Anal. Chem. 1981, 53, 909-911
9Q9
Paired Ion Chromatographic Separation of Neutral Species Sir: Paired ion chromatography has extended the application of high-performance liquid chromatography (HPLC) to the separation of ionic species. Schill and co-workers have written on both fundamental and applied aspects of this interesting separation prlocess (1). Generally, use of a TJV-absorbing counterion provides a convenient mode of detecting components in the eluate. Recently, DiNunzio and Freiser (2) extended this idea to the visible range, using cationic dyes as part of the stationary phase and a mixture of hexane and dichloromethane as eluant, and separated the aliphatic acids with significantly enhanced sensitivity. Using catioinic dyes in reversed-phase mode, we found (3) that we were able to separate aliphatic acids with marked sensitivity at either 254-nm or 651-nm wavelength detection. One of the interesting aspects of ion-pair extraction is the role of neutral species combining with the ion pair to form an “adduct complex” (4). Generally the adducting neutral species has been employed in great excess over the ion pair. In the course of an investigation of the implication of adduct formation in paired ion chromatography, we observed an unusual separation of neutral species such as alcohols and ketones. The injection off submicrogram quantities of various alcohols and ketones on an ODs-methylene blue (chloride form) column gave well-defined characteristic dye-containing peaks that were well separated. EXPERIMENTAL SECTION A modular chromatographicunit consisting of an Altex Pump (Model 100A),a variable-wavelength UV-VIS detector (Schoeffel Instruments No. 770), and a. Fisher Recordall Series 500 strip chart recorder was used. The wavelength used in the study was at 254 nm. A 25-cm Whatman Partisil-5 ODS column, described by the manufacturer as having 10% carbon loading with greater than 95% surface coverage, was employed. The mobile phase used was methanol-waiter in 15:85 v/v % containing 1 X M methylene blue (as chloride). Before the chromatographicseparations were undertaken, the column was conditioned by passing sufficient mobile phase to “saturate” it with methylene blue, as evidenced by the appearance of the dye in the effluent at its initial concentr&ion. This required 1.2 X mmol of dye. The flow rate used for both column saturation and subsequent chromatography was 0.5 mL/min. Solutions of the alcohols and ketones (separately or in admixture) in the mobile phase were added through the 1 0 - ~ Lloop sampling valve (Rotary valve injector Sp-419-0410). As may be seen from the data presented in Table I, good separation of the various alcohols and ketones was obtained at tlhe detection limits of about lo3 g. (Signal/noise = 5 for butanol at. this level.) These
Table I. Retention Volume and Capacity Factors of Alcohols and Ketones on Methylene Blue Columnsa Rv, mL
K
Alcohols ethanol 4.5 2-propanol 6.3 1-propanol 6.9 2-methylpropanol 10.8 2-bU tan01 12.9 1-butanol 16.2 Ketones acetone methyl ethyl ketone Z-pentanone
0.9 1.6 1.9 3.5 4.4 5.8
5.7 10.5 13.1
1.7 4.0
5.1
a Mobile phase 85:15 H,O/MeOH (v/v) with (methylene blue).
low4M dye
detection limits are far lower than those attainable for alcohols using refractive index detectors. The work clearly demonstrates the feasibility of using the small differences in adduct formation in the dye. After approximately 30 mL, a shallow, negative depletion band was observed in every case. This work represents the first separation of neutral compounds as associates of an ion pair compound (methylene blue) and should permit significant extension of paired ion chromatography. Although further work is required to clearly establish the mechanism of the separation of neutrals, it would appear that the neutrals are forming adducts with the methylene blue of sufficiently different stability or partitioning characteristics to give useful separations. Further work along these lines is under way in this laboratory. LITERATURE CITED (1) Schill, G. In “Advances in Ion Exchange and Solvent Extraction”;Marinsky, J. A., Marcus, y., Eds; Marcel Dekker: New York, 1974 Vol. 6 , P. 1 (2) DiNunzio, J.; Freiser, H. Talanta 1970, 26,587-589. (3) Gnanasambandan, T.; Freiser, H, unpublished results, 1980. (4) Modin, R. Acta Pharm. Suec. 1971, 8,509.
T. Gnanasambandan Henry Freiser* Department of Chemistry University of Arizona Tucson, Arizona 85721
RECEIVED for review December 22,1980. Accepted February 23, 1981. This research was supported by the U.S. Environmental Protection Agency.
