Anal. Chem. 1002, 6 4 , 502-506 Masschelein, W. J.; Rice, R. G. Chlorlne LWOXChemistry M and Environmental Impact of Oxychhine Compounds; Ann Arbor Science: Michigan, 1979. Wood, D. W. 111. Determination of Disinfection Residuals in Chlorine Dioxide Treated Water Using Flow Injection Anaiysis. Ph.D. Dissertation, Miami University, OH, 1990. Gordon, G.; Yoshino, K.; Themelis. D. G.; Wood, D. W.; Pacey, G. E. Anel. Chlm. Acta 1888, 224, 383-391. Themelis, D. G.;Wood, D. W.; Gordon, G. Anal. Chim. Acta 1888, 225, 247-444. Gordon, G.; Siootmaekers, 8.; Tachiyashiki, S.;Wood, D. W. 111. J . Am. W8tW Works ASSOC. 1880, 82, 160-165. Aieta, M. E.; Roberts, P. V.; Hernandez, M. J . Am. Water Works A s sOC. 1884, 76. 64-70. Pfaff, J. D.; Brockhoff, C. A.; ODeii, J. W. Test Method: The Determination of Inorganic Anions in Water by Ion Chromatography-Meth-
(20) (21) (22)
(23)
ods 300.0 A 8 B. USEPA Environmental Monitoring and Systems Laboratory, Cincinnati, OH, 1989. Bolyard. M. U.S. Environmental Protection Agency, Cincinnati, OH; personal communication, October 1990. Joyce, R., Dionex Corporation, Sunnyvale, CA; personal communications, September and October 1990. Jersey, J. A.; Johnson, J. D. American Chemical Society, Dhrlsion of Environmental Chemistry, Preprints of Papers Presented at the 199th ACS National Meeting, Boston, MA, April 22-27, 1990, Voi. 30, No. 1, pp 57-60. Gordon, G., Miami University. Oxford, OH. Personal communication, Aug 1991.
RECEIVED for review August 28, 1991. Accepted December 10,1991.
Temperature Programming in Capillary Zone Electrophoresis Chen-Wen Whangt a n d Edward S . Yeung*
Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011
A new type of seiectivlty in capillary zone electrophoresis (CZE) based on temperature programming is presented. By using a buffer system wlth a large temperature coefficient, e.g. Tris buffer, the pH within the capillary can be adjusted in situ by simply controlling the temperature of the capllary. The electromigration behavior of weak acids is found to be influenced by both temperature-induced pH changes and vkcosity changes. The effect of pH on the electrophoretic mobillties of analytes is most significant if the range of pH change matches the acidity constants, pK,, of the analytes. The implementation of temperature programming in both temporal and podtional modes In CZE are illustrated by the separation of a mixture of fluorescent organic acids.
INTRODUCTION Over the past decade, capillary zone electrophoresis (CZE) has proven to be one of the most powerful techniques for the separation of complex mixtures, particularly in the area of biology and biochemistry. CZE is generally characterized as a separation method of extremely high efficiency and fast speed; generation of more than lo6 theoretical plates in less than 20 min has been Separation in CZE is achieved mainly via differences in mobilities of analytes under a high electric field. Both electroosmotic flow of the bulk solution and electrophoretic mobilities contribute to the observed migration behavior of each analyte in the ~apillary.~J Since the electrophoretic mobility of an ion is directly proportional to ita charge? which in turn is strongly affected by pH, manipulation of the buffer pH becomes one of the key strategies in optimizing a separation. This is particularly true in the separation of proteins and peptides by CZE.596 However, for mixtures of acids and bases possessing similar ionic mobilities and/or acidity constants (pKJ, it is sometimes difficut to find a suitable pH which enables a fast and efficient separation. A pH gradient as a function of position, as that commonly employed in isoelectric focusing7 and free-flow electrophoresis: may be used to solve this problem. To date, only a few pH gradient methods in CZE have been reported. Bocek et al.9Jo first developed a three-pole separation column for dynamic (temporal) programming of pH On leave from the Department of Chemistry, Tunghai Univer-
sity, Taichung, Taiwan 400.
