Anal. Chem. 1983, 55, 1814-1815
1814
for atomic spectrometry. In addition to furthering the analytical methodology, these promising preliminary results demonstrate that high-sensitivity LEI measurements are possible in adverse sample environments where traditional methods of optical spectrometry have proven inadequate.
(11) Kingston, H. M.; Barnes, I. L.; Brady, T. J.; Rains, T. C. Anal. Chem. 1978, 50, 2064. (12) Fuwa, K.; Vailee, B. L. Anal. Chem. 1963, 3 5 , 942. (13) Veiiion, C.; Margoshes, M. Spectrochlm. Acta, Part 6 1968, 236, 553.
Joan E. Hall Robert B. Green*
LITERATURE CITED (1) Travis, J. C.; Turk, G. C.; Green, R. B. Anal. Chem. 1982, 54, 1006A. (2) Green, R. 8.; Havriiia, G. J.; Trask, T. 0. Appl. Spectfosc. 1980, 3 4 , 561. (3) Turk, G. C. Anal. Chem. 1981, 53, 1187. (4) Vlckers, G. H.; Trask, T. 0.; Parll, J. D.; Wisman, J. A,; Durham, 6.; Green, R. B. Chem., Blomed. Envlron. Instrum. 1983, 12, 289. (5) Smith, B. W.; Parsons, M. L. J . Chem. Educ. 1973, 5 0 , 679. (6) St. John, P. A.: McCarthv, W. J.: Winefordner, J. D. Anal. Chem. 1967, 39, 1495. Turk, G. C.; DeVoe, J. R.; Travls, J. C. Anal. Chem. 1982, 5 4 , 643. Alkemade, C. Th. J.; Herrmann, R. “Fundamentals of Analytical Flame Spectroscopy”; Wiiey: New York, 1979; p 21. Gilbert, P. T., Jr. “Analytical Flame Photometry: New Developments, Symposium on Spectroscopy 3rd Pacific Area National Meeting: San Francisco, CA, 1959”; ASTM: Philadelphia, PA, 1959; ASTM Special Technical Publications No. 269. Trask, T. 0.; Green, R. B. Spectrochlm. Acta, Part B 1983, 388, 503.
Department of Chemistry University of Arkansas Fayetteville, Arkansas 72701
RECEIVED for review March 18,1983. Accepted June 9,1983. The authors acknowledge the support of the National Science Foundation under Grant CHE-8105000 and the EPSCOR program. This work was presented in part at the 9th Annual Federation of Analytical Chemistry and Spectroscopy Societies, Philadelphia, PA, Sept 1982, and the 184th National Meeting of the American Chemical Society, Kansas City, MO, Sept 1982.
AIDS FOR ANALYTICAL CHEMISTS Ultrasonic Preparation of Fat-Free Blologlcal Materials for Elemental Analysis Tee-Siaw Koh Veterinary Sciences Division, South Australian Department of Agriculture, c/o The Institute of Medical and Veterinary Science, Box 14 Rundle Street Post Office, Adelaide, South Australia 5000, Australia The ether-Soxhlet extraction method is commonly used in the determination of crude fat in biological materials and the preparation of fat-free samples for elemental analysis. The Soxhlet method is time-consuming as it requires extraction periods of up to 16 h (1). T o reduce the extraction time, the applicability of ultrasound in the determination of crude fat content in biological materials was investigated. Ultrasound has a frequency range of 20000 Hz to 50000 Hz. The ability of ultrasound to produce the energetic phenomena of cavitation and streaming (2) in a liquid has been employed in cleaning labware and in disintegrating/ homogenizing cells and tissues. To the author’s knowledge, the application of ultrasound in the preparation of fat-free biological materials for elemental analysis has not been documented.
