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mercury pool. Once the pressure had been set to a desired value. no other adjustments were necessary. Procedure. The silica gel was first dried overnight at 170 "C. It was then slowly poured into the column which had previously been filled with n-pentane. The top reservoir was then attached and also filled with n-pentane. The routine operation of the column consisted of four steps: (a) Filling the reservoir, (h) cleaning the receiver of vapor by blowing filtered house air into it, (c) clamping the receiver to the hottom of the column, and (d) adjusting the flow rate of liquid to a desired level. Once those steps had been taken, the column required no further maintenance.
RESULTS AND DISCUSSION IVe have repeatedly used this technique for cleaning u p technical grade n-pentane. The advantages of this system are. first. that slow flow rates can be used without vaporization losses of the n-pentane becoming a problem. We have used up to 12 h to pass 2 L of n-pentane without appreciable loss. Second, more efficient columns were maintained since bed-disrupting bubbles were prevented from forming. Third, safety was increased because an increase in ambient teniperature could not generate either higher coiumn pressures or noticeable amounts of gaseous n-pentane. (Nevertheless. this system was operated in a good fume hood!) Fourth. the pseudo-closed system eliminated the access of water vapor to the outlet of the column. Finally, accidental degradation of the column material by water adsorption was reduced. Even in cases where the column was accidently allowed to run dry and to remain in that condition for several hours, there was no evidence that atmospheric water had penetrated the system to an extent that deactivated or seriously impaired the operation of the column. Obviously, this type of technique could also be used to exclude atmospheric gases if ian appropriate purge gas were employed. The system was ideal for solvent cleanup, and it can be readily adapted to fraction collection by incorporating several containers inside the receiver flask. In this mode of operation, one would (most probably) use the optional ballast tank.
Figure 1. Column with upper and lower (2-L) reservoirs
60 cm X 2.54 cm (o.d.1 and 2.24 cm (i.d.). The sintered-glass frit was of medium porosity. The top joint of the reservoir was fitted with a tubing-adapter. A latex rubber tube was used to connect the reservoir tubing adapter, through a glass "T", to the venting arm that followed the column. The third port of the "T" was connected by latex tubing to a 4-mm o.d. glass tube which had been inserted through a two-hole rubber stopper and into the mercury reservoir. (An optional ballast tank could be placed as a branch at any convenient location along the latex tubing.) Thr pressure was regulated by the mercury reservoir, and it was adjusted by raising or lowering the height of the glass tube in the
LITERATURE C!ITED (11 "Separatory funnels,addition, pressure equalizing." Item No. 5237-H 10, A r t h u r H. Thomas Co. Catalog, 1976.
RECXIVEI) for review August 5, 1977. Accepted November 14, 1977. This work was supported by the U.S. Energy Research and Development Administration through Contract No. E(38 -1)--854.
Preparation of Wet Fish Reference Material from Shark Meat Yukiko Dokiya, Masashi Taguchi,' Shozo Toda, * and Keiichiro Fuwa2 Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Bunkyoku, Tokyo, Japan, 1 13
Standard reference materials or certified reference materials for metal analysis of biological or environmental samples have recently attracted the attention of analytical chemists who deal with those "soft" materials. Since H. J. M. Bowen ( I ) prepared his Kale Powder in the early 1960's, several trials for preparing such materials have been performed, including grass samples by J. B. Jones (21, Orchard Leaves and Bovine
Liver by NBS research groups ( 3 , 3 ) ,Cd-rice by N. Yamagata ( 5 ) and Oyster Powder by R.Fukai ( 6 ) . Among these works, those of NBS research groups are considered t o be the most systematic and comprehensive. An unprecedented demand for Orchard Leaves and Bovine Liver is currently reported by J. P. Cali (7). T h e authors, in a cooperative study with h'BS research groups, have performed some researches for new biological reference materials, and the work includes the preparation of Tea Leaves and Pepper Bush samples (8). No standard reference materials of fish meat have been successfully
IPresent address, Department of Fisheries, Faculty of Agriculture, The University of Tokyo, Bunkyoku, Tokyo, Japan, 113. Present address, Department of Chemistry, Faculty of Science. The University of Tokyo, Bunkyoku, Tokyo, Japan, 113. 0003-2700/78/0350-0533$01.00/0
C
1978
American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978
Table I. Experimental Conditions for Atomic Absorption and Flame Emission Spectrometry Element K Na Ca Mg Zn Analytical 766.5 589.0 422.7 285.2 213.9 line, nm Mode FE FE FE AAS AAS 0.4 0.4 0.4 1.0 1.0 S.B.W., nm Flame Air flow rate 15 L/min C,H, flow rate 2.4 Limin Position 1.5-1.8cm above the burner
Fe 248.3 AAS 0.4
Table 11. Water and Elementary Content of the Raw Material (Squalus mifswkurii)
H,0 , Shark
T.L., mma B.W., kgb 1131 8.45 1010 6.15 1055 8.48 968 5.24 1045 6.85 1087 6.63 1193 10.37 1080 7.80
1 2 3 4 5 6 7 8
Av Re1 std dev, % a
Total length.
