Freeze Concentration of Organic Compounds in Dilute Aqueous Solutions Phil A. Kammerer, Jr.,l and G . Fred Lee Water Chemistry Laboratory, University of Wisconsin, Madison, Wis. 53706
Solutions of 14C-labeled glycine, glucose, citric acid, and phenylalanine in distilled water and lake water, and distilled water solutions of lindane, were freeze-concentrated by freezing them in stainless steel buckets with constant agitation. Samples of various volumes (800 ml. to 16 liters) with initial solute concentrations ranging from 0.01 to 1.O mg. per liter were concentrated by factors of 3 to 22. Recoveries of the organic compounds approached 100%. Solute losses in freeze concentration seem to be due, at least in part, to nonspecific entrapment that can be controlled by careful choice of operating parameters for the freeze concentration process used.
I
n recent years, there has been increasing interest in dissolved organic substances in natural and waste waters. Usually, a water sample must be concentrated before dissolved organic substances may be studied. In investigations involving solutes whose chemical and physical properties are poorly defined or not completely understood, nonspecific solute concentration is often desirable, and is best achieved by solvent removal. Freeze concentration is a desirable means of solvent removal, since the low temperatures used should minimize the loss of volatile compounds and reduce the possibility of chemical o r biological alteration of the compounds being concentrated. Laboratory scale freeze concentration processes have been carried out both under quiescent conditions and with agitation (Kammerer, 1967). Freezing under quiescent conditions generally only enriches solutes in the last portion of the solution to freeze; recovery of the concentrated solutes is not quantitative. Freezing with agitation is more useful where high concentration factors and recoveries are desired. Evaluations of freeze concentration with agitation as a means of concentrating aqueous solutions of organic compounds have been carried out o n distilled water solutions (Baker, 1965, 1967a; Kobayashi and Lee, 1964; Shapiro. 1967; Wilson, Evans, et al., 1964) and solutions containing various inorganic salts (Baker, 1967b). With the exception of the two recent papers by Baker (1967a, b ) , previous studies have been fragmentary, and the recovery efficiency Present address, U. S. Geological Survey, Water Resources Division, Madison, Wis. 53705. 276 Environmental Science & Technology
reported for various solutes has been variable. No evaluations have been carried out on untreated fresh water. The purpose of this study is to evaluate the recovery of trace amounts of certain organic compounds from distilled water and from natural fresh water by freeze Concentration.
Experimental The freeze concentration apparatus used was similar to one previously described (Shapiro, 1961). A chest-type freezer was filled with a mixture of ethylene glycol and water as a heat exchange medium. A plywood platform. with holes to support sample containers, was placed in the freezer; vertical rods adjacent to the holes supported the stirring motors. Lucite covers were placed over the sample containers during freeze concentration runs. The temperature of the cooling bath was maintained at -20" to -25°C. Samples, in stainless steel pots. ranging in volume from 800 ml. to 16 liters, were placed in the freezer. and stirring was started immediately, The level of the cooling bath was maintained at 1.5 to 2.5 cm. below the surface of the sample. After about 10 minutes, most samples supercooled. causing sudden crystallization that formed a thick slush in the sample pot. The slush prevented adequate mixing of the sample and was dispersed by immersing an electric heater in the sample for about 1 minute while stirring was continued. Runs could be carried to a maximum concentration factor of 6 to 8 before it became necessary to transfer the concentrate to a smaller pot. On completion of a run, the sample pots were removed from the freezer and the concentrates were poured off. The evaluation of the recovery of trace organics by freeze concentration depends on the availability of sensitive, specific analytical methods for the compounds studied. Conventional spectrophotometric methods for the determination of organic compounds are generally subject to interferences and other complications when applied directly to the analysis of concentrated samples of natural water. For this reason, the analyses in this study were carried out using 14C-labeled organic compounds or using gas chromatography as a means of separating the compound to be analyzed from interfering substances. The I4C analytical procedure used is a modification of a procedure described by Bomstein and Johnson (1952). Aqueous solutions of radioactive material were mixed with a gelatin solution, and an aliquot of the mixture was transferred to a planchet, dried, and counted. Glycine. glucose. phenylalanine. and citric acid were the 14C-labeled compounds chosen to evaluate the recovery of
