Table 11. Use of Normalized Measures of Similarity Takes into Account the Information Provided by the Length of the Vectors. The "Overlap"Coefficient Can Be Seen to Provide Less Discrimination than Some of the Alternative Measures
Table I. Unnormalized Measures of Similarity (AND) or Dissimilarity (XOR) Fail to Represent the True Measures of Association of Binary Vectors of Different Length A
11110000
B
11111000 11100000
C D E
11101000 11101111
F
Overlap Dice Cosine
11111111
Upper right half of matrix gives I U XOR matrix gives I U AND q A A B C D
0
4 3 3 3
E F
C
D
E
F
1
1 2
2 1 1
4
0
5 4 4 3
4 4
0
1
7
0
3 4 4 5
4
0
3 3 3
(A,C) 1.0 0.86 0.86
(AJ) 0.75 0.75 0.75
ME) 0.75 0.54 0.57
(AJ) 1.0 0.67 0.71
q, lower left half of
B 0
(AP) 1.0 0.89 0.89
Differences in performance resulting from the use of different similarity coefficients are likely to be of second order in comparison with differences due 'to other possible variations in the analysis procedures employed. However, there is some evidence to suggest that measures such as the cosine coefficient are superior to the overlap coefficient in recall/precision performance (7). The figures given in Table I1 also demonstrate that the overlap coefficient provides less discriminating power than some of the alternative measures.
3
5 4
Hence: Dmin
LITERATURE CITED
= 1-41 - 31BI
Substituting these terms into the definition of the normalized dissimilarity measure and using the relation IAl
+ IBI
= IA XOR BI
S.L.Grotch,Anal. Chern., 42, 1214(1970), S. L. Grotch, Anal. Chem., 43, 1362 (1971). S. L. Grotch, Anal. Chem., 45, 2 (1973). S. L. Grotch, Anal. Chem., 48, 526 (1974). (5) S. L. Grotch, Anal. Chem., 47, 1285 (1975). (6) C. J. van Rijsbergen, "Information Retrieval", Butterworths, London, 1975. (7) G. Salton, "Automatic Information Organisationand Retrieval", McGraw-Hill, New York, N.Y., 1968. (1) (2) (3) (4)
+ 21A A N D BI
one obtains:
D* = ( D - Dmin)/(Dmax - D m i n )
- ] A XORBI
- 2)A A N D B J - IAl + 31BI
N . A. B. Gray
41BI 41BI
=1
King's College Research Centre King's College Cambridge CB2 1ST England
- 41A A N D BI
- Overlap coefficient
RECEIVEDfor review March 22,1976. Accepted May 4,1976.
AIDS FOR ANALVTICAL CHEMISTS Extraction of Mercury from Fish for Atomic Absorption Spectrometric Determination Kazuyoshi Matsunaga,' Tatsuo Ishida,' and Takuzo Oda" Department of Biochemistry, Cancer Institute, Okayama University Medical School, Okayama 700, Japan
An increasing awareness of the problems associated with the redistribution of mercury in the environment has been seen in the past year. The problem, however, is not entirely new. The Minamata poisonings (1, 2) with irreversible neurological damage occurred between 1953 and 1960 in Minamata and Niigata, Japan. The causative agent in both incidents was shown to be fish contaminated with an alkylmercury compound, monomethylmercury. Willis ( 3 ) reviewed recent advances with regard to the means of introducing the test solution of biological materials in atomic absorption techniques. Wet digestion methods using Present address, Institute for Public Health, Okayama Prefecture.
oxidizing reagents such as HzS04, "03, HBr, HC104, and H 2 0 2 have been developed to facilitate determinations for total mercury in most tissues (4-6). Magos determined total mercury in biological samples using cadmium chloride and violent conditions (7). The present study was undertaken to liberate mercury completely from fish samples with a solution of 1 N hydrochloric acid and cupric ion. The mechanism for this action seems to be based on the degree of stability of the chelated compounds. It was found that mercuric ion was set free in the exchange of cupric ion with the mercury combined with tissue material. The new method described here was rapid and simple, and its accuracy was checked with results obtained by other analytical techniques using samples of fish. ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
1421
Table I. Effect of Cupric Chloride on the Release of Mercury Total Cupric Sample,a volume, No. chloride, g g ml 1 0.1 10 100 2 0.5 10 100 3 1.0 10 100 4 5.0 10 100 5 10.0 10 100 a Same homogenized fish sample.
