1531
Anal. Chem. 1981, 5 3 , 1531-1534
Reversed-Phase Liquid Chromatographic Determination of Phenols in Auto Exhaust and Tobacco Smoke as p-Nitrobenzmeazophenol Derivatives Kazuhiro Kuwata, * Michiko Uebori, and Yoshiaki Yamazaki Environmental Pollution Control Center, 62-3, 1 Chome, Nakamlchl, Hlgashinarl-ku, Osaka City 537, Japan
Traces of phenols In auto exhaust and tobacco smoke were collected by using a frlltted bubbler wlth 10 mL of 0.12% sodlum hydroxide soluticin and determlned by reversed-phase high-performance llquldl chromatography via derivatization wlth p-n1trobenrenedia;ronlum tetrafluoroborate In aqueous medlum at pH 11.5. The analytical columns used (20 cm X 4.6 mm 1.d.) were packed with LiChrosorb RP-18 (5 pm) and with Polygosil 60-5C,8, and the mobile phase was 85% methanol/l5% water. Excellent separation of a number of the p-nltrobenrenearo derlvatlves of water-soluble phenols was obtalned. The deitection llmlts of the phenols were 0.05-2.0 ng. Phenol valpors in synthetic air samples, except for p-chlorophenol, could be determined in the low partsper-million or parts-per-billion levels wlth less than 6.5 % of relative standard devlalions.
Phenols have received much attention as photochemical products, and as odorous substances or metabolites in environmental, food, and biological studies. The previous article (I) discusses numerous methods of determining traces of phenolic compounds in such studies and describes a new method of determining traces of phenol in polluted air by reversed-phase high-performance liquid chromatography (LC) via derivatization with p-nitrobenzenediazonium tetrafluoroborate (NBDATFEI) in a weak alkaline aqueous medium. In the method, various phenols are well separated as pnitrobenzeneazo (NBA) derivatives by LC. However, quantitative determination is limited to phenol. Phenolic compounds other than phenol give complex peaks that are difficult to identify. Recently, published are several articles for determination of traces of phenols in polluted water by gas chromatography (2-4) and for determination of phenols in biological sample by LC via derivatization with diazotized p-phenylaniline or 3-metlhyl-2-benzothiazolinone (5)or without derivatization (6, 7). Separations of traces of numerous alkylphenols are achieved by LC without derivatization (8,9). These methods, however, seem to give poor separation for phenol isomers, and effects on the analyses of other organics in complex samples are not discussed in detail. So far, there are few published methods for separately determining trace levels of phenols in polluted gas sample by LC. This article describes an improved method to simultaneously determine a number of water-soluble phenols in low nanogram levels by LC via the derivatization with NBDATFB. Successful applications of the technique for determination of phenols at the low parts-per-million (v/v) or parts-per-billion (v/v) levels in synthetic air samples, auto exhaust, and tobacco smoke are presented.