in the capillary. The actual operational buffer inside the capillary was generated by the simultaneous electromigration of various ionic species of the same polarity from two separate buffer chambers. Using a syringe-type pump, Sustacek et al." created a dynamic pH gradient in CZE through the continuous addition of a modifying electrolyte into the buffer chamber at the injection of the capillary. This method was applied to the separation of nucleic bases and their derivatives. Recently, Foret et al.12 used the same technique to generate a step change of pH in the separation of proteins by CZE. A substantial improvement on the resolution of protein mixtures was demonstrated. All pH gradient methods reported involve the continuous variation of electrolyte composition in the buffer chamber at the injection end of the capillary through an electrical or a mechanical means. In this paper, a new method for manipulating selectivity in CZE is presented. Using a buffer system with a large temperature coefficient (dpH/dT), e.g. Tris buffer, a pH step or gradient can be generated in situ simply by varying the temperature of the capillary as a function of time or as a function of position during electrophoresis. Temperature programming has been a routine technique to solve general elution problems in gas chromatography for many years. Improvement of both speed and efficiency in liquid chromatographic separations through temperature-controlled or gradient techniques has also been dem~nstrated.'~J~ In CZE, temperature control was often used to provide efficient heat r e m ~ v a l . ~ "Manipulation ~~ of chemical equilibria such as metal chelation and micelle partitioningl8Jgwithin the capillary through temperature control has also been reported. The generation of a positional pH gradient by using a buffer system with a large dpH/dT has been demonstrated in isoelectric focusing.20 In this research, effects of temperature-induced pH changes and viscosity changes on the electromigration behavior of analytes in CZE are studied. Improvements in the efficiency of separation by employing a dynamic (time dependent) or static (position dependent) temperature step or gradient technique are also demonstrated. EXPERIMENTAL SECTION The CZE system is similar to that described previously.21 A high-voltage power supply (Glassman High Voltage, Inc., Whitehouse Station, NJ; Model PS/EH40R02.5) was used to generate the potential across the capillary. Fused-silica capillaries (Polymicro Technologies,Phoenix, AZ) of 50 pm i.d. and 360 pm 0.d. were used in this study. Of the 60-cm total length, a 40-cm region
0003-2700/92/0364-0502$03.00/00 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992
was withii a water jacket built in-house (3 mm i.d. X 6 mm o.d.), leaving a 10-cm length of capillary outside each end of the water jacket. Before use, the capillary was washed (pressurized flow) with 0.1 M NaOH for 15 min, followed by a 2-min rinse with water and a 2-min flush with the running buffer. The capillarywas then equilibrated with the buffer under an electric field of 200 V/cm for 12 h. The same cleaning procedure was employed whenever a running buffer with a different initial pH was used. Temperature control of the capillary was performed by rapidly circulating water through the water jacket using a four-channel Minipuls 2 peristaltic pump (Gibson Medical Electronics, Middleton, WI)in conjunctionwith a thermostated water bath (Grant Instruments, Ltd., Barrington, Cambridge). To enhance homogeneity of the temperature along the capillary, the outside wall of the water jacket was wrapped with heating tape which was connected to a variable transformer. At a water circulation rate of 40 mL/min and a temperature of 55 OC, the temperature difference of the water between the inlet and the outlet was 51 "C, as measured