Liquid Chromatographic Determination of Picomole Quantities of Aromatic Amine Carcinogens 1. Mefford, R. W. Keller, and R. N. Adams' Department of Chemistry, University of Kansas, Lawrence, Kansas 66045
L. A. Sternson Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66045
M. S. Yllo Jackson Laboratories, E. 1. DuPont de Nemours & Company, Wilmington, Del. 19898
The trace level analysis of various aromatic amines and diamines in waste waters and other environmental samples is a matter of practical importance since many of these compounds are classified as carcinogens ( I , 2). The direct analysis of compounds of this nature by differential pulse voltammetry (where no separations are required) was recently reported (3). High performance liquid chromatography (LC) with electrochemical detection has been applied with considerable success to picomole levels of readily oxidized species like catecholamines ( 4 ) . It was evident that such an approach could be applied to the amine carcinogens. The present note shows that the LC analysis can be applied to a variety of such mixtures and provides simple, accurate analyses at the picomole level. It was not the intent of this work to design specific analyses since the workup (extraction, pre-concentration, etc.) will vary with each type of environmental sample.
A 28 p1remol.r
3 5 p1como1e6
1-twphthylamlnc
2-naphthylamine
B VI
c a
s
EXPERIMENTAL The aromatic amines and diamines were reagent grade chemicals used as received. All solutions were prepared from distilled water that had been redistilled from alkaline permanganate. Stock solutions were prepared in 0.1 M perchloric acid that had been thoroughly deoxygenated. The LC equipment, including the design of the electrochemical detector, was laboratory-built and has been described in detail in the literature ( 4 , 5 ) .Similar LC equipment with electrochemicaldetectors is available from Bioanalytical Systems, P.O. Box 2206, West Lafayette, Ind. 47906. Alternatively, an electrochemical detector may be added to the end of the column on most commercial liquid chromatographs. Separations were performed on 25-35 cm glass columns packed with DuPont SCX cation-exchange resin. The detector electrode was pre-set a t +0.8 V vs. SCE which suffices to oxidize any of the compounds mentioned herein.
RESULTS AND DISCUSSION As an example, a mixture of benzidine and o-dianisidine was easily separated on the 25-cm SCX column using 0.1 M ammonium acetate as eluant. A flow rate of 0.35 mL/min was satisfactory. Calibration curves for each component were prepared and were linear over the range 0-10 pmol. Equimolar mixtures of 1-and 2-naphthylamine were readily separated on a 35-cm SCX column using an acetate-citrate buffer, pH 5.2 (0.027 M citric acid, 0.06 M NaOH, 0.05 M sodium acetate, 1.05 mL glacial acetic acid) a t a flow rate of 0.94 mL/min. Calibration curves of each separate component a t even lower levels (0-2 pmol) are linear. Since small amounts of the 2-isomer in commercial samples of the 1-naphthylamine are a matter of practical importance, we were interested to see if the LC method could detect 2naphthylamine in a 1000-fold greater concentration of the 1-naphthylamine. Figure 1 A shows the clean separation of equimolar ratios of the two isomers. The results for a mixture of 1.7 pmol of 2-naphthylamine in 1.7 nmol of l-naphthylamine (0.1%of the 2-isomer) are seen in Figure 1B. No further attempts to optimize the chromatographic conditions were
Figure 1. LC separation of naphthylamine mixtures (A)Chromatogram of ca. equimolar concentrationsof 1- and 2-naphthylamines. SF = solvent front. (B)Chromatogram of 1.7 nmol of I-naphthylamine plus 1.7 pmol of 2-naphthylamine (0.1% of 2-isomer). SF = solvent front. 1-NAPH = 1-naphthylamine. 2-NAPH = 2-naphthylamine
made, but it is obvious that the 2-isomer can be analyzed in this fashion. LC separation coupled with the extreme sensitivity of electrochemical detection can offer a simple and inexpensive approach to the analysis of amine carcinogens. Various extraction or pre-concentration steps are obviously required as in any other type of analysis on environmental samples like waste water, air samples, etc. It should be noted that the graphite paste detector used is not suitable for any chromatographic separations (reverse phase, etc.) which employ more than 15-20% nonaqueous solvents. The use of glassy carbon or platinum electrodes for this purpose may be possible.
LITERATURE CITED (1) C. E. Searle. Chem. Br., 6, 5 (1970). ( 2 ) Fed. Regist., 39,3756 (1974). (3) W. M. Chey. R. N. Adams, and M. S. Yllo, J. Nectroanal. Chem., 75, 75
11977). (4) k. Keiler, A. Oke. I. Mefford,and R. N. Adams, Life Sci., 19, 995 (1976). (5) P. T. Kissinger, C. Refshauge, R. Dreiling, and R. N. Adams, Anal. Lett., 6, 465 (1973).
RECEIVEDfor review October 25,1976. Accepted January 17, 1977. The support of this work by the National Science Foundation via grant MPS75-11330 is gratefully acknowledged. ANALYTICAL CHEMISTRY, VOL. 49, NO. 4, APRIL 1977
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