EXPERIMENTAL SECTION Reagents and Materials. Phenols used as standards were special grade from Wako Pure Chemical Industries (Osaka,Japan) and Tokyo Kasei Kogyo (Tokyo, Japan). The other chemicals, 0003-270O/81/0353-153 1$01.25/0 . ~
the solvents, the 0.1% NBDATFB solution and the buffer solution (0.51% sodium bicarbonate/0.21% sodium carbonate) for pH 11.5 used are previously described ( I ) . The 200-mL stock solutions containing 1000 pg/mL of the phenols were made by dissolving the compounds in 0.5 mL of methanol and adding distilled water. Standards of, lower concentrations were made by appropriately diluting the solutions with distilled water. The sampling solution of the phenol vapors was 0.12% sodium hydroxide aqueous solution. The analytical columns used were 20 cm X 4.6 mm i.d. seamless stainless steel tubes slurry-packed with LiChrosorb RP-18 (5 pm) (E. Merk, Darmstadt, West Germany) and with Polygosil 6O-5Cl8 (Machery-Nagel, Dueren, West Germany). Apparatus. A Water Associates (Milford, MA) ALC/GPC 244 liquid chromatograph equipped with a U6K injector and an ultraviolet (UV) absorbance detector adjusted to 365 nm was employed. The mobile phase was 85% methanol/l5% water, and the flow rate was 1.0-1.3 mL/min. Analytical Procedure. Five to one hundred fifty liters of a gas sample was bubbled at 1-2 L/min through a 30-mL fritted bubbler with 10 mL of the sampling solution. In the case of tobacco smoke, 140 mL of smoke from four puffs were filtered with a Toyo Roshi (Tokyo, Japan) No. 5A paper filter to cut particulate and tar substances off, and bubbled with a carrier of 1L/min of clean air (I, IO). The solution was brought up to 20 mL with distilled water. Five milliliters of the solution was taken in a 15-mL tube, and 1 mL of the buffer solution and 3 mL of the NBDATFB solution were added. After 30 min of standing at room temperature, 1mL of 1% sodium hydroxide solution was added. One milliliter of carbon tetrachloride was added to the sample, and the mixture was shaken and centrifuged. Then 2-40 pL of the aqueous layer was analyzed by LC for the para-unsubstituted phenols, while 2-10 pL of the organic layer was analyzed for the para-substituted phenols. Identification of the phenols was made by retention time on the LiChrosorb RP-18 and the Polygosil 6O-5Cl8 columns, and the quantitation was performed by peak height on either of the columns.
RESULTS AND DISCUSSION Eighteen water-soluble phenolic compounds were derived with NBDATFB in weak alkaline aqueous solutions and the derived products determined by LC. The derivatization conditions for LC analysis were examined as previously described ( I ) . Adjustment of pH for reaction was made by adding the sodium bicarbonate/sodium carbonate buffer to a sample solution containing 0.06 % sodium hydroxide. Optimum pH range for reaction was determined as pH range where maximum UV absorbance for a phenol member of interest was achieved within 15 min after the reaction began. Table I indicates optimum p H ranges of the reaction medium prior to addition of the NBDATFB solution. All of the compounds reacted rapidly with NBDATFB around pH 11.5 in the aqueous medium although the suitable pH ranges for the derivatization were somewhat different for different phenols. The quantitative derivatization was performed with the mole ratio of NBDATFB to phenols being higher than 6:l for all of the compounds. The reaction of p-chlorophenol with NBDATFB was rapid and quantitative but was extremely delayed in the presence of other phenols. The reaction seemed 0 1981 American Chemlcal Society
1532
ANALYTICAL CHEMISTRY, VOL. 53, NO. 9, AUGUST 1981
Table I. Optimum pH Ranges of the Reaction Media for Quantitative Derivatization phenol
PH rangea
phenol
PH range a
o-chlorophenol phenol rn-cresol m-chlorophenol or-naphthol o-cresol rn-ethylphenol 3,5-xylenol o-ethylphenol
10.5-1 1.6 10.5-11.6 10.5-1 2.0 10.5-11.6 10.5-11.6 10.5-12.0 10.5-1 2.5 11.3-1 2.5 10.5-1 2.5
2,5-xylenol 2,6-xylenol 2,3-xylenol p-chlorophenol p-cresol p-ethylphenol 3,4-xylenol 2,4-xylenol
11.3-12.5 11.3-12.5 11.3-12.5 10.5-11.6 10.5-12.0 10.5-11.6 11.5-1 2.0 11.5-12.0
a pH of the reaction medium prior to addition of the NBDATFB solution.