by insertion of a chromel-dunel thermocouple into the water jacket tubing. On-column detection was performed using laser-induced fluorescence detection. A small region of the polymer coating was burned off 7.5 cm from the cathodic end of the capillary to form a detection window. An argon ion laser (Cyonics,San Jose, CA; Model 2213-150ML) operating in the light-regulated mode at 13-30 mW for all lines was used for excitation. The 488-nm beam (about 40% of the total power) was selected with a prism and two knife edges and was focused into the capillary with a 1-cm focal length lens. The capillary was mounted at Brewster's angle to reduce scattered radiation. Fluorescence from the sample was collected with a 1OX microscope objective and passed through a colored-glassfilter (Corning Glass, Corning, NY; Model 3-69). The fluorescent image was focused onto a silicon photodiode detector (Hamamatsu Corp., Middlesex, NJ; Model HC220-01) which has a built-in current amplifier. The voltage output from the detector was recorded using either a strip chart recorder (Measurement Technology, Inc., Denver, CO; Model CR452) or an integrator (Spectra-PhysicsInc., San Jose,CA; Model SP4600). AU fluorescentdyes used were laser grade while other chemicals were reagent grade. Fluorescein and fluorescein-5isothiocyanate (FITC) were obtained from Molecular Probes, Inc. (Eugene, OR). Coumarin 343 and 2',7'-dichlorofluorescein (2',7'-DCF) were obtained from Eastman Kodak Co. (Rochester, NY).(-)-Riboflavin was purchased from Aldrich Chemical Co. (Milwaukee, WI). Tris(hydroxymethy1)aminomethane (Tris) was obtained from Sigma Chemical Co. (St. Louis, MO). Deionized water (Millipore Corp., Bedford, MA, Milli-Q system) was used for the preparation of buffer solutions. Tris buffer was prepared by adjusting the pH of a 0.01 M Tris solution with dilute hydrochloric acid. All fluorescent dyes were prepared in 0.01 M Tris buffer.
509
75'c
29'C
2
1
a
4
0
2
12
a
4
0
TIME (min) Flguro 1. Influence of temperature on the separation of fluorescent dyes in 0.01 M phosphate buffer at pH 8.5. Conditions: column 50 pm 1.d. X 360 pm 0.d. X 60 cm total length, 52.5 cm effective length; thermostated region 40 cm; voltage applied 20 kV (20-27 MA); 1-s electromtgratbn inJectbn; laser-induced fluorescence detection. Peak identities: (1) coumarin 343; (2) fluorescein; (3) 2',7'-DCF.
68'C
18OC
4
31
RESULTS AND DISCUSSION In order to generate a useful temperature-induced pH change within the capillary, an electrophoresis buffer with a large temperature coefficient, i.e. dpH/dT, is needed. Among commonly used buffer systems, Tris buffer is particularly notorious for its large pH shift with temperature. A plot of pH valuea versus temperature using literature data= for a Tris buffer ranging from 20 to 60 "C is linear with a correlation coefficient of 0.999 and a slope of -0.0248 pH/OC. This large temperature coefficient suggests that a change of one pH unit can be generated simply by varying the buffer temperature by 40 "C. If the pH of a Tris buffer a t a certain temperature is known, the temperature coefficient can then be used to estimate the pH of the buffer at a different temperature through the equation where pH1 and pH2 are characteristic of the buffer at temperatures T1 and T2,respectively. Temperature-induced effects on CZE are evident in the electropherograms shown in Figures 1and 2. In Figure 1, three weak acids, i.e. coumarin 343 (peak I), fluorescein (peak 2), and 2',7'-dichlorofluorescein (peak 3), were readily sepa-
0
3
6
9
0
3
6
TIME (min) Flguro 2. Influence of temperature on the separation of fluorescent dyes in 0.01 M Tris buffer at an initlal pH of 8.3. Condition: vottage appW, 15 kV (1.5-2.3 FA). Peak k%Mes: (1) rlboflavln; (2) covnarkl 343; (3) fluorescein; (4) 2',7'-DCF. Other conditions are as In Figure 1.
