Anal. Chem. 1991, 63,273-276
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Anion Chromatography Using Octadecylsilane Reversed-Phase Columns Coated with Cetyltrimethylammonium and Its Application to Nitrite and Nitrate in Seawater Kazuaki Ito,* Yasunobu Ariyoshi, Fumio Tanabiki, and Hiroshi Sunahara Department of Environmental Science, Faculty of Engineering, Hiroshima University, 1-4-1Kagamiyama, Higashi-Hiroshima 724,Japan
A procedure Is descrlbed for the separatlon of inorganic anIons using two types of C,, reversed-phase columns (polymer-coated and -uncoated silica packing materlals), which were coated prior to treatment with 1 mM cetyltrlmethylammonium (CTA') in water (H,O) and H,O-methanol (MeOH) mixtures at 20 OC. It Is suggested from the determination of the anion-exchange capacity by three methods that the sorbed CTA' exerts as an anion-exchange site. The mobile phase for anion separation was composed of an aqueous solution of 0.1 M sodium chloride-5 mM sodium phosphate buffer (pH 5.8). The correlation between anlonexchange capaclty and the retention of anlons was good for both columns. The advantages of the CTA'-coated columns and the mobile phase are dlscussed. Uitravlolet (UV, 225 nm) and amperometrlc (AMP, +1.0 V, glassy carbon (GC) working electrode vs Ag/AgCI) detection of an Ion chromatographic system, which consisted of a column with polymercoated packing material (coated with 1 mM CTA' In a H,O(80)-MeOH( 20) mixture; exchange capacity, 0.25 mequiv/ column), could be successfully applied to the direct determlnatlon of nitrite and nitrate In seawater, without interference and Br-. The determination by a large amount of CI-, SO:-, was carried out by the peak area method because of the decrease In peak height with sallnity. The detection limit was 4 pg/L (UV) and 2 pg/L (AMP) for nitrite and 8 pg/L (UV) for nitrate.
INTRODUCTION There has been an increasing interest in the separation of inorganic anions with reversed-phase columns and ion-pair reagents (1-14),as an alternative to conventional anion-exchange columns (15-18).Two methods have been conducted to accomplish anion separation on the reversed-phase columns, for example, with CI8silica-based and polymer-based packing materials. One is the use of a dynamic ion exchanger produced by the sorption of a hydrophobic cationic modifier in eluents, which results in the formation of a charged double layer: the primary layer (hydrophobic cation) and the secondary diffuse layer (inert anion) (1-7).The other approach is to use the reversed-phase columns permanently coated with ion-pair reagents, which gives a charged surface ( I , 8-14). In the latter case, cationic surfactants and dyestuff molecules (12,13)with large and hydrophobic quaternary ammonium ions such as cetyltrimethylammonium (9-11) and cetylpyridinium (8,14) have been employed because the mobile phase for anion separation does not include the ion-pair reagents. These approaches possess several advantages over conventional "fixed-site'' anion-exchange columns: (a) greater chromatographic efficiency and lower price of reversed-phase columns, (b) no need for special equipments, and (c) greater flexibility with regard to choice of columns, eluents, and ion-pair reagents 0003-270019 1/0363-0273$02.50/0
for optimum anion separation. Of these factors, the ability to control the anion-exchange capacity is the most important advantage. However, this advantage has hardly been utilized. In this study, using two types of CI8reversed-phase columns that were permanently coated with cetyltrimethylammonium (CTA+),the efficiency for anion separation was examined in conjunction with the exchange capacity. Then, the columns coated with CTA+ were applied to the determination of nitrite and nitrate in seawater. Anion analysis is one of the most important subjects used to investigate the situation of eutrofication. For the determination, it is necessary to remove the interference by an excess of salts. In previous studies (17, 18),we have applied an anion chromatographic method to the direct determination of parts per billion (ppb) levels of iodide in seawater by using a low-capacity anion-exchange column, ultraviolet (UV) and amperometric (AMP) detection, and 0.1 M sodium chloride as a mobile phase. The system, however, could not be applied to the determination of nitrite and nitrate in seawater because of the interference of an excess of C1-, S04z-,and Br-. This can be mainly attributed to the low capacity of the