Table 11. Stability of the NBA Derivatives of the Phenols
phenol
concn as phenol: pg/mL
o-chlorophenol phenol rn-cresol rn-chlorophenol a-naphthol o-cresol rn-ethylphenol 3,5-xylenol o-ethylphenol 2,5-xylenol 2,6-xylenoI 2,3-xylenol p-chloro henold p-cresol p-ethylphenold 3,4-xylenold 2,4-xylenol
11.7 2.54 11.3 10.2 11.9 13.3 4.75 9.60 6.75 8.99 4.18 9.28 17.8 3.83 5.12 4.86 7.46
2
absorbance,? X 4 OdayC daysC
7 daysC
0.402 0.880 0.732 0.507 0.398 0.732 0.518 0.597 0.536 0.392 0.300 0.333 0.223 0.253 0.282 0.349 0.232
0.464 0.887 0.731 0.524 0.314 0.731 0.500 0.552 0.523 0.354 0.274 0.299 0.227 0.246 0.287 0.330 0.227
0.461 0.880 0.710 0.500 0.373 0.710 0.496 0.579 0.515 0.374 0.310 0.321 0.203 0.237 0.262 0.302 0.214
Table 111. Retention Times of the NBA Derivatives of the Phenols on the Analytical Columns retention time, min LiChrosorb Polygosil RP-18a 60-5C,,b
phenol o-chlorophenol phenol rn-cresol rn-chlorophenol or-naphthol o-cresol m-ethylphenol 3,5-xylenol o-ethylphenol 2,5-xylenol 2,6-xylenol 2,3-xylenol p-chlorophenol p-cresol p-ethylphenol 0-naphthol 3,4-xylenol 2,4-xylenol
3.46 3.89 4.60 4.72 4.94 4.97 5.09 5.58 5.70 5.89 6.16 6.18 6.66 7.34 8.79 9.59 10.81 12.68
3.60 4.44 5.20 5.00 5.94 5.54 6.00 6.24 6.42 6.60 6.42 6.70 7.20 7.66 9.08 9.90 10.82 12.16
a Column pressure, 2000 psi; flow rate of the mobile phase, 1.3 mL/min. Column pressure, 1500 psi; flow rate of the mobile phase, 1.1 mL/min.
a Concentration of phenol in an aqueous sample prior to derivatization.. Calculated from peak height for 2 L of a final sample injected. Storage time (at 25 "C). Ct*Absorbance was determined by using the organic layer.
to occur after the other phenols reacted. An excessive amount of NBDATFB was required to accelerate the reaction. @Naphthol reacted with NBDATFB but could not be accurately determined for the derivatization products. Addition of 0.1% of sodium hydroxide to the aqueous medium was useful to stabilize the NBA derivatives of the phenols while still ensuring protection of the analytical columns. Table I1 shows that the UV absorbance of the derivatives remained constant at least for 4-7 days although the absorbance for o-chlorophenol and rn-chlorophenol tended to increase with time. The analytical columns packed with LiChrosorb RP-18 (5 pm) and with Polygosil 6O-5Cl8 offered 11600 and 12500 theoretical plates (17.2 and 16.2 pm of HETP), respectively, for phenol. Table I11 indicates retention times of the NBA derivatives of the phenols on the columns. The relative standard deviations of the retention times on the columns were less than 0.6% more than on a day when the analytical conditions were not changed. Good separations of a number of the NBA derivatives were obtained on the columns. Figure 1shows typical liquid chromatograms of the NBA derivatives of the phenols on the LiChrosorb RP-18 column. Separation aspects on the two columns were similar. The LiChrosorb column offered overlapped elutions for 2,3-xylenol and 2,6xylenol and a separate peak for 2,5-xylenol,while the Polygosil column gave overlapped peaks of 2,5-xylenol and 2,g-xylenol and a separate peak for 2,3-xylenol from the two compounds.
