Anal. Chem. 1993, 65, 2204-2208
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Electrostatic Ion Chromatography Wenzhi Hu,’*+ Toyohide Takeuchif and Hiroki Haraguchit Department of Applied Chemistry, School of Engineering, Nagoya University, Chikusa-ku, 464-01, Nagoya, Japan, and Faculty of Engineering, Gifu University, 1-1, Yanagido, Gifu 501-11, Japan
Ion chromatography using “strong/strong” charged zwitterionic micellar-coated stationary phase with pure water as the mobile phase has been investigated for the separation of inorganic solutes and organic zwitterionic solutes. Retention mechanisms of the zwitterionic charged stationary phase in ion chromatography have also been investigated using a conductivity detector. For inorganic analytes, anions and their countercations are always eluted together. The retention times of inorganic solutes are only dependent on the anions. Accordingto experimentalresults, we suggest that analyte anions and their countercations combine to make “ion-pairing-like”forms and are separated by the simultaneous electrostatic attraction and repulsion interactions between the zwitterionic charged stationary phase, analyte anion,and its countercation. The formationof the simultaneous electrostatic attraction and repulsion interactions between the same and opposite charges can also be used to explain the retention behavior involved in the separation of organic zwitterionic solutes by the strong/strongcharged zwitterionic stationary phase with pure water as the mobile phase. The present system was used for the rapid determination of iodide and thiocyanate in human saliva samples. INTRODUCTION Electrostatic attraction and repulsion interactions between charges act at a distance. If positive and negative charges in a stationary phase are fixed in close proximity and charged particles are passed through it, repulsion and attraction forces will occur simultaneously. The combined effect of the simultaneouselectrostatic forces is dependent on the charge and radius of the charged particle. This means that the simultaneous electrostatic attraction and repulsion interactions between the same and opposite charges can be used for the separation of different charged particles (ions). To make a suitable positivelnegative charged stationary phase, we use zwitterionic molecules, because the positive and negative charges are very close together in a single molecule. The zwitterionic molecule has been reported for use as a stationary phase for the liquid chromatographic separation of organic zwitterionic solutes,lp2but the utilization of a zwitterionic stationary phase as an electrostatic provider for the separation of ions seems to have been overlooked. In these experiments, we made several ’stronglstrong” zwitterionic charged stationary phases which were used to provide simultaneous electrostatic interactions with analyte + Nagoya University.
Gifu University. (1) Yu, L. W.; Floyd, T. R.; Hartwick, R. A. J. Chromtogr. Sci. 1986, 24, 177-182. (2) Yu, L. W.; Hartiwick, R. A. J.Chrornatogr. Sci. 1989,27,176-185. 0003-2700/93/0385-2204$04.00/0
ions. Separation of ions was achieved using simultaneous electrostatic attraction and repulsion interactions. The charged stationary phases used in this work were formed by coating stronglstrong positivelnegative charged zwitterionic micelles on reversed-phase ODS surfaces using hydrophobic interactions. To make the study of this retention mechanism as simple as possible, pure water was used as the eluent.
EXPERIMENTAL SECTION Apparatus. In this work, two liquid chromatographic (LC) systems were used. One is a microcolumn LC system, the other is a conventional LC system. The microcolumn LC system used in this work consistedof a MF-2microfeeder (AzumadenkiKogyo, Tokyo, Japan) equipped with a 0.5-mL gas-tight syringe (MSGANOSO; Ito, Fuji, Japan) as a pump, a microvalve injector with an injection volume of 0.02 pL (ML-552;Jasco, Tokyo, Japan), and a 150 X 0.35 mm i.d. separation columnpacked with Develosil ODs-5 (5pm; Nomura Chemical, Seto, Japan) as a supporter to form the charged stationary phase. A Uvidec-100V UV absorption detector (Jasco)with a modifiedflow cell and a Chromatopac C-R4AX data processor (Shimadzu, Kyoto, Japan) for data treatment were also employed. The flow rate of the mobilephase was 2.8 pL/min. The conventional LC system was a Shimadzu LC-6A system (Shimadzu,Kyoto,Japan) consistingof a Shimadzu LC-6A pump, a sample injector with an injection volume of 20 pL, a SPD-6AUV absorption