Anal. Chem. 1989, 61 784-787
784
Table I. Reproducibility of Sucrose and Glucose Electrodes in Quantifying Sucrose in a Soft Drinka
assay no. 1 2 3 4 5 6 7 8 9 10
meanb sd cv, %
current change (nA/min for rate; nA for steadv state) sucrose electrode glucose electrode rate steady state rate steady state 160 168 168 168 160 168 168 172 172 168
55 54 55 53 53 55 54 56 57 52
8.0 8.0 8.5 8.5 8.5 8.5 8.5 8.5 8.5 8.0
18.0 18.0 19.0 20.0 21.0 20.5 20.0 20.0 20.0 20.0
167 4.13 2.47
54.4 1.51 2.77
8.4 0.24 2.89
19.7 1.00 5.10
Dilution ratio = 3
X
400.
Mean of 10 determinations.
Table 11. Comparison Study between Enzyme Electrode and AOAC Method for the Determination of Sucrose in Food Products
food sample cola 1 cola 2 apple juice" apple grape juicen (baby food) mixed fruit juice" (baby food) honeyb
sucrose, mg AOAC electrode method rate steady state method 7.64 19.01 32.14 31.55 29.09 2.53
7.31 20.57 32.65 34.05 27.94 2.37
6.80 19.30 31.92 31.46 27.74 2.38
"For 1 mL. bFor 100 mg.
months and several hundreds of assays. Sucrose was determined in food products by using both the sucrose and glucose electrodes. The amount of sucrose present was calculated by subtracting the concentration of glucose obtained from the reading of the glucose electrode from the total sucrose plus glucose concentration determined from the reading of the sucrose electrode. Samples were prepared as mentioned in the Experimental Section. Table I shows the reproducibility of the sucrose and glucose electrodes when the sucrose content in a soft drink is measured. The standard AOAC method was used to run food samples for sucrose, and the results were compared with those obtained from the enzyme electrode (Table 11). The average correlation coefficients of the two methods for the analysis were 0.9989 and 0.9982,
and the regression equations for data were 3' = 1 . 0 0 0 9 ~+ 0.3755 and y = 1 . 0 4 0 1 ~+ 0.0819 (y = electrode result; x = AOAC method result; n = 6) for the initial rate and steady-state methods, respectively.
ACKNOWLEDGMENT We are grateful to Xiangfang Xie for her assistance in setting up the apparatus needed for the quantitation of sucrose by the AOAC method and performing sucrose analysis in food products, as well as the Glenn J. Lubrano and Graham Ramsay for helpful discussions and for the supply of various membranes and electrodes. The financial assistance of the US.Department of Agriculture (SBIR Grant No. 86-SBIR8-0096) is gratefully acknowledged. Registry No. Sucrose, 57-50-1; D-glucose, 50-99-7; cellulose acetate, 9004-357; glucoee oxidase, 9001-37-0; invertase, 9001-57-4; mutarotase, 9031-76-9; Teflon, 9002-84-0. LITERATURE CITED (1) Guilbault, G. G. Analytical Uses of Imm06lked Enzymes: Marcel Dekker: New York and Basel; 1984, Chapter 3. (2) Kobos, R. K. TrAC, Trends Anal. Chem. (Pers. Ed.) 1987, 6(1), VIII-x. (3) Van Brunt, J. Bio/Technology 1887, 5(5), 437-441. (4) Murray, R. W.; Ewing, A. G.; Durst, R. A. Anal. Chem. 1987, 59(5), 379A. (5) Rechnltz, G. A. PAC, Trends Anal. Chem. (Pers. Ed.) 1886. 