Trace Metal Determinations by Liquid Chromatography and FIuorescence Detection Sir: Liquid chromatography has been applied to the determination of metal ions lby use of atomic absorption (1-4) or electrochemical (5,s)detection. In addition, metal chelates have been employed (7-9)) These chelates can be detected by spectrophotometric detectors in the visible or ultraviolet region. Recently, a method of detecting metal ions by postcolumn formation of colored complexes was reported (10). Most of these methods suffer from one or more limitations, 0003-2700/8 1/0353-0909$0 1.25/0
in particular a lack of uniform sensitivity to the different metals or a basic limitation in overall sensitivity. We have attempted to overcome some of these limitations by synthesizing potentially fluorescent metal chelates using 4-aminophenylethylenediaminetetraaceticacid [4-NH2Ph(EDTA)] (Figure l), separating the chelates by high-performance liquid chromatography (HPLC), and developing the fluorescence by postcolumn derivatization with fluorescamine. 0 1981 American Chemical Society
910
ANALYTICAL CHEMISTRY, VOL. 53, NO. 6, MAY 1981 N(CH2COOH)Z H2N @NICH2COOH12
Flgure 1. (4-Aminophenyl)ethylenediaminetetraacetic acid
1
Cd
W 0 W 0
Zn
z m
(EDTA).
[r W
9 Chelates of lead, cadmium, and zinc were prepared and characterized by combustion analysis, lH NMR, 13C NMR, and ultraviolet spectrometry. EXPERIMENTAL SECTION Apparatus. All liquid chromatographic separations were performed by using a Waters Associates Model ALC 204 liquid chromatograph equipped with a Model 660 programmer and a Model U6-K injector. A Model 440 ultraviolet detector with 254 nm detection was used. Fluorescence detection was accomplished by using an Aminco Fluoromonitor equipped with the standard filters for use with fluorescamine. The fluorescamine solution was pumped with an ISCO Model 314 syring pump and controller. The ion exchange column used for separation of the chelates was a Whatman Partisil SAX (250 mm X 4.6 mm i.d.). For postcolumn fluorescamine addition the mixing “ T was a Swagelok “ T , 1/16 in. i.d. The mixing coil was 5-6 in. of 0.040 in. i.d. stainless steel tubing. All other connections were made by using low dead volume fittings and 0.009 in. i.d. stainless steel tubing. Reagents. Elution buffers were prepared by making the desired concentrations of sodium acetate in water and adjusting to the proper pH with glacial acetic acid. All buffers were filtered through a Millipore 0.22 Fm cellulose acetate filter. Fluorescamine (30 mg/100 mL) was dissolved in Mallinkrodt SpectraAr acetonitrile. Fluorescamine (Fluram) was purchased from Hoffman-LaRoche, Nutley, NJ. 4-NH2Ph(EDTA)was synthesized by using modifications (11) of the methods of Okaku et al. (12) and Sundberg et al. (13). Chelates. The general procedure for a preparation of the chelates was as follows. A stock solution of 4-NHzPh(EDTA)(1.2 mmol in 10 mL of water, pH 5.7) was prepared. A 10-mL aliquot of the stock solution was added to 10 mL of an aqueous solution containing 1.4 mmol of the appropriate metal salt. The pH was adjusted to the appropriate value for each metal, and the solutions were boiled for 1-2 min. Absolute ethanol was added to precipitate the crude chelate. The crude chelates were recrystallized from water-ethanol and dried for 72 h in a vacuum desiccator at room temperature prior to analysis. The lead chelate was prepared by using lead diacetate trihydrate, and the pH was adjusted to 7.0. The yield was 0.51 g (62%). Elemental analysis was consistent Preparation of the with the formula Pb(CleH17N308)Naz.2.5H20. cadmium chelate made use of cadmium diacetate dihydrate and a pH of 6.7. The yield was 0.52 g (68%). Elemental analysis was consistent with the formula Cd(Cl6Hl7N3O8)Na2.5.5HZ0. In a similar manner, the zinc chelate was prepared from zinc diacetate dihydrate at a pH of 6.3. The yield was 0.35 g (55%). Elemental analysis was consistent with the formula Zn(C16H17N308)Na2. 2.5H20. Elemental analyses were obtained from Galbraith Laboratories, Inc., Knoxville, TN. Liquid Chromatography. The metal chelates were separated by using 0.2 M sodium acetate buffer (pH 6.50) at a flow rate of 1.5 mL/min and the Partisil SAX column. Since fluorescamine can react with primary aromatic amines at this pH, it was not necessary to adjust the effluent pH prior to adding the acetonitrile solution of fluorescamine. The fluorescamine solution was added postcolumn at 0.2 mL/min, and the chart speed was 15 cm/h. Chelation from Solution. For evaluation of the utility of the method for solutions of metal salts, an in situ experiment was performed in which the stock ligand solution was added to the metal salts and the resulting solution of metal chelates which was mol of Zn, 7.1 formed was analyzed. To a solution of 8.2 X X mol of Cd (all as their acetate mol of Pb, and 8.3 X salts) was added an aliquot of a stock solution containing 2.5 X lo4 mol of 4-NH2Ph(EDTA)which had been adjusted to pH 5.5. The solution was diluted to 25.0 mL. A 50-fold dilution was made and the resulting solution injected under the conditions described
32
24
16
8
0
TIME (min)
Separatlon of 4-NH2Ph(EDTA) chelates as detected by fluorescence following postcolumn derivatization with fluorescamlne. Flgure 2.
