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Anal. Chem. 1984, 56, 1525-1527
Liquid Chromatography of Phenolic Compounds on a Microbore Anion Exchange Resin Column Krishnat P. Naikwadi, Souji Rokushika,* and Hiroyuki Hatano Department of Chemistry, Faculty of Science, Kyoto University, Sakyo-ku, Kyoto 606, Japan Separation and identification of phenols are of great importance owing to their wide applicability in pharmaceuticals, dyes, pesticides, and foods. Phenols have also been recognized as a major source of pollutants. They are introduced into the environment through the discharge of industrial wastes and the decomposition of pesticides, dyes, and herbicides. Certain substituted phenols are potentially hazardous to aquatic animals as well as to human beings. The separation of positional isomers of substituted phenols by using conventional columns with polar or nonpolar stationary phases by gas chromatography is very difficult because of the high polarity of phenolic compounds and the decomposition or oxidation of some phenols at high temperature. Better separation of isomers can be obtained with derivatives rather than with free phenols (1-4); however, the process of derivatization makes the analytical procedure lengthy. Thin-layer chromatography (5,6),paper chromatography (7, 8),and ring oven techniques (9) have been also used for the analysis of phenols. Recently, a comprehensive review on the determination of phenols by GC and HPLC has been published (IO),and the literature regarding the determination of phenols by TLC, PC, GC and HPLC from 1972 to 1979 is reviewed in a book (11). High-performance liquid chromatography provides a reproducible, sensitive, quantitative, and qualitative method for the separation and determination of phenolic compounds. Both reversed-phase and normal-phase modes have been investigated extensively (12-15). Most of the phenolic compounds interact with anion exchange resin and hence their separation may be achieved on an anion exchange column (16, 17). Recently low capacity anion exchange resins have been developed for ion chromatography, and conventional columns packed with these resins have been applied to the separation of inorganic and organic anions (18-20). The application of microbore packed columns to ion exchange chromatography has been demonstrated, with excellent performance, for very small amounts of sample, eluent, and packing materials (21,22). This paper described the applicability of the microbore packed anion exchange resin column to the separation of various isomeric phenolic compounds.
EXPERIMENTAL SECTION
All phenolic samples and chemicals were obtained from Nakarai Chemicals, Ltd., Kyoto and Tokyo Kasei Kogyo Co., Ltd., Tokyo and used without further purification. Fused silica tubing was obtained from Scientific Glass (North Melbourne, Australia). The packing material used was anion exchange resin TSK-gel IC anion PW (particle diameter 9 & 1 ym, ion exchange capacity 30 yequiv/g) which was developed for the ion chromatography by Toyo Soda Co., Tokyo. The instrument was constructed by assembling a Milton Roy minipump for eluent delivery, a Model PG 350 D pressure gage with pulse damper, a Model ML 422 microinjector whose sample volume was fixed at about 0.05 yL, and a UVIDEC 100 UV detector (Japan Spectroscopic Co., Tokyo). The anion exchange resin was packed into a 45 cm x 0.19 mm i.d. fused silica tubing and used as a column. The column packing method along with the construction of the column and detector flow cell has been previously reported (21). Standard solutions of 10 mM of each phenolic compound were prepared by dissolving appropriate quantities of phenols in 5 mL of acetonitrile. Phosphate buffer and carbonate buffer eluents were prepared by dissolving sodium dihydrogen phosphate or sodium hydrogen carbonate in water, respectively. The pH was
adjusted by adding 0.1 M NaOH solution. The appropriate amounts of ethanol or acetonitrile were added to the buffers and used as eluents. The pH of the eluent was measured after mixing the buffer with organic modifiers.
