further attempts were made tounravel the nature of the conductivity signal at long migra-tion times.

In the case of cationic surfactants a slightly differentprocedure was implemented as they adsorb strongly to theuncoated silica capillary walls and thereby decrease, or evenreverse the EOF. It proved to be a satisfactory solution to coatthe capillary with the positively charged polymer PDMAC(see Section 2.4 for details). The coating procedure resultedin a reversal of the EOF and effectively prevented adsorptionof positively charged surfactants. Sample introduction wasperformed from the short end of the capillary employingcontinuous injection using the electrophoresis voltage of110 kV. Examples of typical CE-FA experiments on thepositively charged TTAB employing contactless conductivitydetection are shown in Fig. 2. In the case of the two positivelycharged surfactants a plateau corresponding to the bromidecounterion (confirmed from a similar plateau profile usingtetraethylammonium bromide in place of TTAB and DTAB)is observed at P1, and the plateau corresponding to TTABappears at P2. A small signal also appears at 1 min whichmay be ascribed to an impurity possibly formed due to anelectrochemically induced transformation of the bromideion. The surfactant counterion is in practice only detectable

Figure 2. Electropherograms corresponding to 1.5, 10.0, 15.0,and 25.061023 M TTAB in 10 mM sodium phosphate buffer atpH 7.0 and 257C. P1 correponds to the bromide counterionwhereas P2 can be ascribed to the surfactant monomer. The EOFis indicated.

when it does not coincide with the ions of the run buffer.Consequently, a plateau corresponding to the sodium ion ofthe negatively charged surfactants was not observed asexemplified in Fig. 1a for SOBS.

The height of the monomer surfactant plateaus is direct-ly proportional to the concentration of free surfactant. Atconcentrations higher than the CMC the relative amount offree surfactant molecules decreases dramatically. Therefore,an inflection point should be observable at the CMC, separ-ating two regions corresponding to surfactant concentrationsbelow and above the CMC concentration, respectively. Thispoint is illustrated in Fig. 3a (SDS, SOBS, and SDeS) and 3b(DTAB and TTAB). The extracted CMC values are listed inTable 1 (averages of at least three independent experiments).

Figure 3. Plateau height vs. concentration for negatively (a) andpositively (b) charged surfactants in 10 mM sodium phosphatebuffer at pH 7.0 and 257C. Contactless conductivity detection wasemployed for SDS (1), SOBS (^), SDeS (n, inserted graph in a),DTAB (s), and TTAB (6). UV detection was also used forSOBS (r).


Electrophoresis 2007, 28, 2975–2980 CE and CEC 2979

Table 1. CMCs ( 6 SD) in 10 mM sodium phosphate buffer atpH 7.0 and 257C obtained employing CE-FA with con-tactless conductivity detection unless otherwise noted

Compound CMC/1023 M

SDS 4.8 6 0.2 (4.9)a)

SdeS 29.3 6 1.3SOBS 8.1 6 0.3 (8.4)a)

SOBSb) 7.7 6 0.2DTAB 17.3 6 0.3 (17.2)c)

TTAB 3.2 6 0.2 (3.2)c)

a) CMC obtained using conductometry.b) CMC obtained employing CE-FA with UV detection.c) CMC obtained using the signal corresponding to the bromide

counterion.

In Fig. 3a is also shown the result obtained using UV detec-tion which is in excellent agreement with the conductivitydata. The different dependence of plateau height observedfor surfactant concentrations higher than the CMCs mayindicate that the contribution from the surfactants to theionic strength in some cases affects the CMC.

The plateaus corresponding to the bromide counterion(DTAB and TTAB) can also be used to determine CMCs. Asillustrated in Fig. 4, a plot of the plateau height versus con-centration results in a similar shaped curve, compared to thecorresponding plot based on the free surfactant (Fig. 3b). Thebromide signal reflects differences in the association of thebromide ion with the free surfactant (which may often beneglected) and the micelle, respectively. Analysis of free

Figure 4. Plateau height corresponding to the bromide counter-ion vs. concentration for DTAB (s) and TTAB (6) in 10 mMsodium phosphate buffer at pH 7.0 and 257C.

counterion concentration has previously been used to deter-mine CMCs, as exemplified in the CMC determination ofCTAB using a bromide selective electrode [35]. The degree ofcounterion binding may also be quantified from the bromidesignal. However, this point was beyond the scope of thepresent work.

