Use of lyophilization in Determination of Moisture Content of Dehydrated Vegetables BENJAJIIS RI.AKOWER AND ELISiBETH XIELSE\ Western Regional Research Laboratory, Albany, Calif. .4 new reference method for the determination of moisture content of dehydrated vegetables involves addition of a large amount of water to a weighed sample of vegetable; freezing and drying in the frozen state (lyophilization); and completion of the drying in a vacuum oven or vacuum desiccator in the presence of an efficient water adsorbent. The last step can be completed in a relatively short time at, or slightly above, room temperature, because of a marked increase in drying rate brought about by lyophilization. Data presented for white and sweet potatoes, beets, and carrots show that the lyophilized materials can be dried unambiguously to constant weight and that the loss in weight may be taken as a measure of the moisture content. As the final dry weight is virtually independent, within wide limits, of the temperature of drying, the new method ohviates the necessity of careful control of drying temperature.
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N .4 recent publication from this laboratory (2) it was shonn that the determination of water in dehydrated vegetables by heating in a vacuum oven a t elevated temperatures requires calibration to establish the proper time and temperature of drying for different vegetables. The calibration is necessary because there is usually no clear-cut end point observed in the drying and there is no simple way of distinguishing R-hether the progressive, slow loss of weight of the sample on prolonged drying is due to thermal decomposition or to slow removal of adsorbed water. Two methods of calibration were described ( 2 ) . One is the primary reference method, which consists of drying to constant weight over magnesium perchlorate in evacuated desiccators a t room temperature. Because this method is exceedingly slow, requiring about 6 months for the samples to reach constant eight, another more rapid, secondary reference method was devised. It involves a so-called "redrying procedure," nhich consists of dctermination of time necessary to remove a knovm amount of water that was previously added to an essentially dry sample. The time required to carry through the redrying procedure amounts to several weeks, and it is evident therefore that a still more rapid method is highly desirable, The factor responsible for the slowness of the two methods is the rate of diffusion of water through the tissues a t temperatures low enough to prevent thermal decomposition. The fact that finer grinding, up to a practicable limit, does not increase the speed of drying sufficiently has been demonstrated ( 2 ) . However, the authors have obtained a very marked increase in the drying rate with samples of dehydrated vegetable first Jaturated with water to form a slurry, then frozen, and subsequently dried in vacuo in t'he frozen state ( I ) (lyophilized) to a low moisture content. The increase in the drying rate may be attributed to two factors. One is the increase in the volume of the lyophilized tissue, For when water is added to dehydrated vegetables the tissue swells and on subsequent drying only very little shrinkage occurs ( 8 ) . The other factor is the increase in porosity caused by leaching of soluble materials from within the tissue. The leached materials, principally sugars, appear after lyophilization as a distinct, loosely packed, porous layer. The effectiveness of lyophilization is demonstrated by the fact that the speed of drying of diced carrots (approximately 0.5-em. cubes) lvas much faster than that of the same carrots, unlyophilized but ground to pass a sieve' It has been found that the lyophilized materials can be dried rapidly t,o constant weight in a vacuum oven a t suitable temperatures,
and that the loss in weight is the same as in drying in a desiccator at room temperature. Thus the procedure may serve as a basis for a rapid reference method for moisture content. The results of measurements on sweet potatoes, white potatoes, beets. and carrots are described in this paper. EXPERI3IEKTAL
Materials. The dehydrated vegetables used had been prepared from ran' materials which were diced, blanched, and dried ivith hot air to a moisture content of about 7% in the pilot plant of this laboratory. Procedure. The experiments consisted of determinations of loss of weight as a function of time, a t various temperatures, on lyophilized and unlyophilized samples of the several vegetable8 under study. The operation involved successive dryings and weighings of the same sample a t various time intervals. Drying at elevated temperatures was conducted in a vacuum oven a t pressures of 0.05 to 0.15 mm. of mercury. A trap cooled in solid carbon dioxide was maintained between the oven and the oil pump. Drying at room temperature was done over anhydrous magnesium perchlorate in evacuated desiccators. All measurements were made in duplicate on dehydrated vegetables ground to pass a 40-mesh sieve, on samples m-eighing approximately 2
Table I.