rated by CZE using a 0.01 M phosphate buffer at pH 8.5 at both temperatures. Since phosphate buffer has a relatively small temperature coefficient (ca. -0,0028 pH/"Cm), the difference in pH of the buffers at 29 and 75 OC can be considered insignificant, and the changes in electromigration time
504
ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992 p H 9.1
p H 6.9 2
I
I
Table I. Electroosmotic Flow Coefficients ( p m ) and ~ ~ ) s)] of Fluorescein Electrophoretic Mobilities ( j ~ [cm2/(V in Tris Buffers of Different Initial pH's and at Different Temperatures temp
27 40 50 62
pH 6.9 104c(,, 1
6.92 (0.03) 7.28 (0.05) 7.86 (0.10) 8.26 (0.09)
pH 9.1 0 4 ~ ~ ~
104r,
1o4reP
2.95 (0.03) 9.80 (0.03) 3.35 (0.02) 3.22 (0.01) 10.67 (0.06) 3.96 (0.03) 3.50 (0.03) 11.88 (0.12) 4.61 (0.05) 3.68 (0.04) 12.87 (0.12) 5.35 (0.06)
"Data are represented as mean (standard deviation); n = 4. Experimental conditions are 88 in Figure 3.
I I
TIME (min) Flgure 3. Electropherograms of riboflavin and fluorescein at 27, 40, 50, and 62 O C In 0.01 M Trls buffers at lnitlal pH's 6.9 and 9.1. Conditions: voltage applied 12 kV; current range 4.4-6.2 p A for pH 6.9 buffer, 0.7-1.2 pA for pH 9.1 buffer. Peek !<bS: (1) riboflavin; (2) fiuoresceln. Other condltlons are as In Figure 1.
with temperature for the three acids should be predominantly due to temperature-induced viscosity changes of water. Resolution among the three peaks did not change much, showing that they are all influenced to the same extent; i.e. the individual pK, values are not affected. There is however a minor benefit of increased separation speed. In Figure 2, riboflavin, a neutral compound, was included in the sample mixture and separationswere performed at 18 and 68 OC using a 0.01 M Tria buffer at an initial pH of 8.3. The electroosmotic flow velocities, as measured from the migration time of riboflavin, increased by a factor of ca. 1.5 from 18to 68 OC, while the viscosity of water decreases by a fador of 2.5 over the same temperature range. This indicates that the temperature-induced viscosity change of water is only partially responsible for the change in electroosmotic flow with temperature, vide infra. The pH value of Tris buffer at 68 OC should be about 7.0.22 When the buffer pH shifts from 8.3 at 18 "C to 7.0 at 68 OC, the degree of ionization of each of the three weak acids will.also change, because they all have pK, values in the range 6.0-6.4.24*25 Since ionic mobility of an analyte is dependent on its charge, the observed electrophoretic behavior of the three acids in Figure 2 is the result of temperature-induced pH change, in addition to the viscosity change of water. It is evident that such a pH change is useful for separation, as shown in Figure 2 by the resolution of peaks 3 and 4 at the lower but not at the higher temperature. To further study the temperature-induced pH changes in CZE, riboflavin and fluorescein were used as model compounds. Figure 3 shows changes in electromigrationbehavior in CZE of riboflavin (peak 1) and fluorescein (peak 2) with temperature using 0.01 M Tris buffers at initial pH's of 6.9 and 9.1. As expected, both substances migrate faster with increasing temperature. However, pH changes as a function of temperature influence the migration velocity of each sub-
stance differently because riboflavin is neutral while fluorescein is a dibasic acid. Fluorescein is clearly affected more by temperature compared to riboflavin. From Figure 3, values of electroosmotic flow coefficients (pea) as well as effective electrophoretic mobilities (pep) of fluorescein at different temperatures can be calculated. To avoid variations in surface effects among different capillaries, the same piece of capillary was used for all experiments. Before changing to a new buffer, the capillary was cleaned and equilibrated as described in the Experimental Section. With each buffer of a certain initial pH, four runs were made at each selected temperature from which the mean value and the standard deviation were calculated. The results are listed in Table I. As mentioned before, the temperature-induced viscosity change of water should be mainly responsible for the change in electroosmotic flow velocity. However, from the data in Table I, an increase in temperature from 27 to 62 "C only resulted in 19% and 31% increases in electroosmotic flow coefficients, peo, at pH 6.9 and 9.1, respectively. Since the viscosity of water decreases by a factor of 2 over the same temperature range, the observed changes in peo with temperature could not be accounted for solely by the viscosity change of water. Electroosmotic flow in fused-silica capillaries is known to have a strong dependence on buffer pH.