organic compounds using freeze concentration. Since they are water-soluble and nonvolatile, their concentration in aqueous solutions is relatively easy to control, and any losses experienced during freeze concentration runs can be attributed to degradation or entrapment in the ice, rather than to volatility. Collectively, these compounds represent a wide range of structures and functional groups, some of which are likely to be present in the dissolved organic fraction of natural waters. Heiss and Schachinger (1951) reported that acid components of fruit juices (malonic, malic, tartaric, and citric acids) were concentrated in the ice during commercial freeze concentration of the juices because of their capillary activity. The freeze concentration process used by Heiss and Schachinger involved cooling fruit juices until an ice slush formed. The unfrozen liquid (concentrated fruit juice) was then allowed to drain out of the slush. The fruit acids mentioned above were reportedly trapped in capillary spaces in the slush. Citric acid was chosen as a test compound in this study to see if losses of the type experienced by Heiss and Schachinger were typical of freeze concentration processes in general. Two series of freeze concentration runs were carried out using these 14C-labeled compounds, one using distilled water solutions and the other solutions prepared with Lake Mendota water, which had been filtered through Whatman No. 1 filter paper. Lake Mendota water was chosen as a test medium because it is hard and eutrophic, and contains about 10 mg. of dissolved organic carbon per liter. Concentration of the water is necessary to study the dissolved organic carbon fraction, but concentration of Lake Mendota water by freezing presents special problems. Peterson ( 1966) encountered problems with calcium carbonate precipitation while freeze-concentrating Rhodamine B in Lake Mendota water. Turbidity, color, and precipitated solids prevented accurate Rhodamine B analyses on the concentrates. leading to variable recovery data. The 14C analytical procedure used in this study is not affected by turbidity. color. or precipitated solids, and recovery data obtained using lake water should be more accurate. Accurate recovery data for Lake Mendota water may help to define the usefulness of freeze concentration for concentrating hard. eutrophic waters. Distilled water solutions of lindane. a chlorinated hydrocarbon pesticide. were also freeze-concentrated. Lindane analyses were performed on hexane extracts of aqueous samples using a gas chromatograph equipped with an electron-capture detector. Hesitlts and Discirssiori
Distilled Water. Two runs, consisting of three samples each, were made f o r each 14C-labeled compound (Table I ) . In the first run. the initial concentration ranged from 0.01 3 to 0.023 mg. per liter; in the second, the initial concentration was 1.0 mg. per liter. Sample volumes ranged from 800 to 1300 ml. The samples \sere concentrated by factors ranging from 3.3 to 6.3; about 4 hours were required to achieve these concentration factors. One run was carried out on a 16-liter sample containing 0.1 mg. of glycine per liter; this sample was concentrated by a factor of 21.8 i n two steps. The time required for the entire run was 13.5 hours. The calculated counts per minute shown in Tables I and 111 were computed from the initial activity of the samplc and the concentration factor.
Table I. Recovery of '*C-Labeled Compounds from Distilled Water
Compound
Glucose
Glycine
Phenylalanine
Citric acid
Initial Concn., Mg./L.
4 , ,Concn. _ _ _C.P.M. _ Factor Calcd. Found Recovery
0.022 0.022 0.0 I9
4.04 4.56 3.33
367 414 246
340 3 62 216
93 88 88
1 .o 1 .o
1 .o
6.05 5.65 5.7 1
398 495 407
384 504 402
97 102 99
0.023 0.0 17 0.019
6.35 5.42 4.70
5 04 327 308
52 1 33 1 306
I03 101 99
1 .o 1 .o 1 .o 0.10
4.9 I 5.17 4.35 21.8
394 31 1 284 29 I
404 308 282 294
102 99 99
0.01 3 0.0 17 0.013
6.25 4.96 4.54
3 67 388 289
358 389 308
100 1 Oh
1 .o 1 .o 1 .o
5.75 4.00 3.14
338 313 200
303 299 I96
90 96 98
0.0 5 0.0 0 0.0 6
5.42 4.73 4.04
348 405 28 1
338 458 288
97 I13 IO2
I .o
6.02 6.04 4.65
515 388 323
478 403 319
93 IO4 99
1 .o 1 .0
101
98
Recoveries of glycine, phenylalanine, and citric acid approached 100% within the concentration extremes tested. One hundred per cent recovery of glucose was achieved at 1.0 mg., but not at 0.02 mg. per liter. Recoveries for samples with an initial glucose concentration of 0.02 mg. per liter averaged 9 0 % . The low glucose recovery may be due to experimental error rather than to the low initial concentration used, since glucose recovery from lake water at an initial concentration of 0.13 mg. per liter approached 100%. A recovery of 101 % was obtained for the single run on a 0.10 mg. per liter glycine solution. Citric acid recoveries were somewhat variable, but the average recovery was 1 0 0 % . The difference between these results and those reported by Heiss and Schachinger ( 195 1 ) may be a result of differences in the freezing processes used. Five aqueous lindane solutions, with initial concentrations of 13 to 14 pg. per liter. were freeze-concentrated (Table 11). Recoveries were more variable than those obtained with '.T-labeled compounds; they ranged from 88 to 101 52. The average recovery was 9 4 % . Filtered Lake Mendota Water. One run, consisting of three samples. was made for each IT-labeled compound (Table 111). The initial concentrations of the samples were 0.12 to 0.15 mg. per liter. Sample volumes ranged from 800 to 1300 ml. The samples were concentrated by factors ranging from 3.3 to 7.4. with about 4 hours required for each run. One run was carried out on a 16-liter sample Volume 3, Number 3, March 1969
277
Table 11. Recovery of Lindane from Distilled Water Initial Sample Volume, MI.
Concentration Factor
1300" 1300"
5.20 5.42 5.86 5.00 4.85
Final Lindane Concn., /lg./L. Calcd. Found
69.5 72.3 81.5 69.6 67.5
70.5 71.5 71.5 64.8 60.3
%
Recovery 101
99 88 93 89
Initial lindane concentration 13.4 @./I. Initial lindane concentration 13.9 pg./l.