I
0
I
10 Retention time, rnin
5
I
15
Flgure 1. Liquid chromatograms of the NBA derivatives of the phenols. Conditions: column, LiChrosorb RP-18 (20 cm X 4.6 mm id.); mobile phase, 85% methanol/l5% water; flow rate, 1.3 mL/min; pressure, 2000 psi; phenols InJected,10-40 ng. Peak identlty: (1) o-chlorophenol, (2) phenol, (3) m-cresol, (4) m-chlorophenol, (5)a-naphthol, (6) o-cresol, (7) m-ethylphenol, (8) 3,5-xylenol, (9) o-ethylphenoi, (10) 2,5-xylenol, (1 1) 28-, 2,3-xylenol, (12) p-chlorophenol, (13) p-cresol, (14) p-ethylphenol, (15) P-naphthol, (16) 3,4-xylenol, (17) 2,4-xylenol.
On the other hand, the Polygosil column offered a complete separation between o-cresol and rn-ethylphenol that the LiChrosorb column could not produce. The two columns offered a very similar performance for quantitative determination of the phenols. The analysis of the phenols via the derivatization offered two advantages over LC analysis without the derivatization. One was the increased sensitivities by 1&30 times and another was the reduced effects of other organic compounds. Table IV indicates the detection limits (a response to twice the noise level) and the linear calibration ranges for the phenols. Because the NBA derivatives of para-substituted phenols, such
ANALYTICAL CHEMISTRY, VOL. 53,
Table IV.
NO. 9, AUGUST 1981
determination
phenol o-chlorophenol phenol m-cresol m-chlorophenol or-naphthol o-cresol m-ethylphenol 3,5-xylenol o-ethylphenol 2,5-xylenol 2,6-xylenol 2,3-xylenol p-chlorophenolb p-cresol p-ethylphenol 3,4-xylenol 2,4-xylenol
Ranges of the Phenols linear range,a pg/mL detection limit, ng min max 0.5 0.05 0.1 0.5 0.5 0.1 0.2 0.2 0.3 0.2 0.2 0.2 2.0
1.00
0.5 1.0
0.50 0.50 1.00 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 4.00 0.50 2.00
0.5 0.5
1.00 1.00
200 160 80 200 140 80 160 120 160
120 160 120 16OC( 4 0 d ) 160' ( 2 0 d ) 16OC( 4 0 d ) 160' (lod) 160' (lod)
Concentration of phenol in an aqueous sample prior to derivatization; 2 pL of a final sample were analyzed on Examined for samples the LiChrosorb RP-18 column. Calibrated by using containing p-chloro henol only. Calibrated by using the aqueous the organic layer. medium. a
as p-chlorophenol, p-cresol, p-ethylphenol, 3,4-xylenol, and 2,4-xylenol, had poor water solubility, determination of the phenols by using the aiqueous medium was limited to low concentration levels. However, when the aqueous medium was shaken with carbon tetrachloride after addition of 0.1% of sodium hydroxide, tlhe NBA derivatives of the para-substituted phenols were completely extracted into the organic layer while the NBA derivatives of the para-unsubstituted phenols remained in the alkaline aqueous layer and were not
t
I
1
1
0
5
10
15
Retention time, min Flgure 2. Effects of organic substances on determination of phenols in tobacco smoke (Peace). Condltlons: column, Polygosil 6O-5Cl8 (20 cm X 4.6 mm i-d.); mobile phase, 85% methanol/lb% water: flow rate, 1.1 mL/min; pressure, 1500 psi. Aqueous medium before extraction (BE-AM), (1) phenol, (2) m-cresol, (3) o-cresol, (4) o-ethylphenol, 2,6-xylenol, (5) p-cresol; extraction-aqueous layer (E-AL), (1) phenol (2.40 ng), (2) m-cresol (0.29 ng), (3) 0-cresol (0.25 ng), (4) o-ethylphenol, 2,&xylenol; extraction organic hyer (E-OL), (5) p-cresol (1.47 ng), (6)3,4-xylenol, (7) 2,4-xylenoL
-
detected in the organic layer. Thus, calibrations of the para-substituted phenols could be made by using the organic layer, and their linear calibration ranges were in the same levels as those of the para-unsubstituted phenols obtained by using the aqueous layer. The relative standard deviations of
Table V. Determinati& of the Phenols in Synthetic Air Samples phenol
sample 1
concn of phenola sample 2
t
std dev,b ppm (v/v) (RSD,' %) sample 3 sample 4
o-chlorophenol phenol m-cresol m-chlorophenol o-cresol m-ethylphenol 3,5-xylenol o-ethylphenol 2,5-xylenol
0.451 f 0.0120 (2.7) 0.881 t 0.0469 (5.3) 1.01 t 0.034 (3.4) 0.612 ?: 0.0215 (3.5) 0.905 t 0.0305 (3.4)
2,3-xylenol p-chlorophenol p-cresol p-ethylphenol 3,4-xylenol 2,4-xylenol
a
1533
2.59 t 0.089 (3.4) 1.91 i 0.101 (5.3) 1.07 t 0.019 (1.8)
2.87 f 0.081 (2.8) Average conceiitration of phenol in 6 runs.
