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Anal. Chem. 1981, 53,543-544
Extraction of Rubber from Guayule Using Liquid Nitrogen Sir: The development of a fast and accurate method to extract rubber from guayule (Parthenium argentaturn Gray) has eluded researchers for many years. Several different solvent methods have been presented with use of acetone, petroleum ether, ethyl alcohol, benzene, carbon tetrachloride, and sulfuric acid to extract the rubber (1-3), These methods generally utilized various types of fine grinding, blending, or crushing of the shrub to break open the rubber filled cells. Recently, two methods have been developed to determine rubber quantity without solvents. Mehta et al. ( 4 ) utilized trichome and leaf morphology as an indicator of high rubber bearing plants. Bauer (5) makes use of the anatomy of the plant for rubber storage by treating and staining microtomed thin stem sections. Both methods are rapid, but precision and accuracy have yet to be shown. There are two commercial extractions currently being considered. The Mexican pilot plant a t Saltillo, Coahuila, utilizes a water-NaOH-acetone system (6). Firestone Rubber Co. has suggested an acetone-hexane direct solvent process for commercial processing of guayule rubber (7). The use of liquid nitrogen is proposed as a method to eliminate the length of time required for drying in the conventional extraction of guayule. The rapid freezing of fresh plant cells, fracturing of frozen membranes during grinding, and subsequent thawing rupture the cells to allow rapid exposure of the rubber to the solvent without extensive grinding (&IO), and a t the same time the frozen tissue can be easily milled without predrying. The method provides a quick method of sample preparation, and subsequently enhances precision comparable to lengthy chemical extraction. EXPERIMENTAL SECTION A 2-year old guayule plant (Parthenium argentaturn line N563) growing in Tucson, AZ, was cut approximately 8 cm above the root crown in Feb 1980. The plant was parboiled for 15 min, chopped into lengths of 2.5-5.0 cm, and frozen in liquid nitrogen. The frozen material was immediately ground in a Wiley mill with a 2-mm screen, mixed to give a homogeneous sample and stored at -20 "C. Dry weights were determined by drying at 80 "C for 24 h. Replicated fresh weights (5,10,20,or 30 g) were each homogenized for 10 min in 100 mL of acetone (AR) at 65% power in a Virtis "45" homogenizer. The mixture was vacuum filtered through Whatman No. 1 paper and the cake rinsed with 50 mL of acetone. The filtrate was dried to constant weight at ambient temperature and the residue referred to as acetone solubles. The filter cake was air-dried and homogenized with 100 mL of hexane (AR)for 10 min at 50% power with the homogenizer. The homogenate was centrifuged at 13 200g for 20 min and the supernatant fdtered through a Kimwipe (Kimberly-Clark) and dried as described above. This yielded the rubber fraction. The residue was reextracted twice with hexane to determine percent recoverable rubber. The recommended procedure is outlined in Figure 1.