Total mercury, PPm 0.253 0.300 0.293 0.268 0.215
Table IV. Recovery of Methylmercury Methylmercury in sea bream, PPm 0.175 0.175 0.175
Methyl- Theoretical mercury amount, added,ppm PPm 0.20 0.375 0.10 0.275 0.02 0.195
Experimental amount, RecovP P ~ ev,% 0.375 100 0.290 105 0.195 100
Table V. Reliability of Analytical Results of This Method Table 11. Amounts of Total Mercury Measured after Allowing Different Releasing Times into 1 N Hydrochloric Acid Time, h 0.3 1 2 3 4 5 24
Total mercury, pprn 0.245 0.245 0.240 0.240 0.240 0.240 0.240
Table 111. Comparison of Total Mercury in Various Species of Fish by the Present Method and by the Wet Digestion Method Present Wet Species of method, digestion,a No. fish PPm PPm 1 Bass 0.090 0.092 0.028 2 Squilla 0.026 0.028 0.027 3 Shrimp 0.390 0.382 4 Rock trout 0.400 5 Rock trout 0.420 a Wet digestion by oxidation with nitric acid-potassium permanganate-hydrogen peroxide (9).
EXPERIMENTAL Apparatus. All atomic absorption measurements were made with a Shimadzu Model MAF-1 atomic absorption spectrophotometer equipped with mercury hollow cathode lamp and gas flow quartz cell of 10-mmdiameter X 200-mm length. The working conditions were as follows: wavelength, 253.7 nm; lamp current, 10 mA; and spectral band width, 0.73nm. The homogenizer used was a Kinematica GMBH Model Polytron 20 ST. Centrifuge tubes were 50-mlgraduated tubes with stoppers. Reagents. All reagents used were of Wako Chemical Industry, Ltd. super special grade, containing less than 0.01 ppm mercury. Mercury in blank solutions, containing all reagents as described in the following procedure, was not detected, because no absorption was observed in the measurement of the blank solution at the highest sensitivity of the atomic spectrophotometer. Procedure for Fish. Salt-water fish, such as bass, squilla, shrimp, rock trout, and sea bream, was used. All muscle tissues tested came from skinless fillets of fish. Twenty grams of finely cut, mixed fish muscle tissue was homogenized with a little distilled water. The homogenized solution was transferred along with about 100 ml of distilled water to a 300-ml vessel containing 1g of cupric chloride and 20 ml of concentrated hydrochloric acid. This mixture was stirred well and the total volume was adjusted to 200 ml with distilled water. The mixture was shaken again and transferred to 50-mlcentrifugetubes and centrifuged at 3000 rpm until the water layer became clear. After sampling 1 to 5 ml of this clear snlution, total mercury was reduced to elementalmercury with 2 ml of 10%stannous chloride solution in 100 ml of 1 N sodium hydroxide solution,followed by vaporization 1422
ANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
Measurement No. 1 2 3 4 5 6 7 8 Av. Std dev Rel. std. dev
Total mercury, ppm 0.145 0.145 0.145 0.145 0.135 0.155 0.155 0.145 0.1462 h0.006 4.1 %
of volatile mercury with circulating air into the light path of the flameless atomic absorption instrument. RESULTS A N D DISCUSSION Cupric Chloride. When samples are dissolved in 1N HC1, the amount of mercury which will go into solution is different if it is in an inorganic form as compared to an organic compound. The method described here is based on discovery that the mercury combined with tissue material can be released from the sample by adding cupric chloride to the 1 N HCl. Using this method, total mercury and inorganic mercury can be determined by flameless atomic absorption in basic and acidic media, respectively, the corresponding redox potentials of tin(I1) chloride being -0.93 V and 0.15 V. The difference between the two readings gives the amount of organomercury compound in the sample. In the report of Umezaki e t al. (8), 2 mg of cupric ion was used as a catalyst for reduction of organic mercury compounds. The authors speculated that mercury combined with tissue material. A large amount of cupric ion exchanges with the combined mercury, and the copper combined with tissue material more strongly than does mercury and thus liberates mercury into the 1N hydrochloric acid solution. The relationship between cupric ion and mercuric ion was studied by adding cupric chloride to the homogenized fish sample, and mercury was actually released from fish muscle tissues into the 1 N hydrochloric acid solution. As shown in Table I, excellent results were obtained when the mass of cupric chloride added was 0.005-0.01 the mass of the solution. One gram of cupric chloride per 200 ml of 1N HC1 was selected as the proper amount of cupric chloride because the highest yield of mercury was given in this amount of cupric chloride. Total mercury yield decreased when cupric chloride was added a t more than 5 g. A large amount of cupric chloride showed a tendency to precipitate protein. Time. The rate of release of mercury was demonstrated by analyzing the fish sample, As shown in Table 11,20 min was required to release the mercury into the 1 N hydrochloric acid solution containing cupric ion. This proposed method was characterized by the reduction of analytical time compared with existing methods. Analysis of Fish Samples. The mercury content of fish