column. Therefore, it seems to us that one way to overcome this problem is the use of high-capacity anion-exchange columns. Some chromatographic procedures for the determination of nitrite and nitrate have been reported and are based on ion-exchange (19-22),ion-interaction (4,6), and ion-exclusion (23)methods. These sensitive detection methods use UV spectrophotomeric (NOz- and NO3-) (4,7,19-21, 23), amperometric (platinum (Pt) (23)and glassy carbon (GC) (6,19) working electrodes, NOz-), and conductometric (19, 22) techniques. Although the systems have been applied to various food and environmental samples, it was difficult to detect the trace level of analyte ions in seawater: For ionexchange methods with UV and AMP detection (19,20),a small volume of samples (10 pL) or 20-fold diluted samples were injected in order to eliminate the interference by coexisting ions, resulting in a decrease of detectability. In addition, the effects of coexisting ions on the peak shapes and the determination of the ions were not examined. Although the ion-exclusion mode is effective for nitrite, nitrate is eluted in the void volume together with other anions, C1-, Sodz-, etc. (23).In this study, the optimum chromatographic system (a CTA+-coatedcolumn with high anion-exchange capacity, UV and AMP (GC working electrode) detection, and 0.1 M NaCl as a mobile phase) was examined for the determination of nitrite and nitrate by direct injection of 100-pL seawater samples. The higher concentration mobile phase is UVtransparent and electroinactive on a GC electrode and has the possibility of eliminating interferences by a large amount of coexisting ions ( I 7,18).
EXPERIMENTAL SECTION Apparatus. The ion chromatograph comprised (a) a Tosoh CCPM pump (Tosoh), (b) a Rheodyne 7125 injection valve (1OO-pL sample loop), (c) a UV spectrophotometric detector (L0 1991 American Chemical Society
274
ANALYTICAL CHEMISTRY, VOL. 63,NO. 3, FEBRUARY 1, 1991
Table I. Anion-Exchange Capacities of Columns Coated with CTA+ column' Capcellpak Cl8 TSKgel ODS-80TM
exchange capacity, mequiv/column CTA+ sorbed salicylate nitrate 0.274
0.250
0.456
0.421
0.257 0.441
'The columns were coated with 1 m M CTAC in a H20(80)MeOH(20) mixture at 20 "C.
I
0
I
I
20
Methanol Concentrat ion
I
40 ( V / V %)
Flgure 1. Variation of the amount of sorbed cetyltrimethylammonium with the concentration of methanol in the coating solution. C1,, reversed-phase column: (A) Capcellpak Cle: (B) TSKgel ODS-80TM.
Coating conditions are given in the Experimental Section.
4200; Hitachi), (d) an amperometric (AMP) detector with a glassy carbon working electrode (VMD-1O1A and P-1O00, Yanagimoto), and (e) a chromatoprocessor (CP-8O00,Tosoh). The method for pretreatment of the GC was described previously (18). Two types of ClBreversed-phase columns were used: (a) Capcellpak Cia (Shiseido, 150 X 4.6-mm i.d.; 5-gm spherical particle; packing material, octadecyl-bonded silica gel coated with silicone polymer) (24); (b) TSKgel ODS-80TM(Tosoh, 150 x 4.6-mm i.d.; 5-pm spherical particle; packing material, octadecyl-bonded silica gel). The flow rate of 0.1 M NaCl eluent was kept at 1.0 mL/min under pressures of 9C-110 kg/cm2 (Capcellpak Cia) and 110-130 kg/cm2 (TSKgel ODS-80Tv). Reagent and Mobile Phase. All inorganic salts used were of analytical grade. Standard solutions of inorganic anions were prepared from stock solutions (10-50 g/L) of the sodium salts. Artificial seawaters (salinity 0-45%) were prepared according to the Lyman-Fleming formula (25). A mobile phase of 0.1 M NaC1-5 mM sodium phosphate buffer (pH 5.8) was prepared from stock solutions of 2 M NaCl, 0.1 M Na2HP04,and 0.5 M NaH2POk These solutions were prepared in distilled, deionized water and filtered through a 0.45-pm membrane filter, prior to use. Preparation of the Separation Column. Two types of Cla reversed-phase columns, immersed in a water bath thermostated at 20 & 0.05 "C, were coated with 1mM cetyltrimethylammonium chloride (CTAC) in water (H,O) and H,O-methanol (MeOH) mixtures. CTAC and HPLC-grade methanol, used as received, were obtained from Katayama Chemicals. The coating solutions were pumped through the columns at a flow rate of 0.5 mL/min until adsorption equilibrium was accomplished. Completion of the coating of the column was decided by measurement of nitrogen (in CTA+)in the effluent, using a Mitsubishi Kasei TN-02 nitrogen analyzer. The coating solutions were switched to water and then 0.1 M NaCl-5 mM sodium phosphate buffer (pH 5.8). The