1070 6.5
Body weight.
7.50 21.6
NaC 795 740 930 700 770 975 725 725
K 3590 3000 3670 4050 3320 3700 3600 3050
Ca 43 50 57 45 82 43 43 38
Mg 235 21 0 240 250 21 5 225 225 230
Zn
73.9 79.4 74.4 76.3 75.2 75.3 75.3 76.5 75.8 2.2
759 12.9
3500 10.1
50 28.1
229 5.7
3.3 14.8
%
4.1 2.9 2.9 2.8 3.6 3.8 3.6 3.0
Fe 6 7 7 7 6 10 12 5
8 30.7
Hg 1.75 1.74 1.83 1.54 1.40 2.13 2.11 1.83 1.79 14.0
Elements given in pgig wet.
Table 111. Variation of Water and Elementary Composition during the Preparation of “Wet” Shark Reference Material Steps of Production
78.5 t 1.7‘ 68.1 I 0.4 68.5 i 0.2
2. Mixture in silent cutter 3. “Wet” shark reference material
Q
Steps
Kb
Mg
Ca
1. 2. 3.
3500 i 350 3560 + 110 3650 k 100
229t 13 221 i 4 220~ 6
50 F 14 68 i 7 591 3
(x i un.] )).
Na, pg/g wet 759 f 98 13800 i 200 13700 i 450
H,O, %
1. Raw material
Zn 3.3 F 0.5 3.5 F 0.8 3.1 i 0.6
Fe
8+2 30i 7 26* 5
Hg 1.79 f 0.25 1.94 t 0.03 1.96 i. 0.05
Elements given in pglg wet.
prepared u p t o the present, because of t h e difficulty in realizing homogeneous concentrations of elements, especially that of calcium (9). In this study, the white muscle of shark (Squalus rnitsuhurii) was adopted as the raw material, since t h e distribution of metals in the muscle is known to be comparatively homogeneous owing t o the fact that this fish has less bones in t h e muscle parts (10, 14). In the fields of food science, oceanography, environmental science, etc., where fish meat reference materials are required, metals are often determined in “wet” or “fresh” samples. Although the wet weight is rather hard to define as a scientific unit, it can be practically useful to prepare a reference material of a wet weight basis under given conditions. The procedure of producing Japanese fish paste was adopted for this purpose a n d a strong preservative, AF-2, which is now forbidden for use in food, was utilized t o preserve it for a long period.