5(7), 172-174. (6) Cordonnier, M.; Lawny, F.; Chapot, D.;Thomas, D. FEBS Len. 1975, 59, 263. (7) Satoh, I.; Karube, I.; Suzuki, S. Bbfechnol. Bioeng. 1976, 18, 269-272. (8) Barker, A. S.; Somers, P. J. In Topics in Enzyme and Fermentation Blotechnolcgy; Wiseman, A., Ed.; Ellis Hotwood: Chichester, England, 1978; Vol. 2, p 120. (9) Bertrand, C.; Coulet, P. R.; Gautheron, D. C. Anal. Chim. Acta 1881, 126, 23-34. (10) Kulis. Yu. Yu.; Peslyakene, M. V. J . Anal. Chem. USSR (Engl. Transl.) 1880, 3 5 , 786-790. (11) Kulys, J. J. Anal. Len. 1981, 14, 377-397. (12) Scheller, F.; Renneberg. R. Anal. Chim. Acta 1883. 752, 265-289. (13) Masoom. M.; Towshend. A. Anal. Chim. Acta 1885, 171, 185-194. (14) Olsson, 6.; Stalbom, 6.; Johansson, G. Anal. Chlm. Acta 1986, 179, 203-208. (15) Nabi Rahni, M. A.; Lubrano, G. J.; Guilbauit, G. G. J . Agrlc. Food Chem. 1987, 35. 1001-1004. (16) Hart, F. L.; Flsher, H. J. Modem Food Analysis; Sprlnger-Verlag: New York, Heidelberg, Berlin, 1971. (17) Officlsl Methods of Analysis of the Association of OfHc&l Analytical Chemists, 14th ed.;WilUams, S., Ed.; Association of Official Analytical Chemists, Inc.: Washington. DC, 1984. (18) Carr, P. W.; Bowers, L. D. Immoblured Enzymes in Analytical and Clinical Chemlsfty; Wiley: New York, 1980; p 234. (19) Matsumoto, K.; Kamikado. H.; Matsubara, H.; Osajima, Y. Anal. Chem. 1888, 60, 147-151. (20) AWui Hamid. J.; Moody, G. J.; Thomas, J. D. R. Analyst 1888, 773(1), 81-85 -.
(21) Swindlehurst, C. A. (Koerner); Nieman, T. A. Anal. Chim. Acta 1988, 205, 195-205.
RECFJVED for review March 29,1988. Resubmitted November 1, 1988. Accepted December 2, 1988.
Liquid Chromatographic Determination of Theophytline Concentration with Syringe-Type Minicolumns for Direct Plasma Injection Masato Homma and Kitaro Oka* Division of Clinical Pharmacology, Tokyo College of Pharmacy, Hachioji, Tokyo 192-03, J a p a n
Noriyuki Takahashi Department of Pharmacy, Tokyo Medical College Hospital, Shinjuku-ku, Tokyo 160, J a p a n
INTRODUCTION A method for quantitative column extraction has been developed to determine very low concentrations of steroid hormones and certain drugs in biofluids by high-performance 0003-2700/89/0361-0784$01.50/0
liquid chromatography (HPLC) (1-7). This method, termed rapid-flow fractionation (RFF), involves the cleanup of biofluids using diatomaceous earth granules and an organic mobile-phase solvent of minimal polarity for extracting target 0 1989 American Chemical Society
ANALYTICAL CHEMISTRY. VOL. 61, NO. 7, APRIL 1. 1989
(8)
(A)
4-05-k
(C)
785
(D)
I O K-226-30+15+
Flgum 1. Overall view of the syringe-type mlnlcolumn, Extrasbk (A) application needle: (B) packed two-way cock: (C) packed tube. After a 5-pL plasma specimen had bean introduced In the packed tube (C) wlth an ordinaty microsyringe, Extrashot was then attached to Um SYln6-3 loadlng Sample injector. Extraction solvent. 130 &, was then Introduced by the use of another syringe (0).
