Anal. Chem. 1990, 62, 1587-1591
50
1
0
5
10
1’5
20
i5
30
35
1/v (sec/cm)
Flgure 5. Peak area by UV detection for acetophenone as a function of inverse solute velocity.
first injection, suggesting that buffer mixing is not occurring. Apparently, the equilibration of the surface charge on the fused silica surface is a relatively slow phenomena. In fact, the process may take several weeks a t intermediate pH. In Figure 4, the electroosmotic mobility is shown for a pH 4 buffer as a function of the column equilibration time (initially at pH 12). During this experiment, the buffer reservoirs were replenished on a daily basis. The electroosmotic mobility varies by approximately $fold over a period of 2 weeks. On the other hand, reequilibration to a p H where the surface becomes either fully ionized or fully un-ionized appears to be nearly instantaneous. The kinetics of the surface charge equilibration suggests that those investigators developing analytical CZE assays should use caution in the pH 4-6 region. This is particularly true for quantitative purposes, since the peak area ( A )will vary inversely with the solute velocity (v = p,V/L) as follows:
A = kn/u
(2)
where k is a constant dependent on the system and solute absorptivity and n is the amount of solute injected. This relationship is shown for acetophenone under various con-
1507
ditions in Figure 5. Thw, a small change in the electroosmotic flow may have dramatic effects on the peak area. This change may occur under constant field strength due to hysteresis. This is unlike standard chromatographic techniques where the area is constant under constant flow conditions, even though the retention time may vary. For quantitative analyses, it has been suggested that CZE peak arem be normalized by multiplying the peak area by the inverse migration time (3)or by the zone velocity ( 4 ) . These methods are equivalent and assume that the zone concentration profile is symmetrical for all examined peaks. The hysteresis effect may also offer an explanation as to why it is often recommended (and built into most commercial instrument software) to rinse the capillary with an alkaline solution between injections. If this is done reproducibly, more reproducible migration times may be observed. Without the alkaline rinse, a drift will likely be seen at intermediate pH. (Other arguments for alkaline rinsing, e.g., to remove adsorbed protein, are not relevant to the current discussion.) While the findings of the current study focus on a negative aspect of CZE for analytical use, it is important for investigators to understand both the advantages and the limitations of their techniques. The pH hysteresis effect may offer a partial explanation for the “drift in migration times” that have been recently reported for CZE (3,5,6). With an understanding of the relevant variables, it is likely that CZE will become an important complement to high-performance liquid chromatography for quantitative and qualitative use. Registry No. Fused silica, 60676-86-0.
LITERATURE CITED (1) Lukacs, K. D.; Jorgenson, J. W. HRC CC, J . H5h Resolut. Chromatogr . Chromatogr . Commun . 1985. 8 , 407-41 1. (2) Lambert, William J. J . Chem. Educ. 1990, 6 7 , 150-153. (3) Morina. S. E.: Colburn. J. C.: Grossman. P. D.: Laner. H. H. LC-GC ’ 1990,-8, 34-46. (4) Huang, 45-110 X.; Coleman, W. F.: Zare, R . N. J . Chromatogr. 1989, 480,
-- .
( 5 ) Plckering, Michael V. LC-GC 1989, 7, 752-756. (6) Frenz, John; Wu, Shiaw-Lin; Hancock, William S. J . Chromatogr. 1989, 480, 379-391.
RECEIVED for review February 23,1990. Accepted May 2,1990.