3.2 Comparison of CE-FA and conductometry for

determining CMCs

The measured CMC values are in good accordance with lit-erature data [8, 24] although a direct comparison is difficult.It is well known that CMC values are dependent on temper-ature and, particularly in the case of charged surfactants,ionic strength and counterions [8]. In order to make a com-parison with a more established technique we thereforemeasured CMC values of SDS and SOBS in the buffer sys-tem using a procedure based on conductometry [4]. In thismethod the conductivity is measured as a function of sur-factant concentration. As shown in Fig. 5 two linear regionsappear (below and above the CMC). The CMC values aregiven by the intersection points of the straight lines. It maybe noted that the change in slopes of the two lines in Fig. 5 ismuch smaller than in the CE-based method (Fig. 3a), as noseparation of free surfactant molecules and micelles takesplace in this case. Furthermore, the BGE contributes to thesignal in conductometry, in contrast to the CE-FA procedurewhere absolute signals from specific ions or aggregates areobtained. The CMCs determined by conductometry are inexcellent accordance with the results obtained using the CE-based method (Table 1).

Figure 5. Conductivity vs. concentration for solutions of SDS (1)and SOBS (^)in 10 mM sodium phosphate buffer at pH 7.0 and257C.



4 Concluding remarks

In this work we have described a new general method basedon contactless conductivity and UV detection for determina-tion of CMCs of positively and negatively charged surfac-tants. In the case of cationic surfactants a coating procedurewas implemented in order to stabilize the EOF. For UV-absorbing surfactants, similar CMC values were obtainedusing the two detectors. For SDS and SOBS nearly identicalCMCs were obtained using a classical method based on con-ductometry. The key advantages of the developed method arethe small sample volume requirements, the ease of automa-tion and the fact that it does not rely on specific markercompounds. Furthermore, the method is general in thesense that it can be used for any charged non-UV- or UV-absorbing surfactants. Finally, surfactant counterions can bedetected and also used for determining CMCs as well as forcharacterizing the micelles in terms of counterion binding.An important application of the method could, for example,be the characterization of pharmaceutically relevant surfac-tant systems used in the formulation of drug compounds.Conventional methods require a relatively large amount ofsample which will render studies on the influence of smallchanges in environmental factors (such as ionic strength,temperature, and chemical composition) impractical. On theother hand, the developed CE-FA method only requires a few100 nL of sample for a complete analysis and is thus highlysuited for such applications. In conclusion we thereforeenvision that the present methodology will find use inlaboratories in industry and academia which are involved inthe development and characterization of surfactant systemsand aggregation phenomena in general.

The Drug Research Academy (DRA) at the Faculty of Phar-maceutical Sciences, University of Copenhagen is acknowledgedfor financial support for this project.

5 References

[1] Attwood, D., Florence, A. T., Surfactant Systems Their Chem-istry, Pharmacy and Biology, Chapman and Hall, New York1983.

[2] Holmberg, K., Jonsson, B., Kronberg, B., Lindman, B., Sur-factants and Polymers in Aqueous Solution, Wiley, Chiche-ster 2003.

[3] Florence, A. T., Attwood, D., Physicochemical Principles ofPharmacy, Pharmaceutical Press, London 2006.

[4] Dominguez, A., Fernandez, A., Gonzalez, N., Iglesias, E.,Montenegro, L., J. Chem. Educ. 1997, 74, 1227–1231.

[5] Perez-Rodríguez, M., Prieto, G., Rega, C., Varela, L. M., Sar-miento, F., Mosquera, V., Langmuir 1998, 14, 4422–4426.