EXPERIMENTAL Preparation of “Wet” S h a r k Reference Material. Eight fresh sharks of similar size (Table I) were obtained from the fish market of Choshi and the muscle parts of them were dissected and frozen and processed as follows. NaCl and starch were of analytical grade. AF-2(2-furyl)-3-(5-nitro-2-furyl)acrylamide) was obtained from Ueno Pharmaceutical Co. The white muscle of each fish (ca. 500 g wet) was dissected and cut into small pieces by a stainless steel knife and homogenized by a mixer. The fine bones and the red muscle were discarded. The meat (2.5kg) was further homogenized in an ordinary silent
cutter for 40 min, and KaC1 (75 g) and starch (25 g) were then added to enable the paste to clot after steaming. AF-2 (0.1g) which was also added caused the paste to become yellow in color. The resulting paste was steamed in a wooden frame for 15 min. Then the clotted paste was cut into pillars of 2-3 g and put into Pyrex glass bottles with caps and Teflon-coated packings, and sterilized at 120 “C and 1.2 atm for 30 min by a high-pressure sterilizer. Determination of Metals. A random selection of 10 bottles was taken from the products, and the contents were transfered to flasks (300mL) containing HNOB (5mL) and H202(5mL) for digestion. The flasks were heated gently with coolers whose tops were connected to traps of acid ( 1 1 ) . After 10 h, the digested solution volume was adjusted to 50 mL and used for the metal determinations. U’et samples (2 g) of the raw material and the mixture in the silent cutter were digested in the same manner as above. Na, K, Ca, Mg, Fe, and Zn were determined by flame emission and atomic absorption spectrometry under the conditions specified in Table I, using a Seiko SAS 721 atomic absorption spectrophotometer. Hg was determined by the reduction-cold vapor atomic absorption method using a 100-cm quartz absorption cell attached to a Hitachi 207 atomic absorption spectrophotometer (12). Determination of Water Content. The weight change by drying at 90 “ C in an electric oven was used for determining the water content of the raw material, the mixture in the silent cutter, and the products. Determination of Microbial Activities. Fungal activity was determined by counting the colonies. The testing paper
ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978
535
Table IV. Elementary Composition of "Wet" Shark Reference Material (@g/gwet) Bottles
Na
a b C
d e f g
h i j
Av Re1 std dev, %
Ca
K
Mg
13200 13600 13000 13900 13900 14200 14000 13300 13400 13700
3720 3690 3700 3710 3630 3590 3650 3630 3400 3750
60 66 57 59 60 55 64 57 57 57
2 15 225 210 2 15 225 220 215 2 15 230 215
13700 3.3
3650 2.6
59 5.9
220 2.8
Zn 3.0 2.2 3.0 3.1 4.6 3.4 2.5 2.8 2.8 3.2 3.1 20.6
Fe
Hg
25 21 24 21 25 25 35 32 22 28
1.92 1.99 2.05 1.92 2.00 1.99 1.92 1.89 1.99 1.92
26 17.8
1.96 2.6
Table V. Change of Weight and Microbial Activities in Three Months (from Feb. 1977 t o May 1977) Microbial activity Weight change, gn Fungi Bacteria Room temperature (Dark) _____ _ Sterilizedb 0, 0, - 0.1, 0, 0 Not sterilized - 0.3, - 0.2, 0, -+-tc Cold room ca 4 ° C Sterilized ) -____ 0, 0, 0, 0, 0 _____ Not sterilized 0, 0, 0, 0, 0 Freezer (- 2 0 "C) _____ Sterilizedb 0, 0, 0, 0, 0 _____ Not sterilized 0, 0, 0, 0, -0.05 " Starting sample weight: 1.7-3.0 g. Sterilization: 1 2 0 "C, 1 . 2 atm, 3 0 min.
b '
Table VI. Metal Concentration of Some Animal Reference Materials Ovster Powder lAEA-Monaco MA-M-1 ( 6 ) pgig dry
Bovine Liver KBS-SRM 1577 ( 4 ) K Na Mg
Ca Fe cu Zn Mn Hg a
9700 i 600 2430 r 1 3 0 (605) (123) 270 i 20 1932 10 1 3 0 i 10 10.3 i 1 . 0 0.016 i 0.002
Calculated values from p g / g wet.
Average
300 r 2 0
310i 1 0
26302 120 66r 4 0.20 2 0.02 i
un., .