detector, and a EC-6Aconductivity detector. The support column was an L-ColumnODS (4.6 X 250 mm) on loan from the Chemical Inspection & Testing Institute, Tokyo, Japan. The flow rate of the mobile phase was 0.7 mL/ min. The injection volue of samples was always same as the injection loop volume. All of the separations were carried out at room temperature. Reagents. Reagents used in this work were of analytical reagent-grade and were used without further purification. Purified water was prepared in the laboratory using a Milli-Q system (Nihon Mollipore Kogyo, Tokyo, Japan). Zwitterionic surfactants, 3-[(3-cholamidopropyl)dimethylammoniol-l-propanesulfonate (CHAPS), 3-[(3-cholamidopropy1)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO),and Zwittergent 3-14 were purchased from Dojin (Kumamoto, Japan). Inorganicanalyte ions were purchased from Wako Pure Chemical Industries (Osaka, Japan). UV-absorbing organic compounds such as D-tryptophan, ~-fi-3,4-dihydroxyphenylalanine, and D-phenylalaninewere chosen as the organiczwitterionic analytes and obtained from Sigma (St. Louis, MO). Preparation of Zwitterionic Charged Column. A 30 mM solution of CHAPS, CHAPSO,or Zwittergent 3-14 was prepared in pure water and passed through the support column for 30 min at a flow rate of 2.8 pL/min (forthe conventionalsupport column, a flow rate of 0.7 mL/min for 75 rnin). This was followed by a pure water rinse for at least 40 rnin a t the same flow rate. The column was then conditioned with pure water as the mobile phase for the separation. RESULTS AND DISCUSSION Reagents used to form the zwitterionic charged stationary phase were 3-[(3-cholamidopropyl)dimethylammoniol1-propanesulfonate (CHAPS),3-[(3-cholamidopropy1)dimethylammoniol-2-hydroxy-1-propanesulfonate (CHAPSO),and Zwittergent 3-14. The structures of the zwitterionic reagents 0 1993 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1, lSQ3
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R = H, CHAPS R = OH, CHAPSO
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Retention Time (min) Flgure2. Chromatogramof lnorganlcanlons: column, DevekdloOS-5 coated with CHAPS mlcelles;mobile phase, pure water; flow rate, 2.8 pL/mln; detection, UV absorptlon at 230 nm; analytes. 1 mM each of iodate, nitrite, nitrate, iodide, and thlocyanate. C
Zwittergent 3-14
Fbure 1. Structures of CHAPS, CHAPSO, and Zwlttergent 3-14 zwltterionlc surfactants and representatlon of the mlcellar-coated zwltterionlc charged statlonary phase.
and the coated zwitterionic charged stationary phases are shown in Figure 1. As can be seen, all of the zwitterionic reagents are stronglstrong positivelnegative charged zwitterionic surfactants. Above critical micellar concentrations (cmc), the monomers form micelles. Zwittergent 3-14 is a linear surfactant; therefore, its micelles are linear. CHAPS and CHAPSO are steroidal surfactants and their micelles are reversed helices. Practical Testing. In this section, all of the separations were achieved using a microcolumn LC system with a UV absorption detector, and all of the analyte anions are UVabsorptive. Figures 2-4 show the chromatograms of inorganic anions obtained using CHAPS, CHAPSO, and Zwittergent 3-14 micellar coated stationary phases, respectively, with pure water as the mobile phase. Under the separation conditions shown, only two types of forces are present. One is the simultaneous electrostatic attraction and repulsion interactions between the analyte ions and the zwitterionic charged stationary phase. The other is the transporting force of the pure water mobile phase. From this, we can conclude that the simultaneous electrostatic attraction and repulsion interactions between the same and opposite charges cause the analyte ions to separate. Retention Mechanisms. The first experiments only investigated the separation of anions. Therefore, a UV absorption detector was employed. For the study of retention behaviors of the countercations, a conventional LC system with a conductivity detector was employed. Figure 5 shows the chromatogram of a mixture of inorganic solutes obtained using the conventional LC system and
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i
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Retention Time (min) Flgurr 9. Chromatogramof lnorganlc anlons: column, Devebsll oOS-5 coated with CHAPSO micelles; mobile phase, pure water; flow rate, 2.8 pLlmin; detection, UV absorption at 230 nm; analytes, 0.1 mM each of iodate, nitrite, nitrate, iodide, and thlocyanate.