RESULTS AND DISCUSSION To study the effect of organic solvents on the retention behavior of phenolic compounds, carbonate buffers with 50% acetonitrile or 50% ethanol were employed. The results are summarized in Table I. Capacity factors for phenolic compounds using carbonate buffer with ethanol were higher when compared with those obtained with acetonitrile. Under these conditions, acetonitrile may prevent, more effectively than ethanol, hydrophobic interactions between the hydrophobic regions of the resin matrix and the phenolic compounds. The pH of the eluent has a profound effect on the retention and the separation of positional isomers of substituted phenols. The retention behavior of phenolic compounds depends upon the type of the group substituted on the phenolic ring. Phenoxy1 anion formation will be more pronounced when a ring deactivating group is present on the phenolic ring. This can be seen from the results of methylphenols, chlorophenols, and nitrophenols, where the capacity factors are in the order of the deactivation effects of the substituted groups, i.e., NOz > C1 > CH3 a t pH 10.2. The k’ values for all phenolic compounds increase with eluent pH up to their maximum dissociation owing to the increasing negative charge which resulted in more and more interaction with the anion exchange resin. After the complete dissociation of the phenolic group is attained, the increase in pH of the eluent effected a decrease in k’values, which can be seen for nitro and chloro derivatives of phenol. However, the k’ values for the compounds with a higher pK, such as methylphenols, naphthols, and aminophenols, have not decreased with an eluent p H examined here. The effect of lower eluent pH on the retention behavior of various phenolic compounds was also studied by using phosphate eluent and summarized in Table I. The capacity factor and selectivity of all phenolic compounds were affected by the concentration of organic modifiers which reduced the retention of all phenolic compounds; however, the extent of the effect was dependent on the structure of the solute molecules. The optimum concentration of the organic modifier for the maximum selectivity of positional isomers depended on the pH of the eluent and the pK values of phenolic isomers. The logarithm of the k’ value of nitrophenols was plotted against the volume percentage of ethanol in the phosphate buffer as shown in Figure 1. At lower concentrations of ethanol, complete resolution of positional isomers were observed, but required a long retention time, and peaks were asymmetrical, a condition for optimum resolution with symmetrical peaks can be obtained by increasing concentration of ethanol. The linear relationship between the log k’ and the concentation of the organic modifiers was usually observed in the reversed-phase partition chromatography on the C18-modified silica gel column. The present results may also be explained by the reversed-phase partition effect through the hydrophobic interactions between the hydrophobic region of the gel matrix and the undissociated solute phenolic molecules (23,24). The slight deviation from the linear curves may be due to the complex effects such as hydrogen bonding, structural change,
0003-2700/84/0356-1525$01.50/0 0 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 8, JULY 1984
Table I. Capacity Factor k’ of Phenolic Compounds at Various Eluents