On-Line Coupling of a Gas Chromatograph to a Continuous Liquid-Liquid Extractor Evaristo Ballesteros, Mercedes Gallego, a n d Miguel Valcircel*
Department of Analytical Chemistry, Faculty of Sciences, University of Cdrdoba, Cdrdoba 14004, Spain
A new analytlcai methodology for sample preparation and analysis comMnlng on-ilne extraction and gas chromatography is reported. The extractlon unit used Is connected to the instrument vla an Injection valve that permits the introduction of 4 pL of vaporized sample directly into the instrument Injectlon port. The proposed methodology can be Implemented in two operational modes aliowlng quantltative extractlon and the simultaneous extraction-derlvatlzatlon of phenol compounds. The esters of phenols, cresols, and chlorophenols were formed by continuous extractlon into acetlc anhldrlde in n-hexane, which allowed between 0.2 and 300 pg/mL of different phenols to be detected wlth relative standard deviations in the range 0.8-3.5 %
.
0003-2700/90/0362-1587$02.50/0
INTRODUCTION The current great interest in chromatographic techniques has fostered the search for new improvements to existing method, particularly in the past few years in response to the growing mechanization and computerization of analytical instrumentation. Thus, gas chromatography (GC) research has been aimed a t accomplishing the automatic injection of viscous or poorly volatile liquids in routine industrial applications ( I , 2). As GC is often used for the determination of very low concentrations, an extraction and/or concentration step must usually be included prior to the separation and quantification operation. The instrumentation and methodology involved in the automated on-line combination of su0 1990 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
percritical fluid extraction (SFE) and GC and its application to sample preparation and analysis were originally developed by Wright et ai. (3). However, although these techniques are of great use in studying supercritical fluids, their analytical applicability is limited to a few specific problems; in addition, it calls for strict pressure control, the use of depressurizing devices, and the fitting of the extraction system to the chromatograph, which usually involves major alterations (3, 4). The advent of liquid-liquid extraction with segmented flow provided a more efficient tool for the mechanization of extractive sample workup (5, 6). Since all manipulations are performed inside narrow-bore tubing, the procedure is efficiently shielded from the transfer of substances from the atmosphere to the sample and vice versa. Furthermore, the very rapid extraction achieved under liquid-liquid segmented flow conditions allows sample and reagent consumption to be minimized. A continuous extraction system is used for sample pretreatment prior to chromatographic analysis. However, the extracted samples are collected in vials and subsequently injected into the injection port by means of syringes (7). Normally, only a fraction of each sample is injected, so sample, time, and reagents are wasted. Two methods using a continuous extraction system connected to a gas chromatograph have been reported. One of them was developed by Roeraade (8) for the automated monitoring of volatile organic trace compounds in waters and relied on extraction in a segmented flow and the use of large on-column injected volumes (150 pL); the system required various alterations to the chromatograph. The other, somewhat simpler, was reported by Fogelqvist et al. (9). The system was isolated from the atmosphere and allowed rapid, precise workup of seawater samples for the determination of the halocarbons chloroform, bromodichloromethane, dibromochloromethane, bromoform, trichloroethylene, tetrachloroethylene, carbon tetrachloride, and l,l,l-trichloroethane. The conventional continuous liquidliquid extraction unit was connected to the chromatograph via an injection valve. The GC column was drawn out through the oven wall and directly connected to one of the valve ports. Along the distance between the oven and the valve, the column was shielded by a ceramic block to maintain a constant temperature gradient. As the valve was switched, the pentane extract (30-130 pL) held in the loop was transferred onto the GC column by means of the carrier gas. The method thus developed was very sensitive (detection limits between 20 and 500 pg/L), though relative standard deviation was higher than that of its manual counterpart. In addition, it had some pitfalls, namely, the injected volumes are rather large (30-130 pL), the internal standard or reagents usually required for precolumn derivatization cannot be added automatically, and the chromatograph must be altered (e.g., by piercing the furnace wall, connecting two columns serially, or changing the carrier gas inlet) and thus rendered unusable for routine syringe injections. Because of the high polarity and low vapor pressure of phenols, they often have to be derivatized to improve their chromatagraphic performance or their extraction from aqueous samples. Derivatizing reagents such as diazomethane (10), heptafluorobutyrylimidazole ( I 1), silanizing compounds (12-151, 2,4-dinitrobenzene (16, 17), pentafluorobenzoyl derivatives (18, 19),and acetic anhydride (20-26) have been used for this purpose. However, derivatization has some drawbacks such as intensive sample manipulation, problems arising from potential decomposition of the derivatives during their storage, and the toxic, carcinogenic, or explosive nature of many reagents (27, 28). This work was aimed at the reducing human participation in the analytical process, particularly in sample treatment, analytical reaction development, and transfer of the treated
W.