% At low pH, the ionization of the surface silanol groups is suppressed and the electroosmoticflow approaches zero. Under high pH conditions, the silanol groups will be fully ionized and the flow reaches a plateau value. The largest change of electroosmotic flow with pH is in the pH 4-6 region.27 The degree of ionization of surface silanol groups will affect the t potential (t,) of the inner wall of the capillary, which in turn will influence the electroosmotic flow because plea is directly proportional to tC.l6 Variation of {, withiin the pH range studied is therefore primarily responsible for the discrepancy between the observed temperature-induced changes in pe0 and the viscosity change of water. Small variations in ionic strength of the buffer solution as a function of temperature may be a minor contributor to the observed discrepancy. In CZE, the relationship between the buffer pH and the effective electrophoretic mobility (pep)of a monobasic substance with acidity constant pK, can be expressed asz8 p H = pK,
+ log
(
pre:'pep)
where prepis the ionic mobility of the respective substance in the fully ionized form. In the case of fluorescein, which can be represented as HzFLwith pK,, = 4.44 and pKa2 = 6.36,=vB a modified version of eq 2 is needed. In order to simplify the derivation, we only consider the pH region where HFL- G H+ FL2- is the major equilibrium process. The concentration of undissociated fluorescein, H2FL, can be assumed negligible because of its insolubility in water. In addition, since the ionic mobility of an analyte is directly proportional to its charge, the electrophoretic mobility of HFL- should be only
+
ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992
505
Table 11. pH of 0.01 M Tris Buffer within the Capillary at Different Temperatures ("C) temp 27 40 50 62 a
PH,: 6.90 6.58 6.33 6.03
PHdb 6.86 6.58 6.39 6.14
Estimated from eq 1 using an initial pH 6.90. Calculated from L values listed in Table I and PK., = 6.36.
ea 3 using
half that of FL2-, assuming that HFL- and FLZ have the same ionic radius. According to an analogous derivation, eq 2 can be modified to
where is the electrophoretic mobility of fluorescein measured at a pH at which both HFL- and FL2- exist and clep2 is the electrophoretic mobility of fluorescein measured at a pH where only FL2- exists. Returning to Table I, in a Tris buffer of initial pH 6.9 where both HFL- and FL2- exist, the change in electrophoretic mobilities, clW, with temperature was actually the result of two effects temperature-induced viscosity change and temperature-induced pH change. However since FL2- can be assumed to be the only major species that exists in Tris buffers of pH range 8-9, the change in P~~ with temperature at an initial buffer pH of 9.1 can be mainly attributed to the temperature-induced viscosity change of water. Therefore, the influence of pH on pep as a function of temperature can be estimated from the difference between two pepvalues measured at a selected temperature with buffers of different initial pH. To verify this, the cleP values listed in Table I along with the literature value of pK,,, for fluorescein were substituted into eq 3 to determine the actual pH of the Tris buffer at different temperatures. The pH values of Tris buffer at different temperatures also can be estimated from eq 1. Table I1 compares the results obtained using both methods. The calculated pH values are found to be in good agreement with the estimated pH values, except for a small deviation at the highest temperature. With eq 3, two assumptions were inherently included: (1) the acid dissociation constants of fluorescein do not change with temperature; (2) FL2- is the only species in the buffer of initial pH 9.1. While these two assumptions seemed reasonable at low temperature, they might not hold at higher temperatures, particularly the former, because organic acid/base systems generally show a higher temperature coefficient than inorganic systems.23However, the discrepancy is not significant even at 62 "C. The results in Table I1 show the predictability of