"Wet" Shark Reference Material (Shark Paste) p g / g dry"
p g i g wetb
11600 t 300 43400 i 3 0 0 0 8 9 0 i 13 1 8 7 k 13 83 i 1 5 (3Y 1oi 2 (3Y 6.22 I 0.16
3 6 5 0 i 100 13700 i 450 220 t 6 591 4 261 5 3 . 1 i 0.6
1.96
i
0.05
Approximate value.
"Bactester", purchased from Kanto-Kagaku Co. Ltd., was utilized for the determination of bacterial activities.
RESULTS AND D I S C U S S I O N C h a r a c t e r i s t i c s of t h e Raw Material. T h e total length, the body weight of the eight sharks obtained, their water content, and the K, Na, Ca, Mg, Fe, Zn, and H g contents of the white muscle are summarized in Table 11. T h e most typical characteristic of this fish is t h e high concentration of Hg in the muscle, presumably owing to their eating habits. .4correlation between the total length and the Hg content in the muscle is also known (13). Thus, considering the field of utilization and according to the user's requirements, shark muscle of Hg content ranging from 0.2 to 2.0 pg/g (wet weight basis) can be obtained as the raw material for the reference by chosing t h e appropriate variety and the total length of t h e shark. T h e K, Ka, Ca, Mg, Fe, and Zn contents are similar to those of other fish meat. T h e variation of Ca content (Re1 std dev, 28.1 O/c between the individual sharks was the greatest among those of the elements examined. C h a n g e s d u r i n g P r e p a r a t i o n . T h e changes of water
content and mineral concentrations during the preparation are summarized in Table 111, .4s NaCl was added in the process, the concentration of Na was increased more than ten times that of the raw material. T h e slight decrease of the water content of the mixture in the silent cutter may be attributed to the addition of starch (1%)and to evaporation. T h e increase of Fe concentration observed with the mixture in the silent cutter and with the product was considered to be due to contamination from the material of the silent cutter. This pontamination should be eliminated by chosing a more suitable cutter material or by changing this process to a less contaminating one. T h e variation of metal concentrations was shown t o be reduced after the preparation, except in the case of Zn. T h e situation was best for Ca where the re1 std dev value after preparation was 5.9% relative to 28.1% for the raw material. No significant loss of Hg was observed during the preparation. W a t e r a n d M e t a l C o n t e n t s of "Wet" S h a r k Reference Material. The water and metal contents of samples contained in 10 bottles selected a t random are shown in Table IV. The homogeniety with respect to Na, K, Ca, Mg, and Hg was shown to be within 6 % (re1 std dev value), while that of Zn and Fe
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ANALYTICAL CHEMISTRY, VOL 50, NO. 3, MARCH 1978
were as much as 20%. P r e s e r v a t i o n of the S a m p l e . As the samples are wet, long-term preservation should be one of the most difficult factors to realize. Thus, the preservation test was performed periodically after the completion of the preparation. Fifteen bottles were sterilized by a high-pressure sterilizer and sets of 5 bottles were then kept at (1)room temperature, (2) 4 "C, and (3) -20 "C. T h e weight and microbial activity of each sample was determined. This procedure was repeated for a further 15 sample bottles b u t omitting sterilization. A summary of the results of these tests after 3-month storage are given in Table V. Except for t h e bottles not sterilized and kept at room temperature, microbial activity was not observed, neither fungal nor bacterial, and the change of weight during preservation was shown to be negligible. These tests are being continued for a few years. C o m p a r i s o n w i t h O t h e r A n i m a l Reference Material. T h e elementary composition of this wet shark reference material was compared with NBS-SRM 1571, Bobine Liber, and with the Oyster Powder produced by R. Fukai ( 6 ) of the Marine Research Laboratory (Monaco) of IAEA for the purpose of intercalibration (Table VI). T h e most typical characteristics of this shark reference material are the high concentration of Hg and the low concentration of other heavy