According to the above relationships, the value of N for a given column is reduced as the ratio of inlet to outlet pressures increases. At very high pressure drops, this places an upper limit on the maximum value of N which can be attained, regardless of column length. It is interesting to note that this adverse effect of column pressure drop can in principle be cancelled by the use of coupled-columns of increasing diameter. Thus V J L , can be held constant by proper design of the overall system. A theoretical analysis of some practical situations of this type, however, suggests that this expedient would be only marginally effective. APPENDIX I1 n
It is not obvious that the relationship u 2 =
cut2holds
i=l
when values of ui are defined in terms of eluate volume, and K , is permitted to vary among the various columns. Part of the difficulty in accepting this relationship arises from the fact that u Z values measured in terms of column length (the usual convention) change across column junctions when Kt changes, For example, assume that K I increases by a factor of 2 in going from column 1 to column 2, and u iis equal to 2
cm at the end of column 1 and 5 rnl in the eluate from column 1. As the sample band enters column 2 it initially occupies 1 cm (std dev or value of u ) at the front of the column. That is, the band has been compressed from 2 cm to 1 cm in crossing the junction between columns 1 and 2. But this phenomenon has no effect on the final contribution of ut to u, as long as values of ui are computed in terms of volume. To see this more clearly, assume that column 2 is completely equivalent to column 1 except for the difference in Kt values, An initially narrow sample band will then have u2 equal to 2 cm (or 10 ml) at the end of separation on column 2. Combination of columns 1 and 2 is seen to give a value of u2 equal to (12 22)cmz, or 5 cm2. This is equivalent to 125 m12 in the eluate from column 2. But the same result is obtained by simply adding u i 2 and u t 2 expressed in volume units: u 2 = 5 2 lo2. The additivity of ai2 in terms of volume units (or equivalent time units) in this situation has also been discussed by Giddings (3).
+
+
RECEIVED for review July 24, 1967. Accepted November 14,1967.
Rapid Procedure for the Chloroacetylation of Microgram Quantities of Phenols and Detection by Electron-Capture Gas Chromatography Robert J. Argauer Entomology Research Diaision, Agricultural Research Seroice, U.S . Department of Agriculture, Beltsuille, Md. Aqueous sodium hydroxide solutions containing as little as 0.01 ppm phenol were treated with choracetic anhydride in benzene to prepare a relatively stable phenol derivative sensitive to electron-capture detection and analysis by gas chromatography. The benzene and aqueous phases were mixed for 2 minutes, and a 5-pl aliquot of the benzene phase was injected onto the column of the chromatograph. Retention times were tabulated for the chloracetate derivatives of 32 phenols.
PHENOLS and their derivatives are used as antiseptics, disinfectants, and pesticides, as antioxidants in food:products, and in many other chemical applications. The use of the carbamate derivatives as pesticides has therefore generated renewed interest in a rapid and sensitive means of detecting phenolic compounds. Gutenmann and Lisk ( I ) hydrolyzed microgram quantities of the carbamate insecticide carbaryl, to 1-naphthol, which then was brominated and acetylated to prepare an electron-capture sensitized derivative. However, a procedure that would allow the preparation of a sensitive derivative with a minimum number of physical-chemical manipulations was still desired. When Landowne and Lipsky ( 2 ) prepared various mono-, di-, and tri-haloacetates of cholesterol in tetrahydrofuranpyridine solution and measured the response of the recrystallized derivatives to an electron-capture detector, the chloro(1) W. H. Gutenmann and D. J. Lisk, J. Agr. Food Chem., 13, 48 (1965). (2) R. A. Landowne and S. R. Lipsky, ANAL.CHEM., 35, 532 (1963).
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ANALYTICAL CHEMISTRY
acetate derivative was the most sensitive. In the following procedure, a reaction medium of aqueous sodium hydroxide and chloroacetic anhydride in benzene was used. The prepared chloroacetate derivatives can be injected into the gas chromatograph without further physical-chemical workup. EXPERIMENTAL
Apparatus. A conventional gas chromatograph (Varian Aerograph Model 200) was used. It was equipped with a 5-foot X lfrinch i.d. stainless steel column containing 2 x (w/w) XE60 @-cyanoethyl methyl silicone polymer) coated on 50- to 60-mesh Anakrom ABS (Analabs, Hamden, Conn.) and maintained at 170" C. The tritium detector was held at 205' C, the injection heater at 210" C, and the nitrogen flow at 30 ml per minute at the column outlet. Screening of Phenols. To 10 p1 of each phenol in benzene (1 pg/pl) in a 125-ml Erlenmeyer flask were added 15 ml of 0.25N NaOH and 10 ml reagent [l gram of chloroacetic'anhydride (Eastman White Label) per 200 ml benzene]. The flask was shaken for 2 minutes on a mechanical shaker. A 5-kl aliquot of the benzene layer was injected into the gas chromatograph. Retention Times. A mixture of five chlorophenol chloroacetates were used as references to obtain over 2 days the retention times (expressed in minutes from the benzene front) of the chloroacetate derivative of 32 phenols. In these measurements, 1 pg of each of the respective phenols was treated as in the screening tests. The retention times obtained for benzene dilutions of the chloroacetate derivatives of 10 phenols prepared in 50-gram quantities in our laboratory by S. I. Gertler 15 years ago were identical with those obtained by this procedure.