absorption equilibrium was further confirmed from constant retention volumes of each inorganic anion, after additional flow (ca. 2 h) of each coating solution. The amount of sorbed CTA+ was determined by two methods: (a) elution of CTA+ with H20(30)-MeOH(70) and analysis of the nitrogen content; (b) the difference in the nitrogen content between the influent and the effluent. RESULTS AND DISCUSSION (1) Permanently Coated Column Systems. Determination of the Anion-Exchange Capacity. Figure 1 shows the variation of the amount of sorbed CTA+ on two C18reverse stationary phases, Capcellpak CIa and TSKgel ODS-~OTM, against the coating solutions. The former is further coated with silicone polymer on a silica gel surface. Cetyltri-
methylammonium chloride was chosen as the coating reagent by considering the higher hydrophobicity of the cetyl group and the good water solubility of the reagent. For both columns, the amount of sorbed CTA+ decreases with an increase in methanol, due to the decrease in hydrophobic interactions between the cetyl group and the octadecyl group on the silica surface. Further, the amount of Capcellpak C18 in each solution is ca. 60% that of TSKgel ODs-80TM over the water content range studied. This may probably be ascribed to the decrease of the surface area, due to the use of silica gel with a smaller surface area and its coating by silicone polymer (24). The anion-exchange capacity, which is estimated from the amount of sorbed CTA+, might not be necessarily the same as the intrinsic value, because the CTA+ sorbed by electrostatic interaction through a silanol group does not work as an anion-exchange site (IO). Therefore, for the columns coated in H20(80)-MeOH(20) solution, the anion-exchange capacities were estimated from breakthrough volumes of salicylate (2 mM) and the determination of the amount of nitrate sorbed on anion-exchange sites. The former was obtained from the difference in breakthrough volumes of salicylate between the columns coated and uncoated with CTA+. The determination of nitrate was carried out as follows: The columns coated with CTA+ were converted into nitrate form with a flow (0.5 mL/min, 2 h) of 0.1 M NaN03 and then washed with HzO (0.5 mL/min, ca. 40 min) until the elution of nitrate ceased. The nitrate sorbed on the anion-exchange sites was eluted out with 0.1 M NaCl-5 mM sodium phosphate buffer (pH 5.8; 1 mL/min) and was determined from the nitrogen content. The elution of nitrate and salicylate was confirmed with UV detection. These data are summarized in Table I. For both columns, the data obtained by the two methods are in good agreement with those obtained from the amount of sorbed CTA+. Because the adsorption of salicylate on the uncoated columns was negligible, each CTA+ on the coated columns interacts with salicylate through electrostatic attraction. This indicates that each CTA+ sorbed exerts satisfactorily as an anion-exchange site. Thus, it is possible to prepare the columns with high anion-exchange capacity. The critical micellar concentration (cmc) of CTAC in water at 20 "C is around 1m M the cmc's of CTAC at 30 "C and the bromide salt a t 25 "C are 1.3 and 0.92 mM, respectively, although the data of CTAC at 20 "C are not available (26). The amount of sorbed CTA+ increases with an increase in monomeric CTA+ and is almost constant above the cmc (7,27).In this study, the amounts of sorbed CTA+ in 1.5 mM CTA+ were almost equal to those in 1.0 mM CTA+ for both columns. Thus, the maximum value of the anion-exchange capacity is ca. 0.4 and 0.8 mequiv/ column for Capcellpak Cla and TSKgel ODS-~OTM,respectively. Separation of Inorganic Anions. The effects of the coated columns on analyte retention were studied by use of a mobile phase of 0.1 M NaCl-5 mM sodium phosphate buffer (pH 5.8). For both columns, the retention volumes of the anions increased with an increase in the amount of sorbed CTA+, viz. anion-exchange capacity, with higher correlation. In addition, the elution order of singly charged anions, IO3-, NOz-, Br-,
ANALYTICAL CHEMISTRY, VOL. 63, NO. 3, FEBRUARY 1, 1991
275
(6)AMP
( A ) UV
3
(A)
-a al
>
2
.-P c
a
2 011
5
10
15
20
1
Time ( m i n )
Ion chromatograms of inorganic anions. CondUions: Column, (A) Capcellpak Cla coated with 1 mM CTA’ in H20(80FMeOH (20); (B) TSKgel IC-Anion-PW. Mobile phase, 0.1 M NaCI-5 mM sodium phosphate buffer (A, pH 5.8; 8, pH 6.7). Detection, UV absorbance at 210 nm. Flow rate, (A) 1.0 mL/min, (6) 1.2 mLlmin. Sample volume, 100 pL. Flgure 2.