IE x i R A C T ION^
Fitting of the extraction unit to a gas chromatograph: A, tube stopcock; B, heating system; I V , injection valve; IP, injection pot?; D, detector; I, integrator; W, waste. Figure 1.
sample to the detector. For this purpose, we used a conventional liquid-liquid extractor allowing the simultaneous extraction and derivatization of various organic compounds. Fitting the extraction unit on-line to the chromatograph required no alteration to the latter, which allowed it to be used for manual syringe injections and to be coupled to an extraction unit if required. The performance of this system was tested with phenols, cresols, and chlorophenols. The acetate esters of six phenol compounds were formed by adding acetic anhydride to the n-hexane extractants.
EXPERIMENTAL SECTION Apparatus. The automated on-line extraction-derivatization/gas chromatographyinstrumentation used consisted basically of three sections, namely, an extraction unit, a switching valve (interface), and a gas chromatograph. The flow extraction-derivatization system comprised a peristaltic pump (Gilson Minipuls-e), an A-10 T solvent segmenter (Tecator), and a customized phase separator made from a Fluoropore membrane (l.O-rm pore size, FALP, Millipore), which was described elsewhere (29). Poly(viny1chloride) and Solvaflex pumping tubes were used for the aqueous and organic solutions,respectively; all coils were made of Teflon tubing. A six-port switching valve (Knauer 6332000) was also used. A Hewlett-Packard 5890 A gas chromatograph furnished with a single flame ionization detector was used for chromatographic analyses. A 10 m X 0.53 mm i.d. fused silica capillary column coated with a 2.65-rm film of 100% cross-linked poly(dimethylsi1oxane) (HP-I) was employed. The injector temperature was kept at 130 "C and the detector at 250 "C throughout. The temperature of the chromatographic oven was programmed at either 50 "C (3 rnin), 5 "C/min to 80 "C, 15 "C/min to 200 "C (2 rnin), or 40 "C (1rnin), 8 "C/min to 50 "C, 2 "C/min to 70 "C, 10 OC/min to 200 "C (4 rnin), for the separation of phenols and phenol acetates, respectively. Nitrogen was used at a flow rate of 42 mL/min as carrier gas. Peak areas were measured by a Hewlett-Packard 3392 A integrator. Design of the Interface Unit. A schematic diagram of the system is depicted in Figure 1. We first concentrated on the sample-to-gas chromatograph interface as it seemed to pose the most serious problems to developing a satisfactory unit. The injection interface was constructed by using a injection valve with an inner volume of 2.5 wL. Owing to the large volume of the valve loop (10 wL),a new loop of 1.5 p L was constructed from stainless-steel tubing (30 mm long, 0.25-mm i.d.). The new injected sample volume was 4 pL. A second alteration to the valve involved fitting a 25-cm stainless steel tube (0.5-mm i.d.) to the carrier outlet. The tube had a stainless steel needle soldered to one of its ends. The valve was thus ready for direct fitting to the injection port of the gas chromatograph by inserting its needle into the septum of the instrument port. This tube linking the valve and the injection port was heated of a wire coiled helically around hollow ceramic tube. The wire temperature was controlled through a voltage regulator comprising an electronic circuit featuring triac withstanding currents of up to 15 A. This device allowed the temperature of the tube valve port to be readily set from 25 to 175 "C. The carrier gas inlet (nitrogen, 42 mL/min) to the chromatograph was split into two, which were directly connected to one of the ports of the valve (flow rate 30 mL/min) and the chromatograph injection port (flow rate 12 mL/min), respectively. The inlet was shut by a stopcock, so that the instrument could be used for manual injections by allowing the nitrogen stream
ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990 PUMP
Table I. Optimum Conditions for the Determination of Phenol Compoundsa
.I_
EXTRACTANT SAMPLE
parameter EC
8) 150 cm
* EC
studied range
optimum range
selected value
PS w1
A)
1589
60°C I-----'
w ----_I
EC
Flgure 2. Manifold for the extraction and extraction-derivatization of phenol compounds: S, phase segmenter; EC, extraction coil [(A)for extractlon and (6)for extraction-derivatization] ; PS, phase separator; DC, desiccating column; I V , injection valve; W, waste.