EXPERIMENTAL SECTION Apparatus. A Dawe Soniprobe comprising a generator, type 7530A, and a sonic converter, type 1130/1B, with a tip diameter of 12.7 mm (Dawe Instrument Ltd., London) was used for the ultrasonic extraction of the crude fat. The generator operated at a frequency of 20 000 Hz with a peak power output of 200 W. An ether-Soxhlet assembly consisting of the following units was used: Jobling Quickfit Soxhlet extractor (catalog no. EX 5/55/100), electrothermal heating mantle (240 V, 50 W) with a 250-mL round-bottom flask (catalog no. MS 9503) and Quickfit condenser (catalogno. C1/12), together with a Whatman cellulose extraction thimble, 28 mm X 80 mm (W. & R. Balston Ltd., U.K.). An atomic absorption spectrophotometer (AAS)Varian AA 775 was used for elemental analysis, and a Pye Unicam SP6-550 UV/VIS spectrophotometer with a microflow cell was used for the colorimetric assay of phosphorus. Reagents. All chemicals were of analytical grade. Diethyl ether (May & Baker Aust. Ltd., Australia) was used for fat extraction of samples. An acid mixture of HNO3:HC1O4:HzSO4 1Ol:l (v/v/v)
was used to digest samples for elemental analysis. LaCl,, 0.72 M (BDH Chemicals, Aust. Pty. Ltd., Australia), was used as a releasing agent for calcium determination. Samples. Samples of ovine bone, meat-bone meal, and macadamia nuts were homogenized in a stainless steel grinder and oven-dried at 100 “C for 48 h prior to fat extraction. Procedure. Ether-Soxhlet Method. About 2 g of a dried sample was accurately weighed into a cellulose thimble and ether extracted for 16 h (I). Ultrasonic Method. About 2 g of a dried sample was accurately weighed into a polypropylene centrifuge tube (25 mm i.d. X 100 mm) containing 20 mL of diethyl ether. Ultrasound was applied to the ether/sample mixture for about 30 s and the contents were centrifuged at 1500g for 1-2 min. The ether layer was removed with the aid of a water vacuum pump and the extraction was then repeated three times (five times for macadamia nuts because of the expected higher fat content). The excess ether was then allowed to evaporate, and the defatted samples were dried at 100 “C for 1 h. Percentage of ash in the bone samples was determining by ashing the defatted samples at 750 “C for 2 h. Copper, manganese, and zinc were determined by AAS after acid digestion of the samples. Samples of ashed bones were treated with concentrated HCl and LaC1, prior to the assay for calcium and magnesium by AAS. Phosphorus in the bone samples was assayed colorimetricallyby a method involving the formation of molybdovanadophosphoric acid (3).
RESULTS AND DISCUSSION Figure 1illustrates that the ultrasonic method is about 20 times faster than the Soxhlet method in the extraction of crude fat from the bone sample. With the Soxhlet method, although the AOAC official method recommends extraction periods of up to 16 h ( l ) Figure , 1indicates that complete extraction was achieved within 40 min. Similar rates of fat extraction for the ultrasonic and Soxhlet methods were observed for the
0003-2700/83/0355-1814$01.50/00 1983 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983 /o-o-o-o
1815
Table 11. Ash Content in Bone and Elemental Concentrations in Bone, Meat-Bone Meal, and Macadamia Nuts Extracted by the Soxhlet and Ultrasonic Methodsa, procedure
/
constituent
Soxhlet
ultrasonic Bone
,i,v
P, mmol/kg
,
0
2
4
, 6
, 8
1
1
20
0
11
40 5 o i o l &
Time,min
Flgure 1. The rate of fat extraction from 2 g of dried bone sample by the ultrasonic and Soxhlet methods. Each polnt in the figure represents the mean value of four determinations.