temperature-induced pH control. Like that in chromatographic methods, the main purpose of using temperature programming in CZE is to improve the speed and/or the efficiency of a separation. With a suitable buffer, such as Tris buffer, it is possible to optimize a separation by the simultaneous control of pH and temperature during electrophoresis. Figure 4 gives a demonstration of this application. In Figure 4A, five fluorescentdyes Le., ribotlavin (peak 11, coumarin 343 (peak 21, FITC (peak 41, fluorescein (peak 5) and 2',7'-DCF (peak 6), were isothermally eluted at 20 "C using a Tris buffer at pH 7.3. Peak 3 was due to impurities contained in FITC. A t this pH, fluorescein and 2',7'-DCF could not be separated, and the total analysis time was over 8 min. An attempt to improve the resolution by raising the temperature to 70 "C (which lowers the pH to 6.1) was not totally successful although the analysis time was
0
2
4
6
6
0
2
4
6
TIME (min)
5
0
2
4
6
8
0
2
4
6
8
TIME (min) Flgure 4. Influence of temperature programmlng on the separatlon of fluorescent dyes In 0.01 M Tris buffer at an lnltlal pH 7.3: (A) 20 "C, isothermal; (8)70 "C, Isothermal; (C) 70 "C for 2 mln, then step a m from 53 to 20 OC along the capilary. to 20 " C @) temperatue g Condltlon: voltage applied, 15 kV (5-8 PA). Peak Mentltles: (1) riboflavin; (2) coumarin 343; (3) lmpurlties; (4) FITC; (5) fluoresceln; (6) 2',7'9cF. Other condltlons are as in Figure 1.
shortened by ca. 25% (see Figure 4B). While the two fluoresceins are separated from each other, the FITC impurities merged with the coumarin. The separation can be further improved by employing a step change (dynamic gradient) of temperature (and thus pH and viscosity) during electropho-
606
ANALYTICAL CHEMISTRY, VOL. 64, NO. 5. MARCH 1, 1992
resis, as shown in Figure 4C. CZE was initially performed at 70 "C. After 2 min, the temperature of the entire capillary was rapidly lowered to 20 OC by changing the water flowing into the water jacket from hot water to tap water. The exact temperature gradient was not determined. The resolution among all six peaks was significantlyimproved,in comparison with those in Figure 4A,B. However, the baseline was not stable, probably due to variations in refractive properties of the buffer solution (and thus stray light) caused by the sudden temperature change. A static on-column temperature gradient (pH plus viscosity gradient along the capillary) for the CZE was finally tested. In order to generate a temperature gradient along the capillary, only the half of the water jacket close to the injection end of the capillary was wrapped with heating tape while the other half was left unheated. Water of 20 "C was continuously pumped into the cool end of the water jacket at a flow rate of ca. 2.5 mL/min. The temperature of water flowing out of the water jacket (injection end) was about 53 OC. A temperature gradient was therefore created, which in turn induced a pH gradient from pH 6.5 to 7.3 along the capillary. Figure 4D shows the electropherogram obtained using such a static pH gradient along the capillary. Upon injection, all species are exposed to a buffer of higher temperature (or lower pH). On migration toward the cathodic end of the capillary, each species experiences a gradual lowering of the temperature (or increase in pH), which alters the degree of ionization. There is also a change in the individual electrophoretic mobilities. There are however no refractive index changes a t the detector. The separation in Figure 4D was thus further improved and the baseline was more stable, compared with that in Figure 4C. In conculsion,temperature control in CZE can be used to generate in situ pH changes which may facilitate separations of complex mixtures. Variation in migration velocities of acidic or basic analytes can be attributed both to temperature-induced pH changes and viscosity changes of water. In comparison with other reported pH gradient techniques in CZE, generation of temperatureinduced pH gradient is simpler both in the ease of operation and the apparatus required. In contrast to flow-generated pH gradients, problems associated with inhomogeneity in mixing do not exist. There are also no restrictions on the magnitude or direction of electroosmotic flow. This method should be applicable to complex samples, where inadequate separation is obtained at any single buffer PH.
