ANALYTICAL CHEMISTRV, VOL. 51,
of trace antimony in phosphoric acid. The calibration curve obtained under the optimal experimental conditions shown in Table I has a linear dynamic range of about four orders of magnitude, linear up to 2000 ng of antimony. The detection limit, calculated on the basis of a signal-to-noise ratio equal to three as recommended by IlJPAC (9): was 0.5 ng of antimony, equivalent to 25 pg S b / m L for 20-mL sample volame. The reagent blank had a value of approximately 5 ng of antimony in 20 mL of sample volume. Table III depicts the reproducibilities of measurements with the use of antimony stafiriard solutions of various concentrations. From the interference study of diverse elements and inorganic acids in the antimony determination described previously (1) arsenic interference was mostly expected in the determination of ant,imony in phosphoric acid by the present method. I t has been reported that levels of less than 0.1 pg As/mL are found in ACS reagent grade phosphoric acid (10). In addition, the requirement for the arsenic content of J I S reagent grade phosphoric acid is limited to riot more than 0.5 pg As/mL (2). Such concentration levels of arsenic present in phosphoric acid as an impurity were confirmed in thifi work to give no interference in the antimony determination. For a more accurate determination, however. the method of standard additions in which each phosphciric acid was spiked with antimony in the range 5-20 rig Sb;mL (corresponding to 100-400 ng of antimony) was used in this work. All samples were obtained from several Japanese manufacturers. The results of the determinations are shown in Table 1V. These results (Table IV) show to a high degree good agreement between the proposed method and the ICP-OES. The values by ICP-OES were obtained by using a Nippon Jarrell-Ash Model ICAP-500 under the following conditions: forward RF
NO. 9, AUGljST 1979
1591
power. 1.6 kW: argon flow rates, 1.3, 0.8. and 0.4 L/min for cc:dant. plai.ma. and sample transport gases, respectively; sampie uptake rate, 1.0 m1,imin; height of observation, 15 mm above work coil; wavelength, 217.6 nm; entrance and exit slit widths, 10 pm; integration time for measuring the signals, i 0 s. Oilr data indicate that the sensitivity and precision of the method described here are quite adequate for the deterrnination of trace antimony in reagent grade phosphoric acid.
ACKNOVVLEDGMEKT The authors express their gratitude to Nippon Jarreli-Ash Co., Kyoto, Japan, for loan of an ICAP-500 optical emission
spectrometer equipped with an inductively coupled plasma and to Eiji Yoshimoto for his assistance.
LITERATURE CITED T. Nakahara, S. Kobayashi, and 3 . Musha, Anal. Chim. Acta. 101, 375 (1978). "!+osphOric Acid", JIS K-9005, Japanese Indusaial Standards Cornminee, Japanese Standards Association, Akasaka, Tokyo, Japan, 1972. "Reagent Chemicak", American Chemical soCier{ Comminee on Anatytical Reagents. 5th ed., American Chemical Society, Washington, D.C.. 1974. I May Y . C . Geological Survey, Reston. Va. 22092. personal cornmtinication. January 1978. H. D. Flenxng and R. G. Ide, Anal. Chim. Acta, 6 3 , 67 (1976). J. A. Fiorino. J. N. Jones. and S.G. Capar. Anal. Chem., 48, 120 (1976). H. Haraguchi, W. K. Fowler, D. J. Johnson. and 2 . D. Winefordner, Speitrochinr. Acta, Part A , 3 2 , 1539 (1976). W . K. Fowler anti J. D. Winefordner, Anal. Chem.. 49, 944 (1977). IUPAC Commission on Spectrochemical and Other Optical Procedures for Analysis, "Nomenclature. Symbols, Units and Their IJsage in Speckochemicai Analysis". Part 11, 4. :. Revision 1975, Pvre Appl. Chem., 45, 99 (1976). [Anal. Chem., 48, 2296 (1976); Ap,d. Spactrosc., 31, 345 (1977): Spectrochim. Acta, &rt 8. 3 3 , 241 (1978).] I. May and L. P. Greenland, kilal. Chem.. 49, 2376 (1977).