0.501 f 0.0185 (3.7)
0.524 i 0.0183 (3.5) 0.768 t 0.0227 (3.0) 0.794 i 0.0179 (2.3)
3.01 f 0.152 (5.0) 2.57 i 0.080 (3.1) 2.53 i 0.095 (3.8) 1.74 i 0.089 (5.1) 2.11 i 0.047 (2.2)
5.74 f 0.284 (4.9)
3.78 i 0.243 (6.4)
2.59 f 0.097 (3.7) 2.71 i 0.121 (4.5) 2.24 t 0.130 (5.8)
sample 5
27.3 i 0.96 (3.5) 16.0 f 0.54 (3.4) 18.8 i 0.43 (2.3) 8.49 i 0.382 (4.5)
14.1 t 0.29 (2.1)
6.87 i 1.157 1.20 i 0.172 (14.3) (16.8) 3.11 c 0.112 5.08 t 0.213 (3.6) (4.2) 1.86 i 0.121 1.69 i 0.061 (6.5) (3.6) 1.42 f 0.068 1.21 i. 0.067 (4.8) (5.5) 0.636 f 0.0401 3.03 t 0.167 (6.3) (5.5) Std dev, standard deviation. RSD, relative standard deviation.
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 9, AUGUST 1981 I/
1
Table VI. Phenols in Auto Exhaust and Tobacco Smoke phenol found cresol
Pcresol
Auto Exhaust,a ppm (v/v) Toyota Engine Model HMS87V (1977) no. 1 1.46 0.332 0.231 no. 2 1.24 0.310 0.210 no. 3 1.28 0.263 0.168
NDb ND ND
Tobacco Smoke,c pg/cigarette Hi-lite (Japan) 520 47.5 36.1 Seven-star (Japan) 314 51.7 34.5 Cherry (Japan) 503 42.4 35.2 Peace(Japan) 481 58.1 49.7
49.4 37.4 60.8 59.1
sample
'111
'1
(T -AL)
a Sample volume was 20 L. Average value in three runs.
0
5 10 Retention time, rnin
15
Flgure 3. Analysls of phenols from polluted gases. Conditions, see Flgure 1. Aqueous layer of an auto exhaust sample (A-AL): (1) phenol (9.17 ng), 1.46 ppm; (2) rn-cresol (2.32 ng), 0.322 ppm; (3) o-cresol (1.76 ng), 0.231 ppm; (4) 2,6-, 2,3-xylenol (less than 0.2 ng). Organic layer of the auto exhaust sample (A-OL): (5) p-cresol (less than 0.5 ng). Aqueous layer of a tobacco smoke sample (T-AL) (Cherry): (1) phenol (2.45 ng), 490 pglcigarette; (2) rn-cresol (0.22 ng), 44.4 pglcigarette; (3) o-cresol (0.16 ng), 32.3 Fgkigarette; (4) 2,6-, 2,3xylenol (less than 0.2 ng). Organic layer of the tobacco smoke sample (T-OL) (Cherry): (5) p-cresol(l.12 ng), 60.8 pglcigarette; (6) 3,4-xylenol (less than 0.5 ng); (7) 2,4-xylenol (less than 0.5 ng).