above. Uncomplexed 4-NHzPh(EDTA)was not eluted from the column within 60 min after injection and did not interfere with the analysis. To remove any 4-NH2Ph(EDTA),the column was washed with water, followed by methanol. RESULTS AND DISCUSSION By use of the fluorescamine the 4-aminochelateswere found to exhibit linear fluorescence detector response over about 4 to 5 X IO-‘ g of metal ion). orders of magnitude (5 X When peak height corrections based on relative k’ values were made (14) the response intensity was found to be essentially uniform (&lo%) on a molar basis for the metal chelates studied. The lack of dependence of relative sensitivity to metal identity was anticipated, due to the remoteness of the amino group responsible for the fluorogenic reaction from the chelation center. As a comparison, the relative sensitivities of Pb, Cd, and Zn on a molar basis determined by atomic absorption are 1:20:40 (15). The chromatogram in Figure 2 illustrates the separation under the conditions described above using a 1 0 - ~ Linjection of a solution which contains 1.2 X lo4 M lead chelate, 7.2 X lo4 M cadmium chelate, and 8.9 X M zinc chelate. On the basis of the amount of metal chelate injected into the liquid chromatograph, detection limits for S / N = 2 were determined to be 80 pg of lead, 80 pg of cadmium, and 60 pg of zinc, or 0.38, 0.71, and 0.92 pmol, respectively. The in situ experiment showed that quantitative recoveries (>98%) of metal ions from dilute solutions are feasible and the resulting chelates are amenable to liquid chromatographic analysis. Other substituted EDTA ligands and a wider variety of metal ions are being studied. These results demonstrate that a method for trace metal determinations using liquid chromatography and fluorescence detection following derivatization is possible. Our technique, using a relatively unsophisticated detector, has extended detection limits for metal ions into the sub-picomole range. This approach would provide multielement, rapid sequential determinations at sensitivities as low as the femtomole (1O-l‘ mol) level. Application of more advanced methods, such as laser-induced fluorescence and phase-sensitive detection (16) could lower detection limits even further. LITERATURE CITED (1) (2) (3) (4) (5) (6)
Jones, D. R. I V ; Manahan, S. E. Anal. Chem. 1976, 48, 1897. Jones, D. R. IV; Manahan, S.E. Anal. Lett. 1973, 8 , 745. Jones, D. R. IV; Manahan, S.E. Anal. Chem. 1976, 48, 502. Jones, D. R. IV; Manahan, S. E. Anal. Leff.1975, 8 , 569. MacDonald, A.; Duke, P. D. J. ChfOm8t0gf. 1973, 83, 331. Small, H.;Stevens, T. S.;Bauman, W. C. Anal. Chem. 1975, 47, 1801.
Anal. Chem. 1981, 53, 911-914 (7) Uden, P. C.; Waiters, F. H. Anal. Chlm. Acta 1975, 79, 175. (8) Gaetani, E.; Laurerl. C. F.; ManQia,A,; Parolari, G. Anal. Chem. 1976, 48, 1725. (9) Uden, P. D.; Parees, D. M.; Waiters, F. H. Anal. Lett. 1975, 8, 795. (IO) Jezorek, J. R.; Freiser, H. Anal. Chem. 1979, 57,373. (1 1) Beckett, J. R. Ph.D. Thesis, University of Wyoming, 1978. (12) Okaku, N.; Toyoda, K.; hloriguchi, Y.; Ueno, K. Bull. Chem. SOC.Jpn. 1967, 40, 2326. (13) Sundberg, M. W.; Meares, C. F.; Goodwln, D A.; Diamanti, C. T. J . Med. Chem. 1974, 17, 1304. (14) Snyder, L. R.; Kirkland, J. J. “Introduction to Modern Liquid Chromatography;” Wiiey: New York, 1979; p 556.
(15) “Atomic Absorption Methods Manual”; Instrumentation Inc.: Wiimington, MA, 1977.
Laboratories,
(16) Diebold, G. J.; Zare, R. N.
911
Science 1977,
1439.
J a m e s R. Beckett David A. Nelson* Department of Chemistry University of Wyoming Laramie, Wyoming 82071 RECEIVED for review May 1 9 3 1980a Accepted 5, Taken in part from the Ph.D. Thesis of J.R.B. (May 1978).