RECEIVED for review Januaiy 29. 1979. A-cc.epted Fpbruary 33, 1979.
Noncontaminating, Representative Sampling by Shattering of Cold, Brittle, Biological Tissues John A. Nichols"' Analytical Chemistry Facility, Colorado State University, Fort Collins, Co/oradc 80523
Lynn R. Hageman Department of Chemistry, Montana Stafe University, Bozeman. Montana 597 17
A general sample procedure tor plant and animal tissues must be very versatile to handle soft livers, hard bones, fibrous plant materials, etc., as well as complex cases such as whole fish. Problems with blenders are well-known. An elaborate solution to some of these problems has been described by Iyengar ( I ) , who described homogenizatiozi at liquid nitrogen temperature, using a Teflon-lined ballmill, to prepare bone for neutron activation analysis. However, a need remains, as in atomic spectrometry, for simple and inexpensive sampling procedures to match the stringent requirements of (a) minimal sample contamination and (b) reasonably representative sampling, sometimes with solid-sample aliquots of 50 mg or less. The feasibility of ultratrace metal analysis with furnace atomic absorption has made it tempting to subordinate representativeness to the desire for "minimal handling" of the Present address: D e p a r t m e n t of Chemistry, M o n t a n a State U n i v e r s i t y , Bozeman, M o n t a n a 59717. 0003-2700/79/0351-1591$01,00/0
sampie; this is acceptable only in special cases, when the analyte is known LO be deposited hornogeneously in the tissue of interest. The sample homogenization priw to subsampling can be accomplished with much less risk of contaminants being picked up if the sample is handled "dry". with the water frozen, so that no liquid transfer medium is available (2). This principle can be effectively combined with indirect delivery of energy for brittle shattering, through a clean plastic wrapper, to a sample previously chilied with liquid nitrogen until brittle. Such a procedure has been tested in our laboratories on many diverse animal tissues and assessed in terms of (a) representativeness of 5-g subsamples and (b) absence of contamination. Use of a blender with dry ice to keep the sample cold has been similarly tested, for samples requiring finer pulverization, using subsamples of whole fish down to 8 mg; blank levels for nine elements again document the 'C 1979 American Chemical Society
1592
ANALYTICAL CHEMISTRY, VOL. 51, NO. 9, AUGUST 1979
advantage gained by handling the sample cold and “dry”.
EXPERIMENTAL Procedure A. Shattering with a Hammer or Mallet. Frozen samples weighing from 0.01 to 5 kg are placed in a plastic bag and immersed in liquid nitrogen until adequately chilled. The sample is removed and slipped into a polyethylene bag, which is then folded shut and put into one or several additional poly bags. (The inner bag quickly reaches the sample temperature and may shatter.) The poly bags, 30 by 24 cm, 0.1-mm thick polyethylene, purchased from Fisher, were chosen for flexibility at 250 K. Finally, a layer of canvas protects the outer plastic from possible tearing. Using a blunt side of a hammer, vigorous blows are promptly delivered to the wrapped sample, which shatters by brittle fracture. Large chunks which result are located by feel, and successive blows are delivered to break them up, until no pieces of excessive size can be found. Then the canvas is removed, and the plastic bags are cut or torn open to expose the sample pieces. The sample will most likely be mixed with some broken pieces of plastic, which are visible and easily rejected. Sampling can be done by almost any chosen routine, such as randomly scooping up pieces of sample around several circles a t varying radii from the center. Transfer into a tared flask is done with acid-cleaned plastic SLOOPS and a glass rod. The time available before the sample starts to thaw is determined by its initial temperature, heat capacity, and particle size desired. We found that 0.1 g was ordinarily the lower limit on particle size obtainable by this method when starting with 1-kg samples, although modifications, such as inclusion of dry ice with the sample, might permit finer particles to be obtained. Working rapidly during the shattering of the smaller remaining chunks also decreases the particle size obtainable. Typical Application. Several hundred beef cattle organs have been analyzed using Procedure A, including liver, kidney, bone, fat, brain, spleen, lung, and muscle. Procedure A finds its most advantageous application when large sample weights are used. For the beef organs tested, shattering was continued until a particle size of 1 g or less was obtained and the sample weight was usually 