Results of Vacuum-Oven Drying Experiments on Lyophilized and Unlyophilized Vegetables
Loss in K e i g h t (%) a t Various Times (HoursIa 3 6 22 38 60 100 140 180 Sweet Potatoes Y o t Lyophilized 70 0 5.40 6.27 '7:57 7.89 8.07 8.25 8.33 60 0 4.54 5.28 6.78 7.16 7.45 7.77 7.88 8:bO Sweet Potatoes, Lyophilized 5.05 7.97 7.98 8.27 8.28 8.27 8.27 8.25 70 8.00 8.01 7.88 8.19 8.23 8.23 8.25 8.24 8:2l 60 White Potatoes, Not Lyophilized 70 0 6.56 7.28 8.33 8.54 8.68 8.80 8.86 60 0 5.93 6.61 8.01 8.25 8.47 8.67 8.76 8:84 White Potatoes Lyophilized 70 5.05 8.30 8.33 8.b5 8.77 8.76 8.77 8.77 60 8.71 8.57 8.29 8.82 8.85 8.84 8.87 8.87 8:82 Beets U o t Lyophilized 70 0 3.84 4166 6.00 6.35 6.58 6.80 6.89 60 0 3.13 3.82 5.28 5.76 6.07 6.40 6.56 6:il Beets Lyophilized 70 5.54 6.50 6.52' 6.75 6.82 6.82 6.84 6.84 60 6.10 6.59 6.27 6.83 6.87 6.90 6.95 6.95 6192 Carrots Kot Lyophilized 70 0 5.01 5.80 7.09 7.40 7.59 7.78 7.89 60 0 3.99 4.74 6.26 6.67 6.95 7.22 7.30 7:43 Carrots Lyophilized 70 5.90 7 . 1 8 7.23'7.56 7.63 7.67 7.76 7.82 60 6.42 7.12 7.09 7.37 7.44 7.45 7.50 7.53 7 : 5 l Results represent averages of two samples. Values for lyophilized samples are expressed as peroentages of original weight bqfpre !yophilieation. Values a t zero time are losses t h a t occurred during lyophilization.
Temterature, C.
(1
856
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V O L U M E 20, NO. 9, S E P T E M B E R 1 9 4 8 Table 11. Drying of Lyophilized Vegetables in Vacuum Desiccators over Magnesium Perchlorate, at Room Temperature Loss in Weight at Various Times, 70" Sweet White potatoes potatoes Beets Carrots 0 5.05 5.43 7.96 5.55 1 8.14 6 . 1 4 8.66 6.38 4 8.18 6.36 8.69 6.77 11 8.22 6.52 8.70 6.96 19 8.24 6.57 8.70 7.05 8.23 6.58 27 8.68 7.08 35 8.22 6.61 8.69 7.15 43 8.23 6.62 8.68 7.15 50 8.26 6.63 8.70 7.18 8.22 64 6.65 8.70 7.23 6.67 78 8.67 7.20. 8.23 99 6.67 7.10 8.23 8.66 164 6.71 8.65 7.18 8.23 214 6.70 7.20 8.22 8.66 ' Percentages of original weight of samples before lyophilization. Time, Days
grams each. The differences in the measured losses of weight among duplicates were usually not greater than o.0370. Reproducibility for replicates not dried a t the same time is estimated to be only =+=0.1%. Other details of the oven-drying operations and the precautions taken were the same as described previously ( 2 ) . Lyophilization. The lyophilization procedure was carried out in the following manner. Approximately 2-gram samples of ground vegetables were weighed into cylindrical, ground-glassstoppered weighing bottles, 4 em. in diameter and 5.5 cm. high, 15 to 20 ml. of water saturated with toluene were added to each bottle, and the mixture was allowed to stand overnight in a cold room a t about 5' C. to allow time for rehydration. Toluene and low temperature were required to inhibit microbiological activity. The rehydrated samples (in the form of a slurry) were frozen and cooled to about -70" C. by immersion in a tray containing a slurry of solid carbon dioxide in ethyl alcohol. They were then quickly transferred to a lyophilization apparatus where they remained for about 20 hours (overnight) and dried to a moisture level of about 2 to 3%. This apparatus consisted of a Pyrex desiccator ( 2 5 em. in diameter) connected by glass tubing and ground-glass joints to a 1-liter 3-necked flask which served as a trap for water vapor when immersed in a gallon Dewar flask filled with a mixture of solid carbon dioxide in alcohol. Two spherical ground-glass joints (one horizontal and one vertical) were included in the connecting tube to enable the operator to loosen the desiccator Lid and either to slide away or attach the bottom part of the desiccator without disengaging any connections. The connecting tube also contained a side tube attached to a vacuum gage. One neck of the flask served as a connection to a vacuum pump (mechanical, oil type) and another as an air inlet through a stopcock. .4bout ten sample bottles of materials to be lyophilized were placed on a porcelain plate in the desiccator. After evacuation of the apparatus, the plate was heated by a 6O-watt lamp placed about 20 cm. (8 inches) beloiv the bottom of the desiccator. With this arrangement, the temperature of the samples was between 25' and 30" C. and the pressure in the system was about LO microns, a t the end of the drying. The temperature was read through the wall of the desiccator on a short mercury thermometer which was embedded inside a duplicate sample of one of the vegrt able?. RESULTS