08
06
I
7 -
- + \ 7I >
E 04
G v,
z n. 0
m 02 W
a. a W
a 0
E
04
02
4
8
12
16
20
24
28
32
36
40
44
TIME, minutes
Figure 2. Effect of mixing time on formation of chloroacetate derivative
A . 1 pg p-chlorophenol, B. 1 kg p-isopropylphenol, C. 1 pg p-tert-butylphenol, D . 1 pg p-cresol, E. 1 p g phenol, F. -1, -2, -3. 2.0, 1.0, 0.5-pg naphthol, G . -1, -2, -3. 2.0, 1.0, 0.5 pg 6-Chloro-3,4-xylenol, H. 1.5 PLg phenyl chloroacetate in 10 ml benzene; 15 ml0.25N NaOH added after zero time observation
TIME, minutes Figure 1. Gas chromatograms for five chlorophenols and phenol. Effect of chloroacetate formation
I. 1 pg each phenol per 10 ml benzene; no NaOH or reagent 11. Reagent blank 111. Chloroacetate derivatives-A. phenol, B. p-chlorophenol, C. 2,4-dichlorophenol, D. 2,4,5-trichlorophenoI, E. 2,3,4,5-tetrachlorophenol, F. pentachlorophenol RESULTS AND DISCUSSION
Qualitative. Curves showing the effect of chloroacetylation on 1-pg quantities of five chlorophenols and phenol itself are compared with the reagent blank and the phenols themselves in Figure 1. In addition, a sample of pentachlorophenyl acetate produced an intense response at a retention time of about 2 minutes. However, when an attempt was made to use acetic anhydride as the acetylation reagent for the chlorophenols, no response was obtained. Furthermore, additional mixing of the two phases in the chloroacetylation procedure caused both peaks A and B (Figure 1) to diminish in intensity relative to the other peaks in the chromatogram. Thus the amount of derivative that formed and remained in the benzene layer was related directly to the mixing time of the two phases as well as to the phenolic compound itself. The effects of the length of mixing time on the formation of the chloroacetate derivative are compared in Figure 2. The chloroacetate derivative of phenol (curve E ) appears much more unstable than that of p-cresol (curve 0);thus more controlled conditions would be needed to develop a quantitative method for phenol itself. Many of the other chloroacetates appear sufficiently stable to permit the use of the screening procedure for quantitative purposes--e.g., p-isopropylphenol and 1-naphthol. To determine the GLC response for equivalent quantities of two phenols, direct injections of pentachlorophenyl chloroacetate and 1-naphthyl chloroacetate were compared with
equivalent amounts of the phenols carried through the procedure. Response efficiencies of 52 and 50% were obtained. In addition, when the pentachlorophenol concentration was increased 10-fold, the response efficiency decreased to 37 %. Hence, because of the kinetic factors associated with this procedure, the effects of time and degree of agitation on phenol samples and standards need to be established if quantification is desired. The possibility of side reactions has been established by Koelsch (3) who prepared aryloxyacetic acids in good yield when more concentrated aqueous sodium hydroxidephenol solutions were treated with chloroacetic acid. A decrease in the response associated with several derivatives resulted when the concentration of the reagent was lowered below 20 mg of chloroacetic anhydride per 10 ml of benzene. When the concentration of sodium hydroxide was lowered to approach the neutralization point of the reagent, extraneous peaks and considerable tailing occurred on the chromatogram. The retention times obtained for the chloroacetate derivatives of 32 phenols obtained by this procedure are shown in Table I. No satisfactory results were obtained when aminophenol, several nitrophenols, or several polyhydroxybenzenes were subjected to the procedure. The procedure did not prove suitable for the trifluoro- and trichloroacetate derivatives of six phenols tested. The perfluorobutyl derivative of naphthol prepared in pyridine-benzene solution in microgram amounts was unstable when water was introduced. The ease of screening of phenolic compounds inherent in the procedure allows the researcher to quickly determine the applicability of the technique to the analysis of phenols of specific importance to his research. Retention times for (3) C. F. Koelsch, J . Am. Chem. SOC.,53, 304 (1931). VOL. 40, NO. 1, JANUARY 1968
123