compound p-aminophenol m-aminophenol o-aminophenol p-chlorophenol m-chlorophenol o-chlorophenol p-hydroxybenzoic acid m-hydroxybenzoic acid o-hydroxybenzoic acid p-methylphenol m-methylphenol o-methylphenol 01 -naphthol @-naphthol p-nitrophenol m-nitrophenol o-nitrophenol phenol p-hydroxyphenol m-hydroxyphenol o-hydroxyphenol 2,5-dihydroxytoluene 3,5-dihydroxytoluene 3,4-dihydroxytoluene 1.0
0.1 M carbonate buffer with 50% ethanol at pH 10.2 11.6 12.6 1.50 2.66 3.30 1.91 2.66 3.30 3.00 2.16 3.30 3.08 5.08 4.75 4.58 5.83 5.33 5.50 6.25 5.50 6.83 10.25 7.00 4.58 7.00 7.33 3.58 3.66 3.00 1.66 2.58 3.00 1.58 2.75 3.16 1.58 2.75 3.25 2.16 3.50 5.25 3.50 7.33 6.16 7.41 4.41 5.00 6.66 4.58 5.25 4.16 4.66 5.33 2.00 3.50 3.16
PK, 9.87, 9.92 9.71 9.38 9.08 8.48 4.61, 9.31 3.90, 9.78 2.75, 12.38 10.26 10.09 10.28 9.30 9.57 7.16 8.38 7.21 9.99 10.35 9.44 9.48 9.05, 11.62
0.1 M carbonate buffer with 50% acetonitrile at pH 10.2 11.6 12.6
0.66 0.83 1.00 2.16 3.00 3.33 5.25 3.08 2.08 0.75 0.91 0.83 1.58 1.83 3.79 3.35 3.35 1.08
1.29 1.60 1.70 3.37 4.00 4.30 8.47 5.25 2.64 1.39 1.50 1.39 2.85 3.37 3.10 3.10 2.68 1.80
1.50 2.00 2.00 3.68 3.90 3.90 6.14 5.25 2.23 2.15 2.23 2.00 3.46 4.13 3.08 3.08 2.83 2.56
1
0.1 M phosphate buffer with 40% ethanol at pH
5.4 0.38 0.72 0.86 2.04 2.08 1.83 1.39 1.91 5.97 1.25 1.15 1.25 0.25 2.33 2.33 2.01 2.22 1.18 0.75 0.86 1.07 0.72 0.86 1.07
6.4 0.69 0.97 1.15 2.00 2.38 1.82 2.29 2.70 5.68 1.33 1.41 1.41
1.05 2.95 2.75 1.91 2.29 1.25 0.60 0.88 0.97 0.97 1.15 1.35
7.4
0.16 0.41 0.58 2.12 2.39 2.21 2.48 2.48 3.91 1.50 1.58 1.58 1.05 2.75 4.62 2.48 3.64 1.23 0.87 1.14 1.32 1.05 1.23 1.41
5 1 2
0.8 0.6 x
0.4
I I I
,”
0.2
0
- 0.2 I.
30
,
‘
40
,
.
50
,
I
60
,
*le
Ethanol concentration Effect of concentration of ethanol in phosphate buffer on p -nitrophenol; (0)rn-nitrophenol; log k’values of nitrophenols: (01, (A)o-nitrophenol. The pH values were adjusted to 7.4 after mlxing ethanol. Figure 1.
or solvation of the solute and the gel on the retention mechanisms. At lower pH, since the dissociation of the phenolic-OH group is suppressed, it was observed that the effect of eluent pH on k’was less than that at higher pH. The retention of each compound was lower and selectivities depended mainly on the properties of the substituted group and its position in the phenolic ring. At higher pH, however, the phenolic-OH group is dissociated resulting in large retentions for phenolic compounds on the anion exchange column. Also, the sensitivity of the UV detector is much greater for these species than undhociated compounds. The contribution of the substituted group on selectivity was much less than in the lower pH eluent range due to the large ionic interaction of the phenoxy1 ion with the anion exchange resin. T o obtain separation of a mixture of various phenolic compounds with high selectivity in a short time, it was not advantageous to use a pH higher than the pK of the particular phenolic -OH group. The separation of 12 phenolic compounds was obtained by using a phosphate buffer with acetonitrile as an organic
40
i0 20 T i me ( m i n )
10
i,
Flgure 2. Chromatogram of a mixture of phenolic compounds: column size, 45 cm X 0.19 mm i.d.; packing material, TSK-gel-IC anion PW anion exchange resin; eluent, 0.1 M phosphate buffer with 20% acetonitrile, pH 4.9; flow rate, 2.6 pL/min; temperature, 40 O C ; detection, absorbance at 220 nm. Peaks are as follows: (1) p-aminophenol; (2) rn-amlnophenol; (3) p -hydroxyphenol; (4) 2,5-dihydroxytoluene; (5) m-hydroxyphenol; (6) 3,5-dihydroxytoluene; (7) m methylphenol;(8)rn-nitrophenol; (9)o-nitrophenob (IO)o-chlorophenol; (11) p-chlorophenol; (12) m-chlorophenol.