to follow its normal route through the instrument. Reagents and Chemicals. The standards and reagents used were purchased from Aldrich Chemie and Merck. Standard stock solutions of each phenol compound were prepared at a concentration of 1 g/L in ethanol and stored in glass-stoppered bottles at 4 "C. The standard solution was an aqueous mixture of four phenol compounds (phenol, 3,4-dimethylphenol, 2-tert-butylcontaining between 0.5 phenol, and 2-tert-butyl-4-methylphenol) and 300 mg/L of each; the extractant was ethyl acetate containing 300 mg/L naphthalene as internal standard. For simultaneous extraction and derivatizations,the standard solution was prepared by adding appropriate amounts each of 1g/L stock solution and 0.5 mL of 2 M sodium bicarbonate to 25-mL calibrated flasks and diluting to the mark with water to obtain final concentrations from 0.5 to 300 mg/L of each phenol compound (phenol, 3,4dimethylphenol, o-cresol, rn-cresol, 2-chlorophenol, and 4chlorophenol) in 0.04 M bicarbonate (pH 8.5);the extractant was n-hexane containing 300 mg/L naphthalene (internal standard) and 6% (V/V) acetic anhydride (derivatizing reagent). Procedure. The extraction unit used is depicted in Figure 2. The sample solution was continuously mixed with the extractant (ethyl acetate for extraction only and n-hexane for simultaneous extraction and derivatization). The extracts were used to fill the loop of the injection valve. As the valve was switched, the loop contents were transferred to the chromatograph port by the nitrogen carrier. Loop volumes of 4 pL were used. The section of the tube valve port was heated to 70 "C to prevent the sample from being adsorbed in the loop or the connection tube valve port. The nitrogen flowing to the injection port favored transfer of the vaporized analytes to the chromatographic column. To prevent any water trace from entering the column, the extraction system included a desiccating column (50 mm long, 3-mm i.d.) filled with sodium aluminosilicate pellets (pore diameter 4 A).
RESULTS AND DISCUSSION Three organic solvents (ethyl acetate, toluene, and n-hexane) were assayed as extractants for the phenol compounds. Ethyl acetate was found to be the most efficient for underivatized phenols (extraction yields ranged between 65 and 80%). However, phenolate ions in the alkaline solution were more efficiently extracted and derivatized with n-hexane containing 6% acetic anhydride. The need for phenolate ions for successful derivatization has been emphasized by some authors (21,23),and the optimal pH for their formation has been estimated to be in the range 8-10. As we found ethyl acetate and n-hexane to be extremely efficient in the extraction and extraction-derivatization of the phehols, we used them in all subsequent experiments. The extracted underivatized phenols could not be fully resolved; in fact, the peaks of phenol and o-cresol and of 3,4-dimethylphenol and 4chlorophenol overlapped. However, conversion of the phenols to their corresponding esters allowed the sequential separation of the phenols, cresols, and chlorophenols. Optimization of t h e Extraction Unit. This system was optimized by collecting the ethyl acetate or n-hexane extracts of underivatized and derivatized phenols, respectively, in a
PHa PHb NaHCO,,b M acetic anhydride,b% reactor temp,b"C extraction coil length: cm extraction coil length: cm flow rate:vb mL/min aqueous phase organic phase nitrogen flow rate,"SbmL/min tube valve port temp: "C temp,b"C
2-10 7-11
2-8
8-10 0.01-0.05 0.03-0.05
5.0 8.5 0.04
0.2-7.0
4.0-7.0
6.0
20-75 50-300 50-500
50-75 50-300 150-450
60 150
0.5-4.6 0.4-1.0
3.0-4.0
3.6 0.7
20-100
0.5-0.8 35-55
35-105 25-90
70-100 55-85
85 65
300
42
a a and b denote the extraction and extraction-derivatization method, respectively.