Table I. Retention Times for the Chloroacetate Derivatives of Several Phenols Retention time as chloroacetate, Corresponding minutes from pesticide (as Compound benzene front methyl carbamate) m-Isoprop ylphenol 1 .o Hercules 5727 o-Methoxyphenol 1.4 Bayer 39006 o-Isopropylp henol 1.5 Bayer 39007 2,4-Dichlorop hen01 1.7 Niagara NIA-8586 2,3-Dihydro-2,2-dimethyl-7benzofuran01 2.0 ’Jiagara NIA-10242 6-Chloro-3,4-xylenol 2.3 Banal@ 4-(Dimethylamino)-rn-cresol MatacilD 2.4 4-(DimethyIamino)-3,5xylenol 2.5 ZectranD o-(2-Propynyloxy)phenol 2.9 Hercules 9699 4-(Methy1thio)-rn-cresol 5.4 Bayer 32651 1-Naphthol 6.3 Carbaryl Benzo[b]thiophene-4-01 7.0 Mobil MC-A-600 pChloropheno1 1.2 2,4-Dichlorophenol 1.7 2,4,5-Trichlorophenol 3.2 2,3,4,5-Tetrachlorophenol 4.2 Pentachlorophenol 8.5 Phenol 0.56 0-, m-,and pCresols 0.7 Thymol 1.2 Carvacrol 1.3 3,4-Xylenol 1.3 p-rert-Butylphenol 1.7 o-Isopropylphenol 0.8 rn-Isopropylphenol 1.0 p-Isopropylphenol 1.3 o-Methoxyphenol 1.4 rn-Methoxyphenol 1.9 2.0 p-Methoxyphenol 4-Allyl-2-methoxyphenol 4.2 (Eugenol) 2-Methoxy-4-propenylphenol
(Isoeugenol) o-Phenylphenol pPhenylpheno1
6.4 4.7 15.6
derivatives may be obtained on other types of column packings to establish or confirm the presence of specific phenols. Quantitative. A linear response range of 0.1 to 3 ng for the detector was obtained for the chloroacetates injected. Since only a 5-pl aliquot of a 10-ml benzene reagent solution was injected into the chromatograph, an attempt was made to concentrate the benzene phase 10-fold after reaction and thus to increase the lower limit of detection for the same amount‘injected. The peaks caused by the reagent (Figure 1, curve 11) were found, upon concentration, to interfere with those phenyl chloroacetates with retention times of less than 2 minutes. The only promising results were obtained for the several phenyl chloroacetates tested that had retention times longer than those of the impurities still present because of the reagent. To determine the effect of the ratio of water to the benzene reagent, a mixture of several representative phenols was prepared and diluted to as low as 0.0033 ppm in water. Concentrations on the order of 0.01 to 0.1 mg per 1000 ml (0.010.1 ppm) are detectable by taste and odor tests. Trace amounts approaching 1 pg per liter (0.001 ppm) can impart objectionable taste to water after marginal chlorination (4) (4) American Public Health Association, Inc., “Standard Methods for the Examination of Water and Wastewater,” 12th ed., New York, 1965, p. 769.
124
ANALYTICAL CHEMISTRY
W v)
z 0
a
v)
w U
U
w
0
U 0 V W
U
I
l T
hb---------
11
I
(00033 pprn
3X
( 0 0 0 6 7 pprn 1
TIME, minutes
Figure 3. Gas chromatograms of chloroacetate derivatives prepared a t low concentrations of phenolic compounds in water A . phenol, B. p-Cresol, C . 3,4-xylenol, D. 4-(di-methylamino)3,5-xylenoI, E. eugenol, F. 1-naphthol, G. pentachlorophenol 1. 0.5 p g each phenol in 15 ml0.25NNaOH, 11. 1.0Gg each phenol in 15 ml 0.25N NaOH, 111. 0.5 p g each phenol in 150 ml 0.025N NaOH, IV. 1.0 p g each phenol in 150 ml0.025N NaOH
Figure 3 shows the effect of increasing the voltime of the aqueous phase 10-fold on the quantitative relationship between 0.5 and 1.0 pg of seven phenols. No attempt was made to concentrate the caustic aqueous phase before reaction or t o concentrate the benzene phase after reaction before injection of the usual 5-pl aliquot into the chromatograph. Thus quantitative results may be expected a t concentrations that contain less than 0.01 ppm of phenolic compound in water. Even lower limits may be obtained by concentrating phenols in the aqueous phase by the usual means before reaction. Some success was attained with concentrations as little as 0.5 pg of phenolic compounds per 500 ml of aqueous phase per 10 ml of reagent. The determination of the lowest limit of detection for each of these compounds has not been undertaken because this limit would depend on the specific problem for which the researcher needs or desires to apply the technique. Because of its sensitivity, the procedure should be applicable to the determination and confirmation of phenols that may appear in drinking water and streams, in automobile gas exhaust, in tobacco smoke, and in waste products of various chemical producing facilities. RECEIVED for review September 20, 1967. Accepted November 2. Presented before the Division of Analytical Chemistry, Fall Meeting, ACS, Chicago, Ill., 1967. Mention of proprietary products is for identification only and does not necessarily imply endorsement of these products by the U. S . Department of Agriculture.