samples was determined by the wet digestion method and the proposed method using conventional flameless atomic absorption in order to show that this method is as good as the wet digestion method. The results are given in Table 111,Eind close agreement in the measured values by two different methods was obtained. In comparison with the wet digestion method (9),the present method afforded an excellent yield in high mercury levels in fish. In this method, methylmercury content is obtained by subtracting the value measured in acidic medium from total mercury value. Recovery of methylmercury added to fish was studied. As shown in Table IV, complete recovery was obtained. Precision and Accuracy. In order to examine the feasibility of this method, the reliability of the technique was tested and the results are shown in Table V. The relative standard deviation was acceptable for fish sample analysis. A survey of the mercury content could readily be accomplished,and this method could be applied to monitor changes in mercury
content during storage. With modifications of sample preparation, the present method may be applicable to determine mercury content in blood, hair, and foodstuffs. LITERATURE CITED (1) T. Takeuchi, “Mlnamata Disease-A (2)
(3) (4) (5)
(6) (7) (8) (9)
Study on the Toxic Symptoms by Organic Mercury”, Univ. of Kumamoto, Report of Department of Medical Sciences (1966). K. Irukayama, Adv. Water follut. Res., 3, 153 (1967). J. B. Willis, Endeavour, 32, 106 (1973). F. D. Deitz, J. L. Sell. and D. Bristol, J. Assoc. Offic. Anal. Chem., 56,387 (1973). K. Eichner, Lebensmkelchem. Gerlchtl. Chem., 26 240 (1970); Anal. Abstr., 25, 2598 (1973). S. H. Omang, Anal. Chim. Acta, 63, 247 (1973). L. MaQOS,Analyst(London), 06, 847 (1971). Y. Umezaki, Jpn Analyst, 20, 173 (1971). C. Uklta, Chief Editor, “Standard Methods of Analysis for Hygienic Chemists-with Commentary”, authorized by the Pharmaceutical Society of Japan, Kanahara Publ. Co., Tokyo, 1973, p 275.
RECEIVEDfor review October 28,1975. Accepted March 29, 1976.
Direct Coupling of Glass Capillary Columns to a Mass Spectrometer Frederick A. Thome* and George W. Young R. J. Reynolds Tobacco Company, Research Department, Winston-Salem, N.C. 27 102
The rapid advances made in the production of very good glass capillary columns for chromatographic work have led to the need for development of an interface to directly couple such columns to the mass spectrometer. The availability of large capacity pumps for mass spectrometers now makes possible the design of such an interface to accommodate up to 6 to 8 ml/min of carrier gas (helium, for example) to be taken directly into the ion source of the mass spectrometer. A variety of coupling techniques have appeared in the literature (1-5). In designing such an interface, several prerequisites are to be met. The interface should be capable of reducing the pressure from 1 atm a t the column outlet to less than Torr in the ion source of the mass spectrometer without affecting column performance. It should also produce an inert pathway by which sample molecules can be transferred into the ion source without loss of chromatographic resolution, at the same time offering some mechanical stability to the glass system. Although higher flow rates can be accommodated, the interface volume must be carefully controlled to avoid peak spreading from columns operating at less than 1ml/min flow rates. The interface, of course, must be situated so that it is readily accessible to the operator and permit ease of column change. The interface which we have designed and have been using for over a year now must be considered a variation of the “direct connection” type as described in the paper by Henneberg et al. (I).The interface incorporates an idea published by Neuner-Jehle et al. (2), that of a platinum capillary at the coupling point. The use of platinum a t this point gives good mechanical stability and favorable surface behavior. Our variation in the use of platinum as a buffer for the atmosphere