4-mL glass vial containing anhydrous sodium sulfate and performing manual injections of 1-pL fractions into the chromatograph by a syringe. The variables studied included the sample pH, amount of acetic anhydride, extraction coil temperature, flow rate of the aqueous and organic phase, and residence time. Table I summarizes the optimum conditions for preparation of the samples and extractants and the operation of the flow extraction system. The acetylation yields were not influenced by the bicarbonate concentration ovr the range 0.03-0.05 M; however, they were considerably lower for the phosphate buffer than for the bicarbonate buffer at each pH. The influence of the acetic anhydride concentration on the yield of acetate derivatives was investigated by using several solutions prepared in n-hexane. A 4% solution was found to be adequate for derivatization purposes; lower concentrations resulted in incomplete derivatization of the phenols and in chromatograms including the peaks of both the underivatized and the derivatized phenol. An ionic strength up to l M (as KNOB)did not appreciably affect the performance of the method. The effect of the extraction coil temperature was studied in the range 20-75 "C. This variable affected the extraction yield only when extraction and derivatization were performed simultaneously. This was a result of the formation of the esters being favored by heating. Below 40 "C, the chromatogram showed the peaks of both the underivatized and the derivatized phenols. The extraction coil was heated at 60 "C with a thermostated water bath when the phenols were derivatized continuously in n-hexane. Increasing sample flow rates (at a constant organic phase flow rate) resulted in increased peak areas owing to the increased the preconcentration ratio. As expected, the areas also increased with decreasing organic phase flow rates (at a constant sample flow rate) for the same reason. We chose a sample and organic flow rate of 3.6 and 0.7 mL/min, respectively, taking into account the mutual influence of the reproducibility, concentration ratio, and sampling frequency. The influence of the residence time was also studied at different extraction coil lengths. The peak area was not affected by such a length for underivatized phenols. When the phenol compounds were extracted and derivatized simultaneously, the coil length was some what influential as ester formation took some time; therefore, we used a thermostated (60 "C) reaction coil of 300 cm, which resulted in a residence time of 9 s. Experimental Testing of the Interface Unit. The extraction unit described above was fitted to the chromatograph
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
Table 11. Features of the Calibration Graphs and Determinatioo of Underivatized Phenols" compound
regression eq
phenol 3,4-dimethylphenol 2-tert-butylphenol 2-tert-butyl-4-methylphenol phenol 3,4-dimethylphenol 2-tert-butylphenol 2-tert-butyl-4-methylphenol phenol 3,4-dimethylphenol 2-tert-butylphenol 2-tert-butyl-4-methylphenol
A A A A A A A A A A A A
= 3.81 x = 3.95 x = 4.10 x = 4.51 X = 6.50 X = 6.28 x = 5.53 x = 4.84 X = 6.82 x = 6.61 x = 6.16 x = 5.65 X
10-3x + 1.10 x 10-3 10-3x + 1.91 x 10-3 10-3x + 2.39 x 10-3 lOW3X + 3.18 X lo-* 10-3X - 1.70 X lo-* 10-3x - 6.25 x 10-3 10-3x - 2.31 x 10-3 10-3X - 7.94 X low3 10-3x + 3.89 x 10-3 10-3x + 7.67 x 10-3 10-3x - 4.67 x 10-3 10-3X - 9.51 X
r
range, wg/mL
dl, wg/mL
cv, %
0.9994 0.9996 0.9996 0.9994 0.9997 0.9997 0.9998 0.9998 0.9996 0.9999 0.9998 0.9999
0.5-3 0.5-3 0.2-3 0.2-3 3-30 3-30 3-30 3-30 30-300 30-300 30-300 30-300
0.5 0.4 0.2 0.2
3.4 3.0 3.0 2.3 2.2 1.9 1.7 1.7 1.5 1.4 1.1 0.8
a A , Analyte area/internal standard area ratio; X , concentration (rg/mL); r , correlation coefficient; dl, detection limit; cv, coefficient of variation.