ACKNOWLEDGMENT Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under Contract No. W7405-Eng-82. This work was supported by the Director of Energy Research, Office of Basic Energy Sciences and Office of Health and Environmental Research. REFERENCES Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. Jorwnson, J. W.; Lukacs. K. D. Science 1983, 222, 266-272. Wallingford, R.; Ewlng, A. A&. Chromtcgr. W8S, 29, 1-76. Levine, I.N. Physical Chemistry, 2nd ed.;McGraw-Hill, Inc.: New York, 1983; p 462. McCormick, R. M. Anal. Chem. 1988, 60, 2322-2328. Lauer, H. H.; McManigill, D. Anal. Chem. 1988. 58, 166-170. Righetti, P. G. Isoelectric Focusing: Theory, Methodology, and Applications, 2nd ed.; Elsevier Biomedical Press; Amsterdam, 1983. Shukun, S.A.; Zav'yalov, V. P. J. Chromfcgr. 1989, 496, 121-128. Bocek, P.; Deml. M.; Pospichal, J.; Sudor, J. J. Chromatogr. W89, 470,309-312. Pospichai, J.; Deml, M.: Gebauer, P.; Bocek, P. J. Chromtogr. 1989, 470,43-55. Sustacek, V.; Foret, F.; Bocek, P. J. Chromatogr. 1989, 480, 271-276. Foret, F.; Fanaii, S.;Bocek, P. J. Chromfcgr. 1990, 516, 219-222. Jinno, K.; Phillips, J. 8.; Carney, D. P. Anal. Chem. 1985, 57, 574-576. Biggs, W. R.; Fetzer, J. C. Anal. Chem. 1989, 61, 236-240. Nelson, R. J.; Pauius, A.; Cohen, A. S.; Gunman, A.; Karger, B. L. J. Chromatogr. 1989, 480, 111-127. Grushka, E.; McCormick, R. M.; Kirkland, J. J. Anal. Chem. 1989, 61, 241-246. Kurosu, Y.; Hibi, K.; Sasaki, T.; Saito, M. J. High Resolut. Chromfogr. 1991, 14, 200-203. Terabe, S.;Otsuka, K.; Ando. T. Anal. Chem. 1989, 61, 251-260. Cohen, A. S.; Terabe. S.; Smith, J. A.; Karger, B. L. Anal. Chem. 1987, 59, 1021-1027. Lochmuller, C. H.; Breiner, S.J.; Ronsick, C. S.J. Chromtogr. 1989, 480, 293-300. Kuhr, W. G.; Yeung, E. S. Anal. Chem. 1988, 60, 2642-2646. Lkle, D. R. CRC Handbook of Chemistry and Physics, 71St ed.; CRC Press, Inc.: Cleveland, OH, 1990; pp 8-30. M e r , P.; Lohrum, A.; Gareiss, J. Ractice and Theory ofpH Masurem n t s ; Ingokl Messtechnik A G Urdorf, Switzerland, 1969. Shibata, M.: Nakamizo, M.; Kakiyama, H. Kyushu Kogyo ol/uisu Shikensho Hokoku 1977, 18, 1015-1022. Diehl, H.; Horchak-Monis, N.; Hefley, A. J.; Munson, L. F.; Markuszewski, R. Talanta 1986, 33,901-905. Lukacs, K. D.; Jorgenson, J. W. W C 8 CC,J. High Resoiut. Chromatcgr. Chromatogr. Common. 1985, 8 , 407-411. Lambert, W. J.; Middieton, D. L. Anal. Chem. 1990. 62, 1585-1587. Beckers, J. L.; Everaefts, F. M.; Ackermanns, M. T. J. Chromfogr. 1991, 537,407-426. Diehl, H.; Markuszewski, R. Talanta 1985, 32, 159-165.
RECEIVFDfor review September 9,1991. Accepted November 27, 1991.