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Table 111. Recovery of 'W-Labeled Compounds from Filtered Lake Mendota Water
Compound
Glucose
Phenl lalanine
Glycine
Citric acid
Initial Concn., Concn. Mg./L. Factor
~
C.P.M. 70 Calcd. Found Recovery
0.12 0. 15 0.12
5.00 5.27 3.33
340 478 246
340 461 242
0.12 0.15 0.12
5.54 4.73 3.48
361 411 246
358 410 237
0.12 0.15 0.12 0.10
7.43 5.65 5.33 4.5
512 519 398 223
522 519 393 237
102
0.12 0.15 0.12
7.03 6.38 5.33
581 701 478
574 680 433
99 97 91
100
96 98 99 100
96 100
99 106
containing 0.1 nig. per liter of glycine. This sample was concentrated by a factor of 14.5 in two steps and about 12 hours were required to complete the run. Recoveries of glucose, phenylalanine. glycine. and citric acid approached 100%. A recovery of 106% was obtained for the single run on a 0.10 mg. per liter glycine solution. Calcium carbonate precipitation took place in about 6 0 8 of the lake water samples concentrated. after they had been removed from the freezer. During the freeze concentration runs, the concentrated solutions were apparently supersaturated with respect to calcium carbonate, but no visible precipitation occurred; therefore, the effect, if any, of precipitation during the course of a run was not determined. Calcium carbonate precipitation would probably occur more readily in freeze concentration runs carried to large concentration factors over an extended period of time. Results of this and previous studies suggest that solute losses in freeze concentration of organic compounds, when they occur, are nonspecific for low molecular weight compounds. Losses may be due to solute entrapment at the ice-water interface or to experimental conditions in the freeze concentration procedure. Baker (1967 b) has reviewed the theoretical aspects of solute entrapment at the ice-water interface. Agitation has been shown to be a n important experi278
Environmental Science & Technology
mental variable in freeze concentration procedures. Baker (1967a, b) supplied agitation by rotating the sample in a flask on a rotary evaporator during freezing. H e found that recovery efficiency of organics was not influenced by mixing (rotation) rate in distilled Mater systems, but that the addition of inorganic salts to samples made the recovery of organics highly dependent on mixing rate. Shapiro ( 1961 ) freeze-concentrated a solution containing methyl violet, and noted that when stirring w'as interrupted, laminations of trapped dye mere visible in the ice. Sample agitation in the present study was supplied by propeller-type stirring motors. The stirrers were run at approximately 375 revolutions per minute; this rate prevented solute losses within the range of solute concentrations and concentration factors tested. Solute losses may also be caused by portions of the sample being trapped and frozen on ice surfaces that are not continuously washed or covered by the sample. Shapiro (1961) prevented solute losses caused by portions of the sample freezing on exposed ice shoulders in the sample containers by placing polyethylene displacers on the stirring rods used to agitate the samples. As freezing progressed, the stirrers (and displacers) were raised, causing the level of the liquid to fall below the ice shoulders. In the present study. the level of the coolant in the freezer was kept below the level of the liquid in the sample containers; this prevented the formation of ice above the surface of the sample. Concl~ision~
On the basis of this and previous studies. freeze concentration appears to be an effective method for concentrating water-soluble, low molecular weight organic compounds in distilled or fresh water. Essentially complete recoveries of solutes ranging in concentration from a few micrograms to milligrams per liter levels may be obtained for moderate concentration factors ( 0 to 2 0 ) . Ackno~~ledgincnt
The authors thank Frank Boucher for performing the lindane analyses. Literature Cited Baker, R. A,, J . Water Pollution Control Fed. 37, 1164-70 ( 1965). Baker. R. A,, Water ReJ. 1, 61-7 (1967a). Baker, R. A.. Water ReJ. 1 , 97-1 13 (1967b). Bonistein, R. A,. Johnson, M. J., J. B i d . Cheni. 198, 14353 ( 1 9 5 2 ) . Heiss. R., Schachinger. L.. Food Technol. 5, 2 I 1-8 ( 195 I ) . Kaninierer. P. A , , Jr.. M.S. thesis, University of Wisconsin, Madison, 1967. Kobayashi. S.. Lee, G . F., Anal. Chern. 36, 2197-8 (1964). Peterson. J. 0.. Water Chemistrv Program. Universitv of Wisconsin, personal communication. '1966. Shapiro. J., Anal. Chein. 39, 280 (1967). Shapiro. J., Science 133, 2063-4 (1961). Wilson, T. E., Evans, D. J.. Theriot, M. L., Appl. Microbioi. 12, 96-9 ( 1964). Received for rebliew January 24, 1968. Accepted Jaiiuury 8, 1969. Investigation supported by Training Grant N o . STI-WP-22 and Rerearch Grant N o . WP-00371, Federal Water Pollution Control Administration. 111 addition, support ~.'a.s given by the University of Wiscon.sin Engineering Experiment $ttitioii rind the Depnrtment of Civil Engineering.