Table 111. Features of the Calibration Graphs and Determination of Derivatized Phenols" compound phenol o-cresol m-cresol 2-chlorophenol 4-chlorophenol 3,4-dimethylphenol phenol o-cresol m-cresol 2-chlorophenol 4-chlorophenol 3,4-dimethylphenol
regression eq
A A A A A A A A A A A A
= 9.67 x = 6.51 x = 7.16 x = 5.49 x = 4.23 x = 7.19 x = 9.41 x = 8.24 x = 9.93 x = 7.94 x = 8.45 x = 9.14 x
10-3x + 3.29 x 10-3 10-3x + 2.43 x 10-3 10-3x + 2.50 x 10-3 10-3x + 2.24 x 10-3 10-3x - 2.67 x 10-3 10-3x - 2.77 x 10-3 10-3x + 4.77 x 10-3 10-3x + 2.16 x 10-3 10-3x - 1.97 x 10-3 10-3x + 1.26 x 10-3 10-3x - 1.60 x 10-3 10-3x - 4.51 x 10-3
r
range, rg/mL
dl, rg/mL
cv, k
0.9998 0.9996 0.9996 0.9997 0.9998 0.9998 0.9996 0.9997 0.9996 0.9996 0.9997 0.9998
0.3-3 0.4-3 0.4-3 0.4-3 0.4-3 0.3-3 3-30 3-30 3-30 3-30 3-30 3-30
0.2 0.3 0.2 0.3 0.3 0.2
2.9 3.1 3.5 3.3 3.2 3.1 1.2 1.5 1.5 1.8 1.5 1.3
a A , Analyte area/internal standard area ratio; X , concentration (rg/mL); r , correlation coefficient; dl, detection limit; cv, coefficient of variation.
via an injection valve originally designed for use in HPLC. The first few experiments were carried out by changing the normal inlet of the carrier gas to the instrument so that it passed through the loop of the injection valve to flush the sample into the chromatograph. The results thus obtained were unsatisfactory as the base line was markedly raised, so there was extensive peak overlap and no relationship between peak areas and the amounts of injected samples. These pitfalls may stem from the following facts: (a) part of each sample may be adsorbed in the loop and the tube valve port, thus being carried over subsequent injections and requiring a higher nitrogen flow rate for adequate flushing; (b) on reaching the injection port, the analytes might be diluted in the mobile phase in the absence of a nitrogen stream helping them to enter the column. The carrier flow rate was increased to its limit to prevent the flame detector from extinguishing, with no appreciable improvement. We thus split the nitrogen stream into two lines as shown in Figure 1. The results were somewhat better, although the peaks were broader and the reproducibility was still low. Finally, we aimed a t getting the analytes to reach the injection port in the vapor phase so that they could be readily flushed through the valve port connecting tube and thus clogging of the tube and dilution of the analytes in the carrier gas prior to the chromatographic column could be avoided. By using the heating system described in the Experimental Section, the tube valve port was heated at different temperatures (Table I). Figure 3 shows the chromatograms obtained by injecting extracts of the four phenols in ethyl acetate. The first chromatogram (Figure 3A) was recorded with automatic injection at room temperature, and the second (Figure 3B) by heating a t 80 "C. Similar results were obtained by extracting and derivatizing the phenols with acetic anhydride in n-hexane; however, the required heating temperature was
Figure 3. Gas chromatograms of the extracted phenols: (A) without heating of the tube valve port and (B)with heating at 80 OC. Concentrations: phenol in the aqueous sample, 15 Mg/mL; internal standard (naphthalene),300 pg/mL in ethyl acetate. Peaks: (1) phenol; (2) naphthalene; (3) 3,4dimethylphenol;(4) 2-teff-butylphenol; (5) 2fert-butyl-4-methylphenol. For GLC conditions, see text.