Table I. Percentage of Crude Fat in Dried Biological Materials Extracted with the Ultrasonic and Soxhlet Methodsa'& % of crude fat ultrasonic Soxhlet sample bone meat-bone meal macadamia nuts
8.5 f 0.07 11.4 f 0.82 116.6 i: 0.49 76.8 c 0.51
(4) (4)' (12) (7)
8.8
f
Ca, mol/kg Mg, mmol/kg Zn, mmol/kg ash, %
1 9 2 f 6 (4) 202 f 6 (4)' 6.9 t 0.29 (4) 6.6 c 0.09 ( 4 ) c 140 I0.01 (4) 1 4 0 f 0.01 (4)' 1.4 f 0.05 (4) 1.5 i: 0.07 (4)' 63 f 1.0 (4) 6 2 c 0.4 (4)'
a No effect of methods could be detected on percentage of crude fat (P > 0.05, Student's t test). Mean values f
standard deviation with number of determinations in parentheses. ' Noncenitrifugation method: ether removed bv decantation. macadamia nuts which have 77% fat content. There is no significant difference (P>* 0.05, Student's "t" test) in the crude fat content as determined by the ultrasonic and the Soxhlet methods (Table I). Of the three specimens studied, the macadamia nuts had the highest fat content (77%) and required two additional ultrasonic extractions over that required by the other two specimens. In view of this, it is recommended that for such type of biological material preliminary ultrasonic extraction should be performed on a selected sample, preferably one with high fat content, to determine the number of ultrasonic extraotions required. To eliminate the centrifugation step in the ultrasonic method of fat estimation, the effect of removing ether by decantation was investigated. After each ultrasonic treatment of bone sample, the ether/bone mixture was left undisturbed for 30 s followed by decantation of the ether. As shown in Table I (results with superscript c), the noncentrifugation version of the ultrasonic method gave a fat content which was about 30% higher than that of the centrifugation version. This difference in fat content could be attributed to the loss of suspended bone fragments during decantation of ether in the noncentrifugation version. The within-batch coeffficient of variation (CV), expressed as a percentage, for the crude fat analysis by the ultrasonic method was 0.8 (4) for bones, 3.0 (12) for meat-bone meal, and 0.7 (7) for macadamia nuts (number of determinations in parentheses). The between-batch CV for the meat-bone meal was 5.6 (37) over a 3-month period. As shown in Table 11, there is no significant difference (P > 0.05, Student's "t" test) in the ash content of the bone sample and the elemental concentrations of the three speci-
6.9 c 0.31 ( 6 ) 1 4 0 f 0.01 (6) 1.4 i: 0.05 (6) 63 c 0.3 (4)
Meat-Bone Meal Cu, pmol/kg Mn, pmol/kg Zn, mmol/kg
6 8 I4 (4) 250 f 27 ( 4 ) 1.7 f 0.03 ( 4 )
0.1 (6)
17.4 t 0.88 ( 7 ) 77.7 ? 0.85 (8)
1 9 7 i 6 (6)
60 I7 (4) 250 c 39 (4) 1.8 f 0.05 (4)
Macadamia Nuts Cu, p,mol/kg Mn, mmol/kg Zn, mmol/kg
260 f 9 ( 7 ) 4.9 t 0.13 (7) 1.0 f 0.03 (7)
240 ? 1 5 (8) 4.9 f 0.36 (8) 1.0 ? 0.06 (8)
a No effect of methods on analytical results (P > 0.05, Student's t test). Results based on dry weight fat-free basis and expressed as mean f standard deviation with the number of determinations in parentheses. ' Noncentrifugation method: ether layer removed by decantation.
*
mens extracted by either the ultrasonic or the Soxhlet methods. In contrast to the earlier finding on the fat content (Table I), Table I1 shows that bone sample extracted by the noncentrifugation ultrasonic method gave similar ash and elemental results as that of the centrifugation method, suggesting that the elements are evenly distributed in the bone fragments. In summary, this study shows that the ultrasonic method is applicable to the determination of crude fat content in samples of bone, meat-bone meal, macadamia nuts, and possibly other fat-containing biological materials. The method is an alternative extraction method to that of the etherSoxhlet for the preparation of fat-free samples for elemental analysis. The method is rapid (capable of extracting 40-50 samples per day), requires little bench space, and minimizes fire hazards as heating of ether is not required.
ACKNOWLEDGMENT The author thanks P. J. Babidge, T. H. Benson, and G. J. Judson for valuable comments on the manuscript. Registry No. P, 7723-14-0;Ca, 7440-70-2; Mg, 7439-95-4; Zn, 7440-66-6; Cu, 7440-50-8; Mn, 7439-96-5. LITERATURE CITED (1) Horwitz, William, Ed. "Officlal Method of Analysis of the Association of Official Analytical Chemists", 1l t h ed.; Association of Official Analytical Chemlsts: Washington, DC, 1981; Chapter 7, p 128. (2) Bailey, S. Lab. Pract. 1980, 29, 531-533. (3) Quinlan, K. P.; de Sesa, M. A. Anal. Cbern. 1955, 27, 1626-1629.
RECEIVED for review January 17,1983. Accepted May 2,1983.