substance(s) and eliminating other polar contaminants. The eluent volume is made minimal by critical frontal ertradion. With column size minimized, an extract can be transferred directly into an ordinary syringe loading sample injector for HPLC. In the present study, a new method was developed for determining the plasma concentration of theophylline using a newly designed syringe-type minicolumn, Extrashot, attached to an ordinary HPLC sample injector. The results ohmined by this method were compared with those from conventional fluorescence polarization immunoassay (FPIA) for the therapeutic drug monitoring (TDM) of asthma. One drawback of HPLC in TDM in the clinical laboratory is tedious sample pretreatment requiring much time. A column switching technique by which a sample is pretreated in a precolumn bypass has been developed and commereialized (Merck, Darmstadt, FRG) to eliminate this problem. However, besides the economical problem, it can be applied only to lipophilic substances easily absorbed on the reversed-phase support in the bypass column. For hydrophilic compounds such as xanthine derivatives, including theophylline and theobromine, ita use has never given good results. T o overcome these difficulties, a sophisticated direct injection technique has been developed recently by Pinkerton et al. (8). Tedious sample pretreatment being eliminated, the method has been applied to determining drug concentrations in human plasma (9-11). However, +use of their complete elimination of the extraction pathway. it seems somewhat difficult to improve void peak spreading for base-line separation. EXPERIMENTAL SECTION Apparatus. Exfrashof. Figure 1 provides an entire new of Extrashot. It is composed of three parts: (A) a stainless steel needle, (B)a two-way cock packed with diatomaceous earth granules. and (C) a tube half-packed with the same support material. The needle (A) and packed tube (C)are connected to the cock (B) with nuts. The nuts, two-way cock, and tube are made of poly(tetrafluoroethy1ene)(PTFE). Sizes of these parts are illustrated in Figure 1. The packed tube retains a plasma specimen, the packed cock is for cleanup, and the needle is for introducing the sample extracts to a syringe loading sample injector for HPLC. Assembly and disassembly can be easily performed manually. Both ends of the nuppon material are capped by cellulose filter tips. A 5-pL aliquot of the plasma specimen can be introduced through a filter into the packed tube ontn the surface uf the support material by an ordinary microsyringe of 10-pL inner volume. The capacity of the support material in Extrashot is 44 pL, and ita loading capacity IS less than IO pL of plasma. The extraction solvent is introduced from the tube end by another syringe (D), which can he bruught into contact with the end of the tube. The packed tube is dinposable, while the packed two-way cock has multiple use by preconditioning. All the parts were obtained from Kusano Scientific (Tokyo. Japan). HPLC. The apparatus consisted of a solvent delivery pump (BIP-I,J w , Tokyo, Japan), a W detector (Uvidee-lWV, Jaw$, a syringe loading sample injector (Model 7125,Rheodyne, Cotsti,
CA) provided with a 100,cL l q p ?and a pen &der ( v Nippon Denld, Tokyo, Japan). The d y t i c a l dUmn waa packed with silica gel (Lichrosorh Si-60, particle size 5 pm; 4-mm i.d. X 125 mm:.Merck, Darmstadt, FRG). Cbemioals ma mterials. ~iatommtji earth g.+&ulm were prepared from Celite powder (No. 545) purchased from JohnsManville. Mean particle size was 5W70 pm, passable through a 200-mesh screen. Other reagents and organic solvents were of reagent grade and obtained from Wako Pure Chemicals (Osaka, Japan). The theophylline standard was purchased from Sigma (St. Louis, MO). FPIA theophylline kit I1 was obtained from Dinabot (Tokyo, Japan). Blood Samples. The control plasma specimen w e coilected from a healthy male volunteer, 24 years old, who had never taken theophylline and had been on a diet containing no caffeine for 1 week before the test. Twenty test plasma specimens were collected from 20 outpatients suffering from asthma and taking theophylline at doses of 5o(tsoo mg/day. The plasma Was s e p arated hy centrifugation and stored at ,A0OC until analysis. Procedure. HPLC. Extrashot was preconditioned hy pawing 200 pL each of ethanol and dichlorometbane tkough the whole system before use. Dichloromethaneremainii,in the system was flushed out with 500 pL of air by using syrilige D. A 5-ILLaliquot of plasma specimen and 2 p L of 3% NaHCO, were introduced onto the surface of the support material by a microsyringe. Extrashot was then attached to the HPLC system of preconditioned by a mobile-phase solvent consisting of water/acetic acid/etbanol/dichloromethane(0.2/0.2/4/95.6)a t a flow rate of 1.5 mL/min A 13BpLportion of the extraction solvent containing ethanol and dichloromethane (3/97) was introduced into the injector of the HPLC through Fxtrdot. Thisrequired only 5 1 0 s. Too rapid injection resulted in unsatisfactory result8 owing to overtlow of the aqueous phase. The HPLC was conducted in the usual manner, with the h m a t u g r u n reading at a wavelength of 275 nm for the theophylline assay. Recovery of theophylline at concentratioM from 1!m?%3 %JmL of plasma was calculated from the ratio of observed peak height to authentic peak height hy the latter being determined hy.du& injection of standard solution not passed tbrough the Extrashot. Whether the authentic specimen was introduced through the Extrashot or not, no,peak height difference was observed. FFIA. Twenty plasma specimens from the patient8 were treated by the theophylline kit I1 using,the TDX (fluorescence polarization immunoassay automated system) according to the instructions of Dinabot Co.
