Separation of Phenolic Compounds from Carbon Chloroform Extract for Individual Chromatographic Identification and Measurement James W. Eichelberger, Ronald C. Dressman, and James E . Longbottom Organic Analyses Group, Analytical Quality Control Laboratory, Federal Water Pollution Control Administration,
US.Department of the Interior, Cincinnati, Ohio 45202.
A procedure is presented for efficient isolation of many phenols from carbon chloroform extracts (CCE) prior to chromatographic identification. The phenols, as weak acids, are isolated by a Florisil column cleanup of the CCE. Techniques for evaporation and preparation of the extract are given. Application of the method to grab samples as well as qualitative and quantitative determinations are discussed. Recovery data and relative retention times for a gas-liquid chromatographic separation are reported.
P
henols are introduced into surface waters as pollutants from a variety of sources such as industrial effluents, sewage disposal, agricultural runoff, and chemical spills. These compounds are considered pollutants when they are present in sufficient concentration to cause undesirable taste and odor problems. To control this type of pollution, it is important to have the ability to identify and measure individual phenols. Specific identification is often required to determine the source of pollution. Specific identifications also make it possible to carry out more effective monitoring programs, The carbon adsorption method (Middleton and Rosen, 1956; Middleton, Grant, et a/., 1956) used by this laboratory is capable of collecting phenols for analysis. This method used the adsorptive capacity of activated carbon to concentrate organic materials from large volumes of water. The phenols present in the water, along with many other organic materials, are adsorbed on the carbon at the sampling point and desorbed from the carbon in the laboratory by extraction with chloroform (Goren-Strul, Kleijn, et a/., 1966). The carbon chloroform extract (CCE) is evaporated to apparent dryness and weighed. Although the method is not quantitative, the concentration of the CCE gives some measure of the degree of organic pollution in a stream. The identification and measurement of individual phenols in a CCE are extremely difficult because of large amounts of other organic materials which are desorbed from the carbon. Further, the high volatility of many phenols results in large losses when the CCE is evaporated to apparent dryness. Thus, an efficient means of concentrating and isolating phenols from the CCE is needed. Phenols have been isolated from CCE in this laboratory by use of a modified Shriner-Fuson separation as described by Breidenbach, Lichtenberg, et al. (1966) and suggested refinements by Goren-Strul, Kleijn, et a/. (1966). With both procedures, partitioning of phenols to various solubility fractions and loss of volatile phenols caused by the many manipulations result in poor recoveries. In addition, fractions obtained for gas and thin-layer chromatographic analysis were still not suffi576 Environmental Science & Technology
Table I. Separation Efficiencies Obtained
Compounds Alkyl phenols o-cresol in-cresol p-cresol 2,3-dimethyl 2,4-dimet h y1 2,5-dimethyl 2,6-dimethyl 3,4-dimethyl 3,5-dimethyl 2,3,5-trimethyl 2,4,5-trimethyl 2,3,5,64etramethyl p-tertbut yl 2,6-ditertbutyl-p-cresol
Dosage, mg.
Per cent" recovery from Evapora- cleanup tion procedure
0.16 0.30 0.40 0.40 0.40 0.40 0.20 0.40 0.40 0.80 0.40 0.20 0.30 0.20
64 78 93 75 75 75 85 79 95 68 87 60 73 75
109 88 95 73 60 85 55 81 88 94 90 18 104 0
Chlorophenols o-chloro m-chloro p-chloro 2,3-dichloro 2,4-dichloro 2,5-dichloro 2,6-dichloro 3,4-dichloro 2,4,5-trichloro 2,4,6-trichloro
0.40 0.20 1.20 1.20 1 .oo 0.60 1 .oo 0.30 0.15 1.20
75 71 90 76 79 78 80 82 67 87
91 92 80 93 103 86 106 95 109 83
Aminophenols o-amino m-amino p-amino m-diethy lamino
0.50 0.90 0.80 2.00
13 8 5 36
0 0 0 0
Miscellaneous phenols phenol o-nitro 4-chloro-2-nitro p-methoxy p-butoxy 1-naphthol 2-naphthol p-phenyl 2-bromo-4-phenyl
0.40 1.20 1 .oo 1.20 0.06 1.40 0.60 0.80 0.90
75 55 44 59 70 62 72 78 83
104 102 91 107 109 77 111 103 94
0
Corrected for evaporation loss.
ciently free of nonphenolic organic material. The above problems are largely overcome by the method presented here. This method omits the evaporation of the CCE to dryness and employs an extraction of phenols as acids, followed by a Florisil column cleanup of the extract. The proposed procedure yields phenolic compounds sufficiently free of extraneous material for subsequent separation and analysis by chromatographic techniques. Experirnetital Aliquots of CCE samples (140 mg. each) were dissolved in 30 ml. of chloroform to simulate concentrated extracts. These aliquots were dosed in triplicate with selected groups of phenols and an undosed aliquot was used as a blank. The dosing quantities of each compound are listed in Table I. The 30-ml. samples were quantitatively transferred to 125ml. separatory funnels with chloroform. Each sample was extracted three times with 15-ml. portions of aqueous NaOH of pH 13. The chloroform layers were discarded and the aqueous layers were combined in 125-ml Erlenmeyer flasks. These extracts were acidified with concentrated HC1 to pH 2. The acidified samples were allowed to cool and were returned quantitatively to the separatory funnels. They were then back extracted three times with 15-ml. portions of ethyl ether and the ether layers were combined in 150-ml. beakers. Glass columns 20-mm. in diameter were packed to 10 cm. with Florisil and topped with 2 cm. of anhydrous sodium sulfate. The columns were washed with 30 ml. of ethyl ether. When the last of the 30 ml. reached the sodium sulfate, the 45ml. extracts were added and 250-ml. beakers were used to collect the eluates. When the last of the 45 ml. reached the sodium sulfate, a 200-ml. portion of ethyl ether was added to each column and collected in the same beakers. The eluates were evaporated in the beaker on a warm water bath (50' C.) in an exhaust hood. When the volumes reached approximately 10 ml., the eluates were quantitatively transferred to 15-ml. centrifuge tubes and carefully evaporated again to 10 ml. by use of a warm water bath (50" C.) and a gentle stream of air. The eluates obtained from this procedure were analyzed by gas chromatography to determine recoveries of the phenols and the effectiveness of the cleanup procedure. Eluates containing phenols whose relative retention times are greater than 4.0 were concentrated to 5 ml. for adequate response on the gas chromatograph. The method was evaluated using 37 phenolic compounds, including alkylphenols, chlorophenols, nitrophenols, and aminophenols. The rentention time of each compound was determined on the gas chromatograph prior to the evaluation of the method. Retention times relative to phenol are listed in Table 11. Six of these phenolic compounds were used to dose four replicate CCE samples to determine the effects of concentration and drying of a CCE for weighing prior to a phenol analysis. The CCE samples (140 mg. each) were dissolved in 3 liters of chloroform, dosed with 0.50 mg. of each of the six compounds, and then concentrated to 150 ml. The concentrated extracts were transferred to 250-ml. Erlenmeyer flasks and evaporated on a warm water bath (70" C.) with a gentle stream of air. Two of the samples were evaporated to apparent dryness according to the normal procedure and two were evaporated to a 10-ml. volume. Results arid Discussioti Figure 1 illustrates the effectiveness of the method for cleanup of a dosed CCE. The removal of the bulk of the extraneous background material permits discrimination of
zz -
2
+
I
4
6 MINUTES
8
AFTER
IO INJECTION
IC
,