RESULTS AND DISCUSSION Results of the extraction with N563 are given in Table I. Weights and percentages were calculated on a dry weight basis due to variation in percent moisture at time of extraction. Low variation was found for acetone solubles and considerably less for total extractable rubber and percent rubber. Total extractable rubber is based on the sum of three hexane extractions. Calculated weights for extraction 3 were small and variable. The residue from hexane solubles of extraction 3 may include substances other than rubber. Percent of total extractable rubber for individual extraction levels at each sample weight is given in Table 11. As sample size increased, 0003-2700/81/0353-0543801 .OO/O
HARVESTED PLANT
I I AIR DRY 60 MIN
PARBOIL 10-15 MIN-LEAVES
I
CHOP INTO 2 5 - 5 . 0 CM-SUBSAMPLE
I
FREEZE IN LIQUID N2
I
HAMMERMILL 3 MM SCREEN STOR
i
- 2 0 C-1-3
I 5 - 3 0 G FRESH WT
GM DRY WEIGHT
I
10MIN 100ML ACETONE IN VlRTlS VACUUM FILTER -FILTRATE
I
FILTER CAKE
I
DRY-ACETONE SOLUBLE5
I
IOMIN 100ML HEXANES IN VlRTlS
I
FILTER 0.5MM SCREEN-RESIDUE
I
-I
DISCARD TRASH-FILTRATE CENTRIFUGE -RINSE 15 ML 13,200 X G 2 0 MIN HEXANES
1-
I
DRY FIBER
SUPERNATANT
I
DRY FOR RUBBER
Figure 1. Recommended procedure using the liquid nitrogen rubber
analysis. Table I. Plant Acetone Solubles and Extractable Rubber in Variable-Sued Samples of N563 Guayule fresh wt, g
dry wt,g
5
3.31
10
6.23
20
12.57
30
16.53
acetone solubles total rubber wt, g wt, g %rubber 0.17 i 0.012 0.34 i 0.024 0.71 f 0.077 1.07 i 0.042
0.24 ?: 0.020 0.46 t 0.013 0.90 t 0.021 1.21 f 0.000
7.68 t 0.006 7.40 t 0.002 7.20 t 0.002 7.40 t 0.000
Table 11. Percent of Total Extractable Rubber for Each Hexane Extraction at Variable Sample Sizes of N563 dry wt, 3.31 6.23 12.51 16.23
extraction 1
78.63 82.05 75.53 71.90
% extractable rubber extraction extraction 2 3
10.65 14.68 19.20 20.65
10.65 3.30 5.60 7.40
total 99.93 100.03 100.33 99.95
~~
percent of hexane extractable rubber decreased in extraction 1 and increased in extraction 2. This may be due to hexane-sample size differential. The observed change in solvent-extracted residues at each sample size were linear. Accountable variation is demonstrated as r2 values: total hexane extractables (r2= 0.999), acetone solubles (r2= 0.990), and the first and second hexane extractions (? = 0.996 and ? = 0.998, respectively). The third hexane extraction was lower with r2 = 0.819. The accuracy of percent hexane extractable rubber was similar to that from benzene extraction (11). Of the three 0 1981 American Chemical Soclety
Anal. Chem. 1981, 53, 544-546
544
hexane extractions made on each sample, most of the rubber (averaging 77%) is removed in the first extraction. Small sample sizes provided higher first extraction percentages (Table 11). Additional improvements in technique have increased that t o an average of 85% with the first extraction of 5-g samples. Also small samples reduce cost, reduce time of analysis, and provide improved extraction percentages.
(8) Meryman, H. T. "Cryobiology"; Academic Press: London, 1966; Chapter I . (9) Bidwell, R. G. S. "Plant Physiology", 2nd ed.; Macmillan: New York, 1979; Chapter 28. (10) Anderson, J. O., University of Arizona, Department of Plant Sciences, Tucson, AZ, 1980, personal communication. (11) Garrot, Donald J., Jr., unpublished data, University of Arizona, Department of Plant Sciences, Tucson, AZ, 1980.
Donald J. Garrot, Jr.* D u a n e L. Johnson D. D. R u b i s
LITERATURE C I T E D (1) Hammond, B. L.; Polhamus, L. G. "Research on Guayule (Parthenium argentaturn): 1942-1959", U . S . Dep. Agric. Tech. BUN. 1985, No. 1327, 109-131. (2) Hanson, G. P. "Breeding Improvement of Rubber Yield in Guayule: 3rd Progress Report"; National Science Foundation: Washington, DC, 1978; pp 88-94. (3) Gresa, E. C., B. F. Goodrich Co., Brechsville, OH, 1980, Dersonal corn mudcation. (4) Mehta, I. J.; Dhillon, S. P.; Hanson, G. P. Am. J . Bot. 1979, 66, 796-804 (5) Baue,-T. "Guayule Natural Rubber Development Project: 1st Year Report"; California Department of Food Agriculture, Division of Plant Ind., Sacramento, CA, 1979; p 24. (6) "Guayule: An Alternative Source of Natural Rubber"; National Academy of Science; Washington, DC, 1977; Chapter 6. (7) Glymph, E. M.; Nivert, J. J., presented at the International Rubber Study Group: 25th Assembly; Offices of the Secretarht, London, 1978.