RESULTS Recovery of Theophylline. A five-point andysis of each plasma specimen containing 1,5,10,and 20 pg/mL of thm phylline in plasma was c o n d u e d , and the results are show? in Table I. To avoid effects from endogenous theophylline, control plasma was obtained from a volunteer who had abstained from caffeine-containing beverages for 1 week. At ¢rations within the therapeutic range of 10-20 &mL, observed recovery essentially averaged quantitatively at about 97%. At lower concentratiqns of 1 and 5 pg/mL, it was aS low as 94%, this being sufficient for direct determination. Figure 2A shows a typical chromatogram of plasma from a volunteer adhering to an ordinary diet including caffeine. The
~
~
786
ANALYTICAL CHEMISTRY, VOL. 61, NO. 7,APRIL 1, 1989
Table I. Extraction Recovery of Theophylline from Plasma at Various Concentrations concn of theophylline,
fiug/mL determined (mean f SD)
given
0.94 f 0.03 4.79 f 0.12 9.79 f 0.46 19.39 f 0.71
1
5 10 20
mean
extraction
coeff of
recovery, %
variation, %
93.6 95.8 97.9 96.9
3.0 2.4 4.6 3.6
3.2 2.5 4.7 3.7
96.1 f 3.4
3.5
B
A
-
4 6 min
f f f f
I!
-
4 6 min Figure 2. (A) Chromatogram of plasma from a healthy subject: a, caffeine; b, theobromine; c, theophylline; d, paraxanthine. (8)Chro0
0
matogram of plasma from an asthmatic patient after administration of theophylline. Theophylline concentration was 10.4 pg/mL of plasma. chromatogram in Figure 2B shows the preferential peak height of exogenous theophylline from an asthmatic patient a t a concentration of 10.4 pg/mL. The influence of endogenous theophylline on total peak height in the therapeutic range was less than 1.5% under the ordinary diet condition. Peak Separation of Xanthine Derivatives. As illustrated in Figure 2A, assignments were made for xanthine derivatives on the plasma chromatogram for a healthy volunteer taking caffeine. Retention times under the present chromatographic conditions were 2.50,4.00,4.68, and 5.52 min in the order of caffeine, theobromine, theophylline, and paraxanthine content. The corresponding capacity ratios, k ’, of these derivatives in the same order were 3.13, 5.00, 5.58, and 6.90. The separation factors, a , of the adjacent peaks were thus calculated to be 1.53, 1.17, and 1.18, which indicate good peak separation of xanthine derivatives in terms of base-line resolution. Calibration Curve. The calibration curve for theophylline by our HPLC was made from direct peak height calibration. The correlation equation was found to be Y = 11.723X - 2.392 (r = 0.9994; p < 0.001), where Y is observed peak height in millimeters a t 0.02 AUFS and X is the concentration of theophylline per milliliter of plasma. The coefficients of variation assumed by five-point analysis in each plasma concentration, 1, 5, 10, and 20 wg/mL, were less than 5% (Table I). Comparison of HPLC with FPIA. The present HPLC results were compared with those obtained by FPIA. These
methods show good correlation obtained by the equation Y = 0.9375X 0.6240 (r = 0.9939; p < 0.001), Y being the concentration obtained by HPLC and X, by FPIA. The concentration measured by FPIA was 1.00-1.03 times that by HPLC in the therapeutic range.