[6] Perez-Rodríguez, M., Varela, L. M., García, M., Mosquera, V.,Sarmiento, F., J. Chem. Eng. Data 1999, 44, 944–947.

[7] Nakashima, T., Anno, T., Kanda, H., Sato, Y. et al., ColloidsSurf. B 2002, 24, 103–110.

[8] Lin, C. E., J. Chromatogr. A 2004, 1037, 467–478.

[9] Terabe, S., Otsuka, K., Ando, T., Anal. Chem. 1985, 57, 834–841.

[10] Jacquier, J. C., Desbene, P. L., J. Chromatogr. A 1995, 718,167–175.

[11] Jacquier, J. C., Desbene, P. L., J. Chromatogr. A 1996, 743,307–314.

[12] Lin, C. E., Lin, K. S., J. Chromatogr. A 2000, 868, 313–316.

[13] Lin, C. E., Chen, M. J., Huang, H. C., Chen, H. W., J. Chroma-togr. A 2001, 924, 83–91.

[14] Lin, C. E., Huang, H. C., Chen, H. W., J. Chromatogr. A 2001,917, 297–310.

[15] Lin, C. E., Fang, I. J., Deng, Y. J., Liao, W. S., Cheng, H. T.,Huang, W. P., J. Chromatogr. A 2004, 1051, 85–94.

[16] Cifuentes, A., Bernal, J. L., DiezMasa, J. C., Anal. Chem.1997, 69, 4271–4274.

[17] Mrestani, Y., Claussen, S., Neubert, R. H. H., J. Pharm.Biomed. Anal. 2002, 30, 869–873.

[18] Strasters, J. K., Khaledi, M. G., Anal. Chem. 1991, 63, 2503–2508.

[19] Busch, M. H. A., Carels, L. B., Boelens, H. F. M., Kraak, J. C.,Poppe, H., J. Chromatogr. A 1997, 777, 311–328.

[20] Guijt-van Duijn, R. M., Frank, J., van Dedem, G. W. K., Bal-tussen, E., Electrophoresis 2000, 21, 3905–3918.

[21] Østergaard, J., Schou, C., Larsen, C., Heegaard, N. H. H.,Electrophoresis 2002, 23, 2842–2853.

[22] Østergaard, J., Heegaard, N. H. H., Electrophoresis 2003, 24,2903–2913.

[23] Schou, C., Heegaard, N. H. H., Electrophoresis 2006, 27, 44–59.

[24] Le Saux, T., Varenne, A., Gareil, P., J. Chromatogr. A 2004,1038, 275–282.

[25] Le Saux, T., Varenne, A., Gareil, P., J. Chromatogr. A 2004,1041, 219–226.

[26] da Silva, J. A. F., do Lago, C. L., Anal. Chem. 1998, 70, 4339–4343.

[27] Baltussen, E., Guijt, R. M., van der Steen, G., Laugere, F.,Baltussen, S., van Dedem, G. W. K., Electrophoresis 2002,23, 2888–2893.

[28] Kubán, P., Hauser, P. C., Electroanal. 2004, 16, 2009–2021.

[29] Zemann, A. J., Trends Anal. Chem. 2001, 20, 346–354.

[30] Zemann, A. J., Electrophoresis 2003, 24, 2125–2137.

[31] Jensen, H., Østergaard, J., Thomsen, A. E., Hansen, S. H.,Electrophoresis 2007, 28, 322–327.

[32] Bendahl, L., Hansen, S. H., Gammelgaard, B., Electrophore-sis 2001, 22, 2565–2573.

[33] Østergaard, J., Hansen, S. H., Larsen, C., Schou, C., Hee-gaard, N. H. H., Electrophoresis 2003, 24, 1038–1046.

[34] Perez-Rodríguez, M., Varela, L. M., Taboada, P., Attwood, D.,Mosquera, V., Langmuir 2002, 18, 562–565.

[35] Gerakis, A. M., Koupparis, M. A., Talanta 1994, 41, 765–773.


ce frontal analysis employing contactless conductivity detection for determination of cmcs of non-uv...

Documents