The results of the vacuum-oven drying experiments a t elevated temperatures on lyophilized and unlyophilized materials are sho~vnin Table I. The samples dried a t 60' C. were kept in the lyophilizing apparatus a few hours longer than those dried a t 70' C. and for that reason the former contained very little residual moisture before oven drying (at zero time). These nearly dry samples, when heated in the oven for only a 3-hour period (see results in Table I for 3 and 6 hours of drying), either did not lose any weight or actually gained some weight. They were, of course, very hygroscopic and picked up some moisture when first placed in the oven. Evidently a 3-hour drying period Tvas not sufficient to remove the regained watrr. The results for lyophilized materials dried in vacuum desiccators a t room temperature are shown in Table 11. The data for sweet potatoes are also shown graphically in
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Figure 1. It is apparent that unlyophilized sweet potatoes undergo continuous weight loss over the whole drying period and there is no clear indication that drying is complete in 140 to 180 hours. After 140 hours there is still a considerable difference (0.6%) between the losses in weigh? a t 70" C. and at 60' C It can only be inferred that the two drying curves might become asymptotic after a much longer drying time. The curves for lyophilized samples show that constant weigh? is reached unambiguously in 38 and 22 hours, a t 60 ' and a t 70 O , respectively. Furthermore, the final loss in weight a t 70" (8.3%7,)is the same within experimental error as that at 60' (8.27,) and that a t room temperature (8.2%) (cf. Table 11). From this agreement and the fact that the loss in weight in a desiccator a t room temperature is taken to be the moisture content of the material ( 2 ) , it follows that vacuum-oven drying of lyophilized sweet potatoes a t 60" or 70" C. may be used as a rapid secondary reference method for moisture determination. Similar conclusions may be drawn from data for beets and white potatoes. As with sweet potatoes, constant weight was attained only with the lyophilized samples. The agreement in the loss of weight a t 70', 60°, and room temperature waq fniind to be JTithin about 0.27, or better.
d-d-
NOT LYOPHILIZED
71 1270'c. c
LYOPHILIZED
b d
6-
50
100
1
-- 60'C.
70%
150
I I 1
eo0
TIME, HOURS
Figure 1 .
Dr?ing Curies for Lyophilized and Unlyophilized S w e e t Potatoes at 60" and 70" C.
From the fact that constant Teight is reached with some lyophilized materials dried a t 70" C., one might assume that decomposition rate a t 70" is negligibly small and that the slow change observed in unlyophilized vegetables at 70 ' C. is entirely due to slow diffusion of water. If that were true, it would follow that unlyophilized would eventually reach the same constant weight as lyophilized materials. It appears from data in Table I, however, that the eventual loss of weight in unlyophilized would be greater than in lyophilized samples. This observation can be interpreted only on the assumption that measurable decomposition docs occur when moisture is present in the vegetable, but is negligible for materials in the dry state. Thus some decomposition would be expected t o occur in the drying of unlyophilized vegetables because they remain in the moist state for a long period a t 70". Further support for this explanation is afforded by the observation (unpublished data) that an increase in the measured moisture content occurs n-hen dehydrated vegetables are stored a t elevated temperatures. Carrots differed from the other vegetables in some respects. Constant !wight was not reached in drying of lyophilized samples a t 70" but was attained at 60" C. Failure to reach constant
ANALYTICAL CHEMISTRY
858 weight a t 70 O C. was probably caused by thermal decomposition, which in this case is apparently appreciable even in the dry state. The final loss in weight for carrots a t 60" C. was, however, greater by about 0.3% than the value obtained in desiccator drying at room temperature. This discrepancy exceeds only slightly the estimated experimental error for replicates (*O.l'%j but if it is real, it might be attributed to loss of volatile substances other than water. The other vegetables are probably subject to the same error, as evidenced by somewhat lower losses of weight at room temperature than a t 60" or 70" C., but the magnitude of this effect is much smaller and is not readily distinguishable from the experimental error. From the behavior observed with carrots it appears that materials containing appreciable quantities of nonaqueous volatile substances can be dried only by the desiccator method. That this method is not prohibitively long with lyophilized materials is shown by the data in Table 11. The actual time necessary to remove all but the last 0.1% of the weight was less than 4 days for white and meet potatoes and about 30 days for beets and carrots. With higher temperatures (40' to 50' C.) the time can probably be greatly shortened without appreciable error from thermal decomposition, as shown by the data for the vacuumw e n experiments. In contrast, the drying time for unlyophilized vegetables, under similar conditions, was found to be 6 months or more ( 2 ) . From the moisture values obtained in lyophilization experiments and from the drying experiments on unlyophilized mate-