modifier a t a lower pH as shown in Figure 2. The newly developed low capacity anion exchange resin column worked well and provided superior selectivity for the separation of various isomeric phenolic compounds when compared with the results from a different type low capacity ion exchhange resin which has all ion exchange sites near the surface of the hydrophobic resin beads. The present anion exchange resin is composed of a totally porous gel matrix of a polyacrylamide which includes hydrophilic sites and ion
Anal. Chem. 1984, 56, 1527-1530
exchange groups are introduced uniformly, this structure may be worked advantageously to separate the isomeric compounds. In a recently published review dealing with the separation of phenols (IO), it is mentioned that anion exchange chromatography is rarely used for separation of phenols because of the difficulty in controlling the pH of the eluent and because of the instability of anion exchange columns. In the present work, we did not encounter such problems. Microbore packed anion exchange columns were operated for a year at pH values from 4 to 13 with various organic modifiers without damage to the column. Excellent reproducibility of the results reported in this paper was obtained. It should be emphasized that a microbore column requires only a very small amount of eluent and, once an eluent is prepared at a desired pH and concentrations of buffer and modifier, it can be used for a long time. For example, 1to 2 mL of eluent was sufficient for an entire day of operation. Registry No. 1,123-30-8;2, 591-27-5; 3, 123-31-9;4,95-71-6; 5, 108-46-3;6, 504-15-4; 7, 108-39-4;8, 554-84-7; 9, 88-75-5; 10, 95-57-8; 11, 106-48-9; 12, 108-43-0; o-aminophenol, 95-55-6; phydroxybenzoic acid, 99-96-7; m-hydroxybenzoic acid, 99-06-9; o-hydroxybenzoic acid, 69-72-7; p-methylphenol, 106-44-5; omethylphenol, 95-48-7;&-naphthol,90-15-3;@-naphthol,135-19-3; p-nitrophenol, 100-02-7; phenol, 108-95-2; o-hydroxyphenol, 120-80-9;3,4-dihydroxytoluene, 452-86-8.
Kuwata, K.; Uebori, M.; Yamazaki, Y. Anal. Chem. 1980, 52, 857-860. Kuwata, K.; Uebori, M.; Yamazaki, Y. Anal. Chem. 1981, 53, 1531-1534. Lepri, L.; Desideri, P. G.; Landini, M.; Tanturli, G. J . Chromatogr. 1976, 729,239-248. Sattar, M. A.; Paasivirta, J.; Vesterinen, R.; Knuutiner, J. J . Chromatogr. 1977, 736,379-384. Rawat, J. P.;Mujitaba, S. G.; Thind, P. S. Z . Anal. Chem. 1976, 279, 360. Matyslk, G.;Soczewinskl, E. J . Chromatogr. 1978, 760, 29-47. Buckman, N. G.; Hill, J. 0.;Magee, R.J. Ana/yst (London) 1983, 708, 573-580. Tesarova, E.;PacBkovB, V. Chfomatographia 1983, 77, 269-281. Hanai, T. “CRC Handbook of Chromatography; Phenols and Organic Acids, Volume I”; CRC Press: Boca Raton, FL, 1982. Ogan, K.; Katz, E. Anal. Chem. 1981, 53, 160-163. Realini, P. A. J . Chromatogr. Sci. 1981, 79, 124-129. Schabron, J. F.; Hurtubise, R. J.; Silver, H. F. Anal. Chem. 1978, 50, 1911-1917. Schabron, J. F.; Hurtublse, R. J.; Silver, H. F. Anal. Chem. 1979, 57, 1426-1433. Jandera. P.; ChuriBk, J.; &slaOsky, J.; SzabB, D. Chromatographla 1981, 74, 100-106. Nilsson, B. F.; Samuelson, 0. J . Chromatogr. 1980, 198,267-278. Okada, T.; Kuwamoto, T. Anal. Chem. 1983, 55, 1001-1004. Rokushika, S.; Sun, 2. L.; Hatano, H. J . Chromatogr. 1982, 253, 87-94. Naikwadi, K. P.; Rokushika, S.; Hatano, H. J . Chromatogr. 1983, 280, 261-269. Rokushika, S.; Qiu, 2. Y.; Hatano, H. J . Chromatogr. 1983, 260, 81-87. Rokushika, S.; Qiu, 2. Y.; Sun, 2. L.; Hatano, H. J . Chromatogr. 1983, 280,69-76. Nahum, A.; Horvhth, C. J . Chromatogr. 1981, 203, 53-63. BiJ, K. E.; Horvith, C.; Melander, W. R.; Nahum, A. J . Chromatogr. 1981, 203,65-84.