15 to 20 g (all on a wet weight basis). Feldman’s adaptation ( 3 ) of G. F. Smith’s digestion technique was found highly suitable; it accommodates samples up to 5 g (dry weight)-often up to 20 g on an as-received basis. Redistilled acids for digestion were obtained from G. F. Smith Co.; in one run, Ultrex acids were used. After the samples have been digested (2 to 4 h) and diluted to volume, aliquots are apportioned for atomic absorption analyses, such as direct flame AAS (Cu and Zn); vapor generation techniques (Hg, SeH,); or preconcentration and matrix removal using ammonium pyrrolidine carbodithioate coprecipitation ( 4 ) ,followed by carbon rod atomization analyses for Pb, Cd, Mo, and other heavy metals. Procedure B. Pulverization in a Blender. A heavy-duty, high-speed Waring blender is suitable. Samples on the order of 3 g, such cs small whole fish, can be handled conveniently in a 100-mL stainless steel blender container. The sample is first chilled with liquid nitrogen (or dry ice), and then placed in the blender with a scoop of dry ice weighing 10 g or so. PRECAUTION: Blend immediately. If dry ice is allowed to sit in the bltnder, the bladelshaft assembly may freeze up within a minute, resulting in “lift-off’ of the metal container or other hazard when the power to the blender is turned on. After the sample has been blended to a powder (usually 10 to 30 s suffice), it is poured into a clean container where the dry ice is allowed to sublime. This can take place a t a low temperature, if it is desired to obtain a solid; or a t room temperature if a paste is desired. Samples as small as 8 to 10 mg have been taken routinely for mercury analysis by direct solid sampling; in this procedure the sample is combusted in a stream of oxygen and filtered through a gold-plated graphite crucible (5) suitable for immediate insertion into an electrothermal atomizer. Combination Procedure. In cases where fairly fine pulverization of a large sample is required, it may be advantageous to use Procedure A first, in order to break up the sample into chunks of about 1 cm3, and then place the frozen chunks in an appropriately-sized stainless steel blender for handling as in Procedure B. This has been implemented for several hundred whole fish samples weighing from 10 to 500 g each, including trout,
sauger, carp, northern pike, walleyes, and others found in Montana streams and reservoirs.
RESULTS AND DISCUSSION The temperature a t which tissue becomes reasonably brittle may be as high as 255 K for a soft tissue (liver or kidney) with 70-80% water content. (Kidneys, taken directly from the freezer a t 253 K, have been split in halves or quarters with a hammer blow.) A much colder temperature is necessary for fat to be brittle. However, liquid nitrogen temperature is colder than necessary for brittleness; dry ice temperature seems satisfactory for most things. T h e main advantages of liquid nitrogen in Procedure A are: it is cheaply and readily available, and unlikely to contaminate t h e sample (if liquid nitrogen should flow into the bag holding the sample); there is rapid chilling of any size samples; and finally, by bringing the sample temperature well below the temperature necessary for brittleness, one has a longer time interval in which to work on the sample before it warms up and ceases to be completely brittle throughout. T h e importance of t h e last can be illustrated by the problem which often occurred when shattering beef muscle tissue. The marbling (fat and connective tissue) became plastic or less brittle while the high-moisture content, proteinaceous material remained brittle; thus shattering resulted in many small pieces of meat hanging onto long pieces (or a network) of marbling. This potential problem, of course, can be turned into an advantage if separation of the lean meat from t h e fat is desired. Large bones posed the greatest problem-thorough and uniform shattering was very time-consuming owing t o their structure and strength. No problems were encountered for any of the fish analyzed (by the Combination Procedure), nor were there any problems for brains, lungs, spleen, kidneys or livers (Procedure A). B l a n k s . Before selecting materials in which to wrap samples for shattering, measurements were made to estimate levels of six suspect contaminant elements on materials which might contact the sample. Lead was found to pose the greatest potential contamination problem. As an example of the results, the amount of lead “fall-out’’ which could be obtained by vigorous scraping from 10 cm2 of canvas was determined t o be less than 5 ng. This amount of lead would be reflected in the final result as a blank contribution of