metals such as Fe, Zn, Cu. and Mn compared with other materials. CONCLUSION As the first trial t o make a fish reference material of wet basis, a Japanese-style steamed fish paste reference material was prepared from shark meat. Provided the prerequisites for reference materials are as follows: (I) Availability in large amount, (2) availability at lorn cost, (3) homogeniety of samples, (4) preservation, ( 5 ) safety in transportation, (6) appropriate concentration of elements, this material can be concluded t o be a good candidate. For the first two items, the price of shark fish is comparatively low and the fish can he obtained in sufficient numbers a t certain markets (about
$2 for a fish). T h e homogeniety with respect to Hg, K, Na, Ca, and Mg was shown to he within 6% of re1 std dev value, using about 2 g of wet sample (ca. 0.6 g dry matter). This material can be preserved for at least 3 months even a t room temperature and may he preserved for longer periods in a cold room or in a refrigerator. Safety in transportation can be readily achieved with careful packing. For the last item, this material can serve as a very good reference material in those fields concerned with the analysis of Hg in fish meat, where Bovine Liver, because it contains too low a concentration of Hg. is difficult to utilize. ACKNOWLEDGMENT The authors express their hearty thanks to T. Taniuchi of the Department of Fisheries, the University of Tokyo, for his help in getting the raw materials and to M. Nose of Tachikawa College of Tokyo for her cooperation in the preparation of this reference material. LITERATURE CITED (1) H. J. M. Bowen, Analyst(London), 92, 124 (1967). (2) J. B. Jones, Proceedings of the Pittsburg Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburg, Pa., 1967. (3) Certificate for Orchard Leaves, U.S. Department of Commerce, National Bureau of Standards, Washington, D.C., 1970. (4) Certificate fw Bovine Liver, U.S. Department of Commerce, National Bureau of Standards, Washington, D.C., 1971 (5) N. Yamagata, Bunseki Kagaku, 20, 515 (1971). (6) R. Fukai, Progress Report No. 13, Intercalibration of Analytical Methods on Marine Environmental Samples, International Laboratory of Marine Radioactivity, Principality of Monaco, 1976. (7) J. P. Cali, Anal. Chem., 48, 802A (1976). (8)K . Fuwa et al, BUN. Chem. Soc., Jpn., submitted (9) H. L. Rook, Anal. Div., NBS-USA, personal communication, 1976. (10) T. Taniuchi, "The Sharks", Diving World Pub., Japan, 1976. (11) S . Hanamura et al., Proc. Forum Jpn. SOC.Ana/. Chem.. 34, (1973) (12) S . Yamazaki et al., Nihon Kagaku Kaishi, 1977, 1148. (13) C. R. Forester, K. S. Ketchen, and C. C. Wong, J . Fish. Res. Board Can., 29, 1487 (1972). (14) M Taguchi et al., Bunseki Kagaku, 26, 438 (1977).
RECEIVED for review August 18, 1917. Accepted November 2 . 1977.
Use of Electron Capture-Induced Products for Confirmation of Identity in Pesticide Residue Analysis Walter A. Aue" and Shubhender Kapila 5637 Life Sciences, Dalhousie University, Halifax, N.S., Canada
T h e products of reactions taking place in the electron capture detector (ECD) are sometimes capable of reacting with electrons themselves; in fact, this secondary reaction has so far been t h e only means of detecting their presence (2-3) Some of these EC-induced products have been tentativel) identified by retention data ( 3 ) . Some pesticides yield distinct product patterns (e.g.. pentachloronitrobenzene yields pentachlorobenzene and the possible tetrachlorobenzenes) and these could conceivably be used to confirm the presence of the pesticide in an analytical sample. The need for confirmation of GC peak identity needs no belaboring; t h e publications on this subject are far too numerous t o cite. They include GC-MS, UV photolvsis, use of multiple selective detectors, and. most prevalent. a wide variety of derivatization reactions. Derivatization usually involves reaction of the total sample rather than reaction of a single GC peak. While the latter would be preferable on theoretical grounds-1.e. the prohability of mistaking another compound for the expected de0003-2700/78/0350-0536$01 0010
P r e s Re;
Figure 1. Flow schematic C 1978 American Chemical Society