rials, it is possible to establish the drying times a t 70" C. that would be required in a routine vacuum-oven moisture determination on unlyophilized vegetables. For example, if the moisture content of sweet potatoes is taken to be 8.2%, the drying time at 70" C. for unlyophilized samples, ground through a 40-mesh sieve, is found from the corresponding drying data in Table I to be about 100 hours. Similarly, the time is 100 hours for white potatoes and beets, and 30 hours for carrots. It is of interest to compare these calibrations with those obtained previously by the primary reference method and redrying procedure, although it is not possible to make the comparison rigorous because different materials were used in the two tests. The drying times found previously were: beets and sweet potatoes, 100 to 120 hours (unpublished data); carrots, 29 to 35 hours ( 2 ) ; white potatoes, 43 to 67 hours ( 2 ) . The agreement with the lyophilization method is good, as the moisture values obtained with either calibration would not differ in any case by more than 0.2%. These drying times are excessively long for routine work, and to shorten the time it would be necessary to employ higher temperatures. LITERATURE CITED
Flosdorf, E. W., J. Chem. Education, 22, 470 (1945). (2) Makower, Benjamin, Chastain, S. M.,and Nielsen, Elisabeth, Ind. Eng. Chem., 38, 725 (1946). (3) Wyckoff, W. G., Science, 104, 36-7 (1946). (1)
RECEIVED December 29, 1947,
Detection of Chlordan (Octachloro-4,7-methanotetrahydroindane) in Insecticide Oli Sprays J. S . ARD', Production and Marketing ridministration, t - .S . Department of Agriculture, Reltscille, M d . Chlordan, under the influence of heat, pyridine, and alcoholic alkali, reacts with ethylene glycol monoethyl ether to give an intense red color that distinguishes it from other common ingredients of insecticide oil sprays, and is a quantitative indication of the amount present. During dehalogenation with sodium and isopropyl alcohol, a characteristic odor and temporary darkening occur which serve as confirmatory evidence.
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EPRESESTATIVES of the companies making the product and of the various federal departments concerned have agreed upon the name chlordane for a new insecticidal ingredient whose chemical structure is said to be represented by 1,2,4,5,6,7,8,8-octachloro-4,7-methano-3a,4,7,7a-tetrahydroindane (f,4). It has previously been distributed in technical forms under the name Velsicol 1068 (Velsicol Corporation. 330 East Grand Ave., Chicago 11, Ill.), and also advertised under the name Octa-Klor (Julius Hyman & Co., Denver, Colo.). The purified material is stated to be a colorless, viscouq liquid boiling at 175" C. under 2-mm. pressure (3). A 20% concentrate of the product was furnished the United dtates Department of Agriculture by the Velsicol Corporation. Most of the concentrate, rvhich is of the nature of a solvent for the chlordan, distilled between 95' and 110' C. under 2-mm. pressure, and the color test described below was negative on this fraction a t a 10% concentration. The viscous oil, which is reported to be the highly active fraction of the commercial concentrate (3), and to which the 20% claim and now the name chlordan refer, distilled a t 175" under 2-mm. pressure. This fraction was found to contain the portion that gives the color reaction in the proposed 1 Present address, Bureau of Agricultural Beltsville. Md.
and Industrial Chemistry.
test, and was positive in a 0.2% concentration. A specimen of the commercial 90% concentrate of chlordan (Velsicol 1068) was later tested and gave an intensity of color approximately equivalent to the latter fraction. Concentrations hereafter expressed are in terms of chlordan; the quantities are divided by 5 whenever the commercial 20y0 concentrate was used. DETECTION OF CHLORDAN IN .MINERAL OIL SOLUTIONS
Reagents. Cellosolve-Pyridine Solution. Mix 1 0 ml. of pyridine with 40 ml. of Cellosolve (ethylene glycol monoethyl ether j. Alcoholic Potassium Hydroxide Solution. An approximately 1 N solution of potassium hydroxide in 95% ethyl alcohol, sufficiently fresh to be colorless. Color Test. In a test tube, mix 1 ml. of the sample oil, 2 ml. of the Cellosolve-pyridine solution, and 1 ml. of the alcoholic potassium hydroxide solution. Heat in a boiling water bath with occasional agitation for 5 minutes. A 1-ml. sample of 0.2% chlordan in a deodorized kerosene base gives a wine-red color of considerable strength, and 1% gives an intense dark red color. Very weak colors should be regarded as possibly due to other substances. QUANTITATIVE APPLICATIONS
The colors are soluble and stable in numerous solvents. Sui& able solutions for photometric comparisons can be obtained by