+
DISCUSSION Disposable cleanup items such as Sep-Pak cartridges, developed by Waters (Milford, MA) (12), and Extrelute, by Merck (Darmstadt, FRG) (13,14),are widely used in clinical laboratories. These items are for off-line sample preparation prior to the conduct of HPLC. In contrast to these and our previously reported method using a rapid-flow fractionation (RFF) column system for cleanup in determining very low concentrations of steroid hormones (2-6), the present technique was developed for the analysis of the plasma drug concentrations in the range of pg/mL. The solvent system of Extrashot was designed to condense the target molecule into minimal solvent volume with the least possible polarity. The analytical accuracy of Extrashot was no less than that obtained by our previous method ( 2 ) using RFF and HPLC in conjunction. Extrashot permits satisfactory sample cleanup and injection a t the same time. The normal-phase mode rather than the well-known reversed-phase mode of chromatography should be used for the following reasons. Our method permits the direct injection of as much as 100 pL of the organic extract, while reversedphase chromatography results were unsatisfactory owing to phase destruction. Silica gel chromatography eluted with a solvent system of higher polarity than that of the extraction solvent gave satisfactory results. The extraction solvent used for Extrashot was less polar than the chromatographic solvent containing water and acetic acid in addition to ethanol. The direct combination of extraction and injection can be conducted for the first time by the use of Extrashot. This method lessened the time of the chromatographic run by as much as a half. A highly efficient silica gel short column such as used in this study decreased the time even more. The most practical method for the TDM of theophylline in asthmatic patients is the fluorescence polarization immunoassay developed by Dinabot Co. (15). The pharmacokinetic background of the therapy has been well established (16). However, such drug monitoring by FPIA is too expensive to permit its general application in a community hospital. HPLC finds multiple applications in spite of the considerable time required for sample cleanup. Fortunately, drug concentrations to be determined in TDM can usually be ranged at micrograms per milliliter. The determination can be made by a HPLC equipped with a UV detector, requiring only microliters of sample. Thus the Extrashot eliminates tedious sample pretreatment, as is also accomplished by Pinkerton’s technique, which involves filtering of plasma followed by direct injection. Our results showed close agreement with those obtained by FPIA and indicate that our method is directly applicable to theophylline determination in asthmatics. Our method is thus convenient and usually inexpensive for TDM of theophylline and possibly other drugs under normal-phase chromatographic conditions. Our method and Pinkerton’s technique are comparable in simplicity, but are in remarkable contrast to each other in terms of their phase mode being normal and reversed, respectively. ACKNOWLEDGMENT We are indebted to K. Kubota for his assistance in TDX analysis, to A. Shimizu, M. Matsumura, and T. Takahashi for their assistance in sample collection of asthmatic patients and to S. Ohara for her assistance in the preparation of the manuscript. Registry No. Theophylline, 58-55-9.
Anal. Chem. 1989, 6 1 , 787-789
LITERATURE C I T E D Oka. K.; Minagawa, K.; Hara, S.; Noguchi, M.; Matsuoka, Y.; Kohno, M.; Irimajiri, S. Anal. Chem. 1984, 56, 24-27. Oka, K.; Ohki, N.; Noguchi, M.; Matsuoka, Y.; Irimajiri, S.; Abe, M.; Takizawa, T. Anal. Chem. 1984, 5 6 , 2614-2617. Oka. K.; Ijitsu, T.; Mlnagawa, K. Hara, S.; Noguchi, M. J. Chromatogr. Biomed. Appl. 1985, 339, 253-261. Oka, K.; Noguchi, M.; Kitamura, T.; Shima, S. Ciin. Chem. 1987, 3 3 ,
1639-1642. Oka, K.; Hirano, T.; Noguchi, M. J. Chromatogr. Biomed. Appl. 1987, 423, 285-291. Oka, K.; Noguchi, M.; Hirano, T. Clin. Chem. 1988, 34, 557-560. Oka, K.; Aoshirna, S . ; Noguchi, M. J. Chromatogr. Biomed. Appl. 1985, 345, 419-424. Pinkerton, T. C.; Miiier, T. D.; Cook, S.E.; Perry, J. D.; Rateike, J. D.; Szczerba, T. J. Biomed., Chromatogr. 1988, 1 , 96-105. Pinkerton, T. C.; Hagestarn, I.H. Anal. Chem. 1985, 5 7 , 1757-1763.
787
(10)Pinkerton, T. C.; Perry, J. A.; Rateike, J. D. J. Chromatogr. 1986, 367, 412-418. (I 1) Nakagawa, T.; Shibukawa, A.; Shimono, N.; Kawashima, T.; Tanaka, H.; Haginaka, J. J. Chromatogr. Biomed. Appl. 1987, 420, 297-311. (12)Fasco, M. J.; Cashine. M. J.; Kaminsky, L. S . J. Liq. Chromatogr. 1979, 2, 565-575. (13)Schweizer, K.; Wick, H.; Brechbuhler, T. Clin. Chim. Acta 1978, 9 0 , 203-208. (14)Brelter, J. J. Clin. Chem. Biochem. 1976, 14, 46. (15)Joiiey. M. E.; Stroupe, S. D.; Schwenzer, K. S . ; Wang, C. J.; LuSteffes, M.; Hili, H. D.; Popeika, S. R.;Holen, J. T.; Keiso, D. M. Clin. Chem. 1981, 2 7 , 1575-1579. (16)Ogilvie, R. I. Clin. Pharmacokinet. 1978, 3 , 267-293.