LITERATURE C I T E D (1) Cook, L. E.; Spangelo, R. C. Anal. Chem. 1974, 46, 122-126. (2) Coutts, R. T.; Hargesheimer, E. E.; Pasutto, F. M. J . Chromatogr. 1979, 179,291-299.
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RECEIVED for review November 21, 1983. Accepted March 2, 1984.
Determination of Selenium by Liquid Chromatography with Spectrofluorimetric Detection Yasuyuki Shibata,* Masatoshi Morita, a n d Keiichiro F u w a Nation41 Institute for Environmental Studies, 16-2 Onogawa, Yatabe, Tsukuba, Ibaragi 305, J a p a n Analysis for selenium is of great importance for health concerns. Selenium has been recognized as an essential element. Its deficiency may be implicated in the development of several diseases (1-3). Various analytical methods have been developed and applied to this field. Some of them show fairly good detection limits, i.e., 22-100 pg at present (4-7). These detection limits, however, do not seem to be low enough for certain cases of interest, e.g., biochemical studies of selenium compounds or determination of selenium in a water sample of limited size. Among these methods, spectrofluorimetric analysis is a commonly used method for the determination of selenium, 2,3-diaminonaphthalene (DAN) being the fluorescent reagent that gives the best results (8). During the analysis of biological samples by this method, we noticed that the detection limit of the method was not restrained by the poor sensitivity of the spectrofluorimeter but rather restrained by the fluctuation of the blank values. Recently, several workers (9-11) employed a thin-layer chromatographic separation of selenium-DAN complex and observed that the chromatographic separation worked for removing several chemical species which appeared in the reaction and interfered in the fluorimetric determination by raising the blank level. High-performance liquid chromatography (HPLC) should be another choice for the separation of the selenium-DAN complex. Presented here is an extremely sensitive analytical method for selenium using 0003-2700/84/0356-1527$0 1.50/0
HPLC and an optimized fluorescence detection system. The detection limit for the pure selenium-DAN complex reached the femtogram range. EXPERIMENTAL SECTION Apparatus and Instrumentation. HPLC was performed by a Waters HPLC system equipped with a WBondapak C18 (4.6 mm i.d. X 300 mm; Waters Limited) or a UNISIL 5C18 (4.6 mm i.d. X 250 mm; Gasukuro Kogyo Inc.) column. Oxygen in acetonitrile was purged by bubbling N2 gas. Fluorescence spectra were recorded with Hitachi 650-10s fluorescence spectrophotometer. Reagents. DAN, EDTA, and cyclohexane (Luminazolegrade) were purchased from Dojindo Lab. Selenious acid and hydrochloric acid (EL-SS grade) were products of Kanto Chemical Co. Acetonitrile (special grade for LC) was obtained from Wako Pure Chemicals. Water was distilled and deionized by a Millipore Q water purification system and finally doubly distilled by an all-quartz glass distiller. Naphtho[2,3-c][1,2,5]selenadiazole(NSD) was synthesized according to Parker and Harvey (8). A commercial standard solution for atomic absorption analysis (Kanto Chemical Co., 0.16% solution of selenious acid) was used as a stock solution of selenium(1V). Chelation and Extraction Procedure. The procedure was essentially the same as that reported previously (12,13). A 1-mL volume of the sample solution was poured into a Pyrex glass tube with 0.5 mL of 0.1 M EDTA solution. The total volume was made to 4 mL by adding 0.1 N HCl. After the solution was warmed for a short time at 50 “C, 1 mL of 0.1% DAN solution (in 0.1 N 0 1984 American Chemical Society