lower as the acetylated derivatives were more volatile, and so was the solvent (n-hexane). The repeatability of the automatic injection system was measured by performing 10 successive injections of a standard
ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
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extraction and extraction-derivatization prior to the introduction of samples into a gas chromatograph, with the aim of accomplishing the typical objectives of sample preparation (i.e., matrix effect elimination, solvent changeover, and andyte preconcentration) and analyte derivatization, yielded better analytical results than their manual counterparts in terms of selectivity, sensitivity, precision, throughput, and economy. The heating system designed for this purposed allows the complete introduction of small volumes of scarcely volatile samples into the instrument injection port. The on-line coupling is characterized by its simplicity and affordability as no alterations to the chromatograph are required. These automatic methods allow the extraction and derivatization of phenol compounds at a sampling rate of up to 30 samples h-l, depending on the furnace temperature program used.
A
Registry No. Phenol, 108-95-2;o-cresol, 95-48-7; rn-cresol, 108-39-4; 2-chlorophenol,95-57-8; 4-chlorophenol, 106-48-9; 3,4dimethylphenol, 95-65-8; 2-tert-butylphenol, 88-18-6; 2-tert-butyl-4-methylphenol, 2409-55-4.
LITERATURE CITED Figure 4. Gas chromatograms of (A) underivatized phenols. Peaks: (1) phenol o-cresol; (2) rn-cresol; (3) 2-chlorophenol; (4) naphthalene; (5) 3,edimethylphenol dchlorophenol. (6) Acetate derivates. Peaks: (1) acetic anhydride; (2) phenyl acetate; (3) o-cresyl acetate: (4) m c r e s y l acetate; (5) naphthalene; (6) 2-chlorophenyl acetate; (7) khlorophenyl acetate; (8) 3,4dimethyIphenyi acetate. The tube vake port was heated at 70 'C, and the phenol concentrations in the aqueous sample were 15 pglmL. The extractant was n-hexane, and the GLC conditions are described in the text.
+
+
phenol solution in ethyl acetate. The relative standard deviation for the four phenols assayed was in the range 0.5-1.0%. This repeatability was similar to that achieved by manual injection (0.8-2.070). Figures of Merit. The GLC separation of the underivatized and acetylated phenols is illustrated in Figure 4, parts A and B, respectively. As can be seen, the peaks of some underivatized phenols and chlorophenols extracted into nhexane were completely overlapped. As shown in Figure 4B, excess acetic anhydride posed no interference. The standard curves obtained by plotting the analyte to internal standard peak area ratio versus the analyte concentrations in the aqueous medium were linear over the tested range (0.5-300 pg/mL) for both underivatized and derivatized phenols, cresols, and chlorophenols. The features of these graphs at three integrator sensitivities and those of the analytical procedure are summarized in Tables I1 and 111. The practical detection limit was calculated as the concentration yielding the minimal detectable signal in the chromatogram. The coefficient of variation was checked on 11 samples containing 1, 15, and 30 pg/mL of each phenol compound over the linear range assayed (0.2-3, 3-30, and 30-300 pg/mL, respectively).
CONCLUSIONS The two methods developed for continuous liquid-liquid
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RECEIVED for review January 18, 1990. Accepted April 19, 1990. We express our gratitude to the Spanish CICYT for financial support received through Grant No. PA86-0146.