NO3-, was the same as with conventional anion chromatography (15-18). Figure 2 shows the chromatograms of mixtures of UV-absorbing anions at 210 nm, using a reversed-phase column coated in H20(80)-MeOH(20) solution and a conventional column. The upper chromatogram (Figure 2B) was obtained with a conventional anion-exchange column (TSKgel IC-Anion-PW, Tosoh; exchange capacity, 0.07 mequiv/column) (18). The iodide peak is well separated from that of the other anions. Thus, a highly sensitive determination of iodide in seawater was achieved (18). The lower chromatogram (Figure 2A) was obtained with the Capcellpak C18column (exchange capacity, 0.25 mequiv/column). These five anions are perfectly separated. The difference in the two chromatograms may be ascribed to the anion-exchange capacities, although they have different packing materials. The addition of 0.1 M NaCl in the mobile phases containing CTA+ at a submicellar region (below 1 mM) brings about an increase in the amount of sorbed CTA+ (27). The “salting out” effect of NaCl is due to the reduction of ionic repulsion among CTA+ ions and the enhancement of hydrophobic interactions with octadecyl groups (27). In this study, the retention times of anions IO3-, NOz-, Br-, and NO3- on the Capcellpak C18 column (exchange capacity, 0.25 mequiv/column) did not change for a successive flow (80 h at a flow rate of 1.0 mL/min) of 0.1 M NaCl-5 mM sodium phosphate buffer (pH 5.8), indicating the stability of the sorbed CTA+, probably due to the interactions. Thus, the mobile phase without CTA+ is suitable for the determination of trace level anions in concentrated salt solutions. For mobile phases with CTA+, an excess of salts in the samples will disrupt the equilibrium between the amounts of CTA+ in the stationary and mobile phases. (2) Determination of NO2- and NO3- in Seawater. Separation of NOz- and NO3- in Seawater. The separation of NOz- and NO; in artificial seawater (salinity 35’350) was examined by use of the columns obtained for coating solutions in Figure 1. For both columns, the separation of NOz- and NO3- could be performed at retention volumes of 5 and 12 mL, respectively, under coating solutions of ca. 20% and 30% MeOH for the Capcellpak C18and TSKgel ODS-80TMcolumns, respectively. The column used for the determination was, however, restricted to Capcellpak C18: (a) The column, which is coated with silicone polymer, can be applied to mild alkaline samples such as seawaters (ca. pH 8) (24),although the column is a little expensive; (b) the peaks of the anions
0
I
1
I
I
I
1
0
20
40
0
20
40
Salinity
(a/“
)
Flgure 3. Variation of the peak area (dashed lines) and peak height (solid lines) of NO,- and NO,- (0.25 mg/L of each) wlth salinlty. Detection, (A) UV, 225 nm; and (B) AMP, i-1.0 V (vs AglAgCI). Other conditions were the same as Figure 2A.