AIDS FOR ANALYTICAL CHEMISTS Organic Matrix Modifiers for Direct Determination of Zinc in Seawater by Graphite Furnace Atomic Absorption Spectrometry Roger Guevremont Atlantic Research Laboratocv, National Resear& Council of Canada, 14 11 Oxford Street, Halifax, Nova Scotia, Canada, B3H 321
Graphite furnace atomic absorption spectrometry (GFAAS) is one of the few analytical techniques which has the sensitivity required for the direct instrumental determination of elements including Cd, Zn, Cu, Mn, Fe, and Co in seawater. However, because of the severity of matrix interference effects observed for seawater, direct analysis by GFAAS is at this time not the method of choice for the routine determination of these elements. Of 36 participating laboratories in an International Council for the Exploration of the Sea (ICES) sponsored intercalibration exercise for the analysis of Cd in seawater carried out during 1979 (2) only two laboratories reported results based on direct GFAAS (using ethylenediaminetetraacetic acid (EDTA) at1 a matrix modifier). On the other hand 8 laboratories reported results by using anodic stripping voltammetry, 22 used complexation-extraction based on ammonium pyrrolidine carbodithioate-methyl isobutyl ketone (APDC/MIBK) followed by atomic absorption spectrometry (AAS), and 3 used coprecipitation preconcentration followed by AAS. In spite of a growing number of reports describing various approaches to selective volatilization (2, 3) and reduction of interference effects using matrix modifiers @-IO), the problems with loss of sensitivity and intense background interference caused by the seawater matrix have only been partly resolved. In recent articles ( I I , I 2 ) we described the application of a number of organic releasing agents to the direct analysis of cadmium in seawater, with estimated lower limits of analysis of 0.01 pg L-l and practical analytical results for samples containing approximately 0.05 pg Cd L-* reported. This work has been extended to the direct determination of zinc in seawater by using citric acid as a matrix modifier. The quantity of reagent, graphite furnace heating programs, and possible sources of interference are described. EXPERIMENTAL SECTION Contamination Control. All operations except the atomic absorption measurement were done in a Class 100 clean room facility. Personnel wore polyethylene gloves while manipulating solutions of low zinc concentration. Sample cups for the autosampler were handled only with cleaned plastic tweezers. No difficulty was encountered in carrying out atomic absorption
measurements outside the clean room. Analysis of low zinc deionized water was done each day before attempting analysis of seawater. Further work was not initiated if poor reproducibility or spuriously high signals were encountered during this test. Reagents. High-purity water was obtained from a Milli-Q (Millipore Corp., Bedford, MA) deionization system. Lower zinc concentrationswere achieved by passing this water through a bed of Chelex-100 ion exchange resin. This low zinc water was stored in conventional polyethylene (CPE) containers which had been washed with HCI, with negligible contamination. Citric acid was “AnalaR reagent grade from British Drug House (BDH) Chemicals Ltd.,Poole, England, further purified by use of Dowex 2-X8 anion exchange resin (J.T. Baker Chemical Go.). This procedure included conversion of the resin to its hydroxide form using “Aristar” ammonium hydroxide (BDH Chemicals Ltd.) followed by thorough washing with Milli-Q water and final rinsing with low zinc water from a Chelex-100 column. The rinse water was checked for zinc by graphite furnace atomic absorption measurements. The anion ion exchange resin was converted to the citrate form with citric acid. Once again the resin was washed until the zinc concentration in the washings was below 0.2 I.cg L-l. The citric acid was eluted from the resin by using hydrochloric acid purified by subboiling distillation. This procedure was repeated until the resin was sufficiently clean to provide a 10 mg mL-l citric acid solution with a zinc content below 0.2 pg Zn L-l; a level necessary to give blanks low enough to measure Zn at a few tenths of a microgram per liter (13). The citric acid solution was stored in acid-washed CPE containers at room temperature. A solution containing approximately 0.1 fig Zn L-l was analyzed 4 weeks after preparation and had not been further contaminated during storage. All containers used for storage or during analysis were conventional polyethylene (CPE) or Teflon, cleaned by a thorough 15-minshaking with subboilingdistilled hydrochloric acid followed by rinsing with water from a Chelex-100 column. Polyethylene sample cups for the AS-1 autosampler were obtained from Evergreen Scientific (Los Angeles, CAI. Equivalent polyethylene sample cups obtained from Perkin-Elmer Corp. were unsuitable for zinc analysis even after the cleaning described above. Pure acids were produced by subboiling distillation in the apparatus shown in Figure 1. This system differs from those used elsewhere (14) in that the vapors are actively transported by air from the heated reservoir to the condenser. The reservoir is Pyrex and the condenser a jacketed Teflon tube. The acid was
0003-2700/81/0353-0911$01.25/00 1981 American Chemical Society