RECEIVED for review May 13, 1988. Accepted December 1, 1988.
Potentiometric pH Detection in Suppressed Ion Chromatography M a r e k Trojanowicz Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland Mark
E. MeyerhofPr
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055 The development of modern high-performance ion chromatography was based on two fundamental achievements presented in the original pioneering work of Small et al. (1). These advances were the preparation of new ion-exchange resins that featured high efficiencies yet very low exchange capacities and the ingenious use of a stripper (suppressor) column in conjunction with conductivity detection. Addition of a stripper column (loaded with appropriate ions) immediately after the separating column effectively neutralizes the high concentration of hydrogen or hydroxyl ions in desired eluents, and this results in a dramatic decrease in the background conductivity of the effluent (i.e. suppressed ion chromatography). As a result of the ion-exchange process in the original packed-bed stripper columns or, more recently, in hollow ion-exchange suppressor fibers, separated cations are present as hydroxides in the effluent while separated anions elute with counter protons. Although the counter hydrogen and hydroxide ions have large limiting conductances that aid in the sensitive detection of the separated ions by conductivity, variations in the equivalent conductances of the individual separated analyte ions influence the ultimate sensitivity toward each. Moreover, conductivity detection is extremely sensitive to temperature variations, and this can result in severe base line drift problems unless the cells are well thermostated or some type of reference cell compensation is used. Recent technological advances in the field of ion-selective membrane electrodes have enabled the fabrication of very sensitive and reliable potentiometric sensors selective for hydrogen ions based on the use of plastic membranes doped with appropriate neutral carrier molecules (2,3). Such sensors have been applied successfully to monitor the pH of flowing systems. Under certain conditions, these polymer membrane type pH sensors can exhibit better dynamic response than conventional pH glass electrodes (2). Surprisingly, despite numerous applications of potentiometric detection in ion chromatography (4),only one report has appeared in connection with using a hydrogen ion electrode as a detector (5). This work focused on monitoring the separation of organic
* Author to whom correspondence should be addressed.
carboxylate anion species with a glass membrane p H sensor by detecting small changes in the pH of a postcolumn reagent buffer continuously added to the effluent stream (due to the basicity of the carboxylate species). Since in modern suppressed ion chromatography separated ions are present in the effluent with equimolar amounts of hydrogen or hydroxyl ions, it seemed worthwhile to examine whether a simple polymeric pH electrode could be used in place of a conventional conductivity detector to monitor the separation of both cations and anions. For this purpose, a poly(viny1chloride) (PVC) membrane electrode prepared with tri-n-dodecylamine (2) was used in a flow-through wall-jet arrangement, and the detection capabilities of this system were directly compared to those obtained with conductivity detection. EXPERIMENTAL SECTION Apparatus. The instrumental setup used in this study (see Figure 1)consisted of a Spectra Physics SP 8700 solvent delivery system (San Jose, CA), injection valve 7124 (Rheodyne, Cotati, CA) with a 1OO-pLsample loop, and a conductivitydetector, Model 213 (Wescam, Santa Clara, CA). The potentiometric detector, placed downstream from the conductivity detector, was connected to an Accumet Model 910 pH/mV meter (Fisher Scientific, Pittsburgh, PA). Analog outputs of both signal transducers were connected to a Fisher Recordall Model D5117-5AQ strip chart recorder. Columns. Separation of cations was performed with an Interaction Chemicals ION-210 cation-exchangecolumn (Mountain View, CA), whereas separation of anions was achieved on a Hamilton PRP-X100 column (Reno, NV). In both cases, an ion Guard polymeric column from Interaction Chemicals was placed between the injector and the analytical column. The suppressor used for the chromatography of anions was a 240-cm length of Nafion 811X tubing (Perma Pure Products, Toms River, NJ), while for cations, a 90-cm length of Raipore T-1030 strong anion-exchange tubing (RAI Research Corp., Hauppauge, NY) was employed. The ion-exchangetubings used were coiled (4-cm diameter) and immersed in a 1-L beaker containing the appropriate regenerant solution and a magnetic stir bar. The regenerant solution was changed periodically, as determined from observing the base-line drift of the detectors. Potentiometric Detector. Potentiometric detection was carried out by using a large-volume wall-jet style membrane
0003-2700/69/0361-0767$01.50/0 0 1989 American Chemical Society