Department of Plant Sciences University of Arizona Tucson, Arizona 85721 Gerald M. Dill Department of Plant Pathology and Crop Physiology Louisiana State University Baton Rouge, Louisiana 70803
RECEIVED for review August 25, 1980. Accepted December 12, 1980. Financial support from the National Science Foundation, PFR-7911723, is gratefully acknowledged.
AIDS FOR ANALYTICAL CHEMISTS Concentration of Dilute Pesticide Solutions Gerd Puchwein Landwirtschaftlich-chemische, Bundesversuchsanstatf Linz, Wleningerstrasse 8, A-4025 Linz, Austria
In pesticide residue analysis the final determination of content very often uses methods such as gas chromatography, high-pressure liquid chromatography, or thin-layer chromatography requiring only a few microliters of a cleaned extract. On the other hand most cleanup procedures end up with a final volume of about 1-10 mL. The potential advantages of a substantial reduction of scale of cleanup procedures are obvious provided the sensitivity of the method is preserved (e.g., cut in costs for highly purified organic solvents, reduced hazard due to manipulating inflammable and/or toxic solvents). A prerequisite for a reduction of scale of cleanup procedures is a method for reliably concentrating very dilute solutions to a small and defined volume. The concentration step can be a major source of error due to uncontrollable losses of pesticides ( I ) . This especially holds true if a reduction of volume below 1 mL is desired. A few devices for concentrating such solutions to low volume have already been described (2-6). Usually special glassware is needed, which either renden these methods dependent upon a good glass blower or calls for the acquisition of special equipment, if commercially available (microconcentrator, Supelco Inc.). Our idea was to make use of commercially available parts as far as possible and to work with inexpensive disposable glass tubes as receiving vessels for the concentrated solutions. EXPERIMENTAL S E C T I O N Apparatus. The heart of the device (Figure 1) consists of a concentration vessel, B, with a bulb of 6 mL content and a Luer
tip end connected to a Hamilton hypodermic needle, N, with CTFE Hub (KF-722). The needle extends into the upper opening of a disposable 200-rL one-way pipet, R (Clay Adams, Micropet 200 pL), which is placed inside a T-shaped tube, T. Two ends are sealed gastight by screw caps, SC (Schott, GL 14), and a septum, S (Teflon rubber laminated disks, 12 mm, Pierce), at the upper end and a Teflon-lined silicon ring at the lower end. The glass tube, G, acts as a sleeve which firmly holds in place the receiving tube, R. It can easily be inserted into and withdrawn from tube T, after loosening the screw cap. The pipets with sealed lower ends serve as the receiving vessels for the concentrated solutions. The side arm of tube T is connected via a Swagelok reducing union (B-400-6-2)(not shown in the figure) and 1-mm Teflon tubing to a three-way ball valve (Whitey B-41XS2V). The two other outlets of the valve are connected to a nitrogen line and to a 10-mL disposable plastic syringe, by l/g in. copper tubing and 1-mm Teflon tubing, respectively. Nitrogen flow can be adjusted by a standard laboratory pressure regulator and a needle valve. An aluminum bar, A, 250 mm long, with square cross section (40 x 40 mm) and suitable bores holds five vessels, B. The aluminum bar is electrically heated at two of its long sides by a heating jacket, H, made from heating wire (length = 2.5 m, R = 50 Q ) sandwiched between mica sheets for electrical insulation. For thermal insulation the two heated long sides are covered with two asbestos (5 mm) and two PVC sheets (5 mm) while for all other sides Teflon foil (1.5 mm) is used. The heating jacket is connected to an ordinary laboratory power regulator (220 V); the aluminum bar is grounded. The temperature of bar A is controlled by a contact thermometer and a relay. The whole =tup comisthg of the heated bar, A, tubes, T, valves, and syringes is mounted on a panel, P, and placed in a hood. Only the concentration vessels, B, and the tubes, T, had to be made to order by a local
0003-2700/81/0353-0544$01.00/00 1981 American Chemical Society