detected by the conductivity detector. For further investigation of countercations, sodium sulfate, sodium chloride, sodium nitrite, potassium bromide, and sodium nitrate were injected independently into the column and the separation was achieved under the conditions described in Figure 5. The chromatograms are shown in Figure 6. Inorganic anions and their countercations always eluted together. The retention time of the inorganic solutes is only dependent on the anions. It was also found that sodium nitrate and potassium nitrate eluted with the same retention time (data not presented). To explain these experimental r e s u b , we suggest that the inorganic anions and their countercations combine to make “ion-pairing-like” forms as shown in Figure 7. When an inorganic solute is passing through the stronglstrong positive1 negative charged stationary phase, the analyte anion is attracted by the positive charge present in the zwitterionic stationary phase. But the negative charge in the stationary phase is very close to the positive charge, and as a result, repulsion of the analyte anion occurs simultaneously. The
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1, 1993 i-
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Retention Time (min) Figure 6. Chromatograms of sodium sulfate (A), sodlum chloride (B), sodium nitrite (C), potasslum bromide (D), and sodlum nitrate (E). Separation conditions are the same as in Figure 5. Ion-pairing like form --.
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analyte cation will have the same electrostatic behavior. Therefore, both analyte ions cannot get close to the zwitterionic stationary phase. In other words, neither the positive nor the negative charge of the zwitterionic stationary phase can work as the ion-exchange site. The analyte anions and their countercations, forced into a state of simultaneous electrostatic attraction and repulsion in the zwitterionic stationary phase, can be termed ion-pairing-like forms. The ion-pairing-like forms can be carried out of the column by pure water eluent, which means that the combination of simultaneous electrostatic forces on the ion-pairing-like forms is smaller than the mobile-phase transporting power of the pure water eluent. Various ion-pairing-like forms can be recognized by different combination of simultaneous electrostatic forces. However, the pairing anion is the key factor in the separation because the retention time of the ion-pairinglike forms is dependent on the anion. In this separation,divalent anionic solutes are always eluted before the monovalent anionic solutes. This may be because the combination of simultaneous electrostatic attraction and repulsion forces on the divalent anionlcation ion-pairinglike forms is weaker than that on the monovalent anion/ cation ion-pairing-like forms.
Figure 7. Simultaneous electrostatic attraction and repulsion lnteractlons between analyte Ions and the zwmerionlc charged statlonary phase.
As mentioned above, by using the stronglstrong charged zwitterionic stationary phase for the separation of inorganic solutes, the analyte anions and countercations cannot be simultaneously separated. However, if the strength of the two charges present in the zwitterionic stationary phase is quite different, the retention mechanism will be changed. In this case, the stronger charge works like an ion-exchangesite, and the weaker charge still works as the electrostatic provider, assisted by the stronger opposite charge. In this situation, the weaklstrong charged zwitterionic stationary phase can be used for the simultaneousseparation of cations and anions. However,to replace the analyta ions attracted by the stronger charge,i.e., the ion-exchange site, addition of ions in the mobile phase is required. A method using weaklstrong charged stationary phase for the simultaneous separation of inorganic cations and anions based on the simultaneous electrostatic attractionlrepulsion interaction and ion-exchanging will be reported elsewhere.3 The void time of the present system was also examined under different conditions because the determination of the void time is too difficult when pure water is used as the mobile phase. T o show the void time of the present system, the mobile phase was changed to 3 mM phosphate buffer. Figure 8 shows the chromatogram of a mixture of inorganic solutes obtained using a 3 mM phosphate buffer eluent and the conductivity detector. The negative peak for water, which appeared at 3.75 min, is the void time of the present system. (3) Hu,W.; Haraguchi, H.,to be submitted.
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Retention Time(min) Figure 8. Determination of void time: column, L-Column coated with CHAPS micelles; mobile phase, 3 mM phosphate buffer; detection, conductlvlty detector; analytes, 2 mM sodium sulfate and 4 mM each of sodium chloride, sodium nitrite, sodium nitrate, potassium iodide, and sodium thiocyanate aqueous solution.