determined values of p-chlorophenol and the other phenols for standard samples were 4.0-6.9% and 1.2-5.6%, respectively, in the linear ranges. The relative standard deviations near the detection limits were 12-15%. Effects on the analysis of background peaks caused by organics in polluted gas were much reduced compared to ones previously reported (I). Even in the case of an extremely polluted gas such as tobacco smoke, background effects were minimized by the extraction technique. Figure 2 shows that unknown organics which appeared around the phenol, rn-cresol and o-cresol peaks were transferred into the organic layer while little background peaks were observed a t the p-cresol peak in the organic layer. For investigation on the collection efficiency in the sampling system and on the accuracy of analysis of gas samples, 50 L of air samples containing 0.4-28 ppm of the phenol vapors were prepared in polyester bags by vaporizing the phenols dissolved in 0.3 mL of methanol a t 80 "C for 1h. Five liters of the synthetic samples were sampled a t 25 "C three times a t 1 L/min and three times at 2 L/min through two fritted . bubblers with 10 mL of the sampling solution (six runs in total), and analyzed by the method presented. Stable vapor of a-naphthol was not obtained because of its low vapor pressure. The phenols of interest were completely trapped in the first bubbler and not detected in the second bubbler.
m-
phenol cresol
0-
ND, not detectable.
Table V reports that the phenol vapors, except for p-chlorophenol, can be determined in the low parts-per-million or parts-per-billionlevels with less than 6.5% of relative standard deviations. Phenols in auto exhaust and tobacco smoke were determined. For auto exhaust, 20 L of gas was directly sampled from the exhaust vent of the engine operated a t 2000 rpm. Identification of the phenols was performed by using the two columns and by changing methanol percentages in the mobile phase from 80 to 90%. Table VI reports analytical data of the polluted gases, and Figure 3 shows typical liquid chromatograms of the NBA derivatives of phenols from the sources. Cresols, other than phenol, were determined in the gases. Other phenolic compounds were difficult to identify or to quantitate because of the limited analytical sensitivities. In closing, it is suggested that the technique by using LC might be applicable to determination of phenols in various samples other than polluted airs.
ACKNOWLEDGMENT Authors cordially thank K. Negoro, Faculty of Engineering, Hiroshima University, for his continuous encouragement and advice in the study.
LITERATURE CITED Kuwata, K.; Uebori, M.; Yamazaki, Y. Anal. Chem. 1980, 52, 857-880.
Prater, W. A.; Slmmons, M. S.; Mancy, K. H. Anal. Lett. 1980, 73, 205-212.
Voznakova, 2.; Popl, M. J . Chromatogr. Sci. 1979, 77, 682-686. Coutts, R. T.; Hargesheimer, E. E.; Pasutto, F. M. J . Chromafogr. 1878, 179, 291-299.
Walter, W. M., Jr.; Purcell, A. E. J . Agrlc. food Chem. 1979, 27, 942-946.
Walter, W. M., Jr.; Purcell, A. E.; McCollum, G. K.
J . Agric. Food Chem. 1979, 27, 938-941. Needham, L. L.; Hill, R. H., Jr.; Sirmans, S. L. Analyst(London)1980, 105, 811-813. Shabron, J. F.; Hutubise, R. J.; Silver, H. F. Anal. Chem. 1979, 51, 1426- 1433.
Organ, K.;-Katz, E. Anal. Chem. 1981, 53, 160-163. Kuwata, K.; Uebori, M.; Yamazaki, Y. J . Chromatogr. Sci. 1979, 17, 284-288.
RECEIVED for review March 2,1981. Accepted May 6,1981.
