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 chromatographic unit consisting of an Altex Pump (Model 100A), a variable-wavelengthUV-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 chromatographic separations 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 - ~ L loop 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 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
5.7 10.5 13.1
a Mobile phase 85:15 H,O/MeOH (v/v) with (methylene blue).
K
0.9 1.6 1.9 3.5 4.4 5.8 1.7 4.0
5.1
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 (methyleneblue) 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