were sharp. UV and AMP Detection. The detection of NO, and NO3(0.25 mg/L of each) in artificial seawater (salinity 35%) was examined with W and AMP methods. The artificial seawater contains 19300 mg/kg for C1-, 2710 for SO,-: 142 for HC03-, and 67 for Br- as major anions (25). For UV detection, the negative dip preceding the nitrite peak at 5210 nm interfered with the determination, which could be attributed to Sod2and not to C1-. The peak height of NO2- at 215 nm was low due to this effect. Thus, the determination at 225 nm is preferable, although both anions in pure water exhibit strong absorptivities at lower wavelengths. For AMP detection, which is active for NO2-and inactive for NO3- (6,17-19), the peak at +1.0 V was higher than those at other potentials, which was in good agreement with the result in pure water. However, a long time period (-2 h) was required to obtain a constant base line. Thus, UV detection, which is simple on operation and can be applied to both anions, is recommended although AMP detection shows higher sensitivity for NO2-. Calibration Graphs, Detection Limits, and Repeatability. Figure 3 shows the plots of the relative values to average peak areas (salinity @45%0)and to peak height at salinity 35%. The peak heights decreased rapidly with an increase in salinity. However, the peak meas were almost constant up to a salinity of 45%: The relative standard deviations (RSD’s) for seven solutions (salinity @45%) were 1.4% and 1.5% for NO, with UV and AMP detection and 2.6% for NO, with W detection. Thus, the calibration graphs (0.01-1.0 mg/L of NO2- and NO3- at salinity 35%) by the peak area method were examined. The addition of salinity in the solutions resulted in base line stability and reduction of noise peaks. The graphs with UV detection were linear through the origin. The graph with AMP detection (NO,) showed a downward curvature, which has been obtained for NOz- on Pt (24) and I- and SCN- on GC electrodes (18). With a 100-pL injection of artificial seawater (salinity 35%), the detection limit (at a signal-to-noise ratio of 2) was 4 and 8 pg/L for NO2- and NO3- with UV detection, respectively, and 2 pg/L for NO, with AMP detection. Good precision data were obtained from the RSD’s for 10 replicate injections (0.25 mg/L of both anions in the artificial seawater): 0.7% and 0.6% for NOz- and NO3- with UV detection, respectively, and 0.5% for NO2- with AMP
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 3, FEBRUARY 1, 1991
T a b l e 11. A n a l y t i c a l R e s u l t s
Seto Inland Sea
of NO2- and NOt- in S e a w a t e r by UV and AMP D e t e c t i o n
added, Gg/L
no. 1 30 (30) no. 2 30 (30)
NO*UV (AMP) found, rg/L 4 (3) 34 (31) ND" (ND) 30 (26)
NOB-
uv
recovery, %
added, pg/L
30 (30) no. 4 50 (50)
46 (41) 15 (14) 65 (63)
recovery, %
88
100 (93)
30
100 (87)
30
113 (103)
200
100 (98)
50
12 (10)
no. 3
found, rg/L 118 12 42 193 397 66 116
100 100
102 100
Not detected. ( A ) UV
(E) A M P
I
NO
Thus, the ion chromatographic system using a reversedphase column coated with CTA+, which has a higher anionexchange capacity, could be applied to the determination of nitrite and nitrate in seawater. However, in order to clarify the greater usefulness of the reversed-phase column, it is necessary to compare the properties between the column with a low-capacity and a conventional column for ion chromatography. This is the topic of a further study. Registry No. NOs, 14797-55-8; NOz, 14797-65-0; water, 7732-18-5.
LITERATURE CITED
Flgure 4. Ion chromatograms of a seawater sample (Table I 1 (no. 1)). Solid lines, a real sample; dashed lines, the sample spiked with 0.03 mglL of NOT and NO3-. Conditions were the same as In Figure 3.
detection.
Determination of NO2- and NO3- i n Seawater. By use of the optimized chromatographic and detection conditions, the determination was examined. The surface seawaters were sampled in the Seto Inland Sea near Hiroshima City. The samples were filtered through a 0.45-pm membrane filter within 24 h after collection, pretreated by passage through a Sep-Pak CIBcolumn (Water Assoc.), and injected. Figure 4 shows the ion chromatograms of a seawater sample (solid line) and the sample spiked with 0.03 mg/L of NOz- and NO3(dashed line). Good chromatograms with the same retention volumes were obtained for both detections (UV = 225 nm, AMP = +1.0 V vs Ag/AgCl), and they were in good agreement with those of artificial seawater (salinity 35%). Further, the small and unknown peak near nitrite at 215 nm (inactive for the AMP method) was not observed a t 225 nm. The results obtained are shown in Table 11,together with other data. The agreement between the UV and AMP methods was good for all samples. Moreover, quantitative recoveries, with good precision, were obtained despite the low concentrations of the anions.