0
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Retention Time (min) Figure 0. Chromatogram of amino acids. Separation conditions are the same as in Figure 2 except the wavelength of the UV absorption detector was 200 nm: analytes, 0.5 mM each of ~-&3,4dihydroxyphenylalanine (l),D-phenylalanine(2), and o-tryptophan (3). Separation of Organic Zwitterionic Solutes. As shown in Figure 7, the formation of simultaneous electrostatic attraction and repulsion interactions between the zwitterionic stationary phase and inorganic analytes can also be used for the separation of organic zwitterionic solutes by the same retention mechanism. Under a wide pH range, amino acids are zwitterionic solutes. Therefore, amino acids could also be separated by the simultaneous electrostatic attraction and repulsion interactions with pure water as the mobile phase. Figure 9 shows the chromatogram of three UV-absorbing amino acids. The amino acids are rapidly separated by the zwitterionic charged stationary phase. For the separation of organic zwitterionic solutes using a strong/strong charged zwitterionic stationary phase, another retention mechanism based on the formation of bipolar or quadrupolar interactions between the zwitterionic forms of the solutes and the zwitterionic stationary phase has been suggested by other authors1V2based on original suggestions of zwitterion-pair chromat0graphy.~~5However, if the sep-
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Retention Time (min) Flgure 10. Chromatogram of dansyl amino acids: (1)Asp, (2) Gly, (3) sarcosine, (4) Val, (5)Nvai, (6) Met, (7) Leu, (8) Nleu, (9)Trp, and (10) Phe. (A) Separation conditions are the same as in Figure 2 except the wavelength of the UV absorption detector was 220 nm. (B)Column, Deveiosii ODS-5, 150 X 0.35 mm i.d.; mobile phase, acetonitrile/ water (27:73); other conditions are the same as In (A). aration is only dependent on the bipolar or quadrupolar interactions, solutes could not be eluted from a column when only pure water was used as the eluent. When the zwitterionic forms of the amino acids are passing through the strong/ strong charged stationary phase, the electrostatic attraction and repulsion interactions occur simultaneously. This prevents the zwitterionic solutes from approaching the charged stationary phase, thus making the bipolar or quadrupolar interactions impossible. According to our experimental results, the simultaneouselectrostatic attraction and repulsion interactions between the same and opposite charges are the dominant factors in the separation of zwitterionic solutes using strong/strong charged stationary phase. In order to investigate the reversed-phase activity of the micellar-coatedcolumn,separation of derivatized amino acids, such as dansyl amino acids, was achieved using a CHAPS micellar-coated column with pure water as the mobile phase. The chromatogram is shown in Figure 10A. The amino acids were eluted from the column within 12 min. In comparison, Figure 10B also shows the chromatogram for the separation of the same dansyl amino acids obtained with a reversedphase ODS column using 27:73 (v/v) acetonitrile/water mobile phase, which required 33 min. These results indicate that the ODS stationary phase shows no observablereversed-phase activity after coating with micellar CHAPS or CHAPSO. Practical Application. The method was used, as described, for the rapid determination of iodide and thiocyanate ions in human saliva samples. The saliva was taken up by syringe to remove any solid particles in the saliva samples. The tip of this syringe was covered with cotton wool, which was removed before the sample was injected into the separation system. (4) Knox, J. H.; Jurand, J. J. Chromatogr. 1981,218, 341-354. (5) Knox, J. H.; Jurand, J. J. Chromatogr. 1981,218, 355-363.
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Retention Time (rnin) Flgure 11. Determination of iodide and thiocyanate ions in human saliva by electrostatic ion chromatography with pure water mobile phase: (A) saliva sample diluted 2-fold with pure water: (B) addition of 0.5 mM iodide and thiocyanate standard solution by the same volume into saliva sample. Separation conditions are the same as in Figure 2.
Figure 11A shows the chromatogram of a saliva sample. Figure 11B shows saliva with the addition of 0.25 mM iodide
and thiocyanate. Good chromatograms with the same retention times were obtained. The concentrations of iodide and thiocyanate ions in the sample were 0.02 and 0.85 mM, respectively. The detection limits of iodide and thiocyanate at signalto-noise ratio of 3 were 0.3 and 1.2 fiM, respectively. A standard sample of 1mM iodide and thiocyanate was injected into the CHAPS micellar-coated stationary microcolumn LC system 10 times; the relative standard error (RSE)was found to be 0.8%. The stability of the CHAPS or CHAPS0 micellar-coated stationary phase was also examined; the retention time and separation coefficientdid not changeover a period of 3-months usage.
ACKNOWLEDGMENT The authors thank Mr. Geoffrey Bland for his valuable assistance in correcting the manuscript.
RECEIVED for review August 24, 1992. Accepted May 5, 1993.