(1) Dasgupta, P. K. I n Ion Chromatography; Tarter, J. G., Ed.; Marcel Dekker: New York, 1987; pp 253-272 and references cited therein. (2) Btdlingmeyer, 8. A.; Santasania, C. T.; Warren, F. V., Jr. Anal. Chem. 1987, 5 9 , 1843-1846. (3) Wheals, B. B. J . Chromatogr. 1987, 402, 115-126. (4) Iskandaranl, 2.; Pietrzyk, D. J. Anal. Chem. 1982, 54, 2601-2603. (5) Rigas, P. G.; Pietrzyk, D. J. Anal. Chem. 1986, 5 8 , 2226-2233. (6) Lookabaugh, M.; Krull, I.S. J . Chromatogr. 1988, 452, 295-308. (7) Okada, T. Anal. Chem. 1988, 60, 1511-1516. (8) Cassidy, R. M.; Elchuk, S. J . Chromatcgr. Sci. 1983, 21, 454-459. (9) Cassidy, R. M.; Elchuk, S. Anal. Chem. 1982, 54, 1558-1563. (10) Takeuchi. T.; Yeung, E. S. J . Chromatogr. 1986, 370, 83-92. (11) Duvai, D. L.; Fritz, J. S. J. Chromafcgr. 1984, 295, 89-101. (12) Mulier, G.; Meisch, H . 4 . J. Chromatqr. 1989, 483, 145-151. (13) Golombek, R.;Schwedt, 0. J. Chromatogr. 1988, 452, 283-294. (14) Barkiey, D. J.; Dahms, T. E.; Villeneuve, K. N. J . Chromatogr. 1987, 395, 631-640. (15) Small, H.; Stevens, T. S.; Bauman, W. C. Anal. Chem. 1975, 47, 1601- 1809. (16) Gjerde, D. T.; Fritz, J. S. Ion Chromatography, 2nd ed.; Hiithig: Heiderberg. 1987. (17) Ito, K.; Sunahara, H. BunsekiKagaku 1988, 3 7 , 292-295. (18) Ito, K.; Sunahara, H. J. Chromafogr. WSO, 502, 121-129. (19) Pastore, P.; Lavagnini, 1.; Boaretto, A.; Magno, F. J . Chromafogr. 1889, 475, 331-341. (20) Takahashi, A. Bunseki Kagaku 1980. 2 9 , 508-512. (21) Jackson, P. E.; Haddad, P. R.; Diiii, S. J. Chromatogr. 1884, 295, 471-478. (22) Murayama, M.; Suzuki, M.; Takitanl, S. J . Chromafogr. 1989, 466, 355-363. (23) Kim, H.J. Kim, Y.-K. Anal. Chem. 1988, 61, 1485-1489. (24) Ohtsu, Y.; Fukui, H.; Kanda, T.; Nakamura, K.; Nakano, M.; Nakata, 0.; Fujiyama, Y. Chromafographia 1987, 2 4 , 380-384. (25) Lyman, J.; Fleming, R. H. J . Mar. Res. 1940, 3, 134-146. (26) Rosen, M. J. Surfactants and Interfacial Phenomena; Wiiey: New York, 1978. (27) Berthcd, A.; Girard, I.; Gonnet, C. Anal. Chem. 1988, 5 8 , 1362-1367.
RECEIVED for review May 30,1990. Accepted October 12,1990. This work was supported in part by a Grant-in-Aid for Scientific Research, Nos. 63740316 and 01540483 (K. I.), from the Ministry of Education, Science and Culture of Japan. This work was presented, in part, a t the 1989 International Chemical Congress of Pacific Basin Societies,02-B7, Honolulu, Dec 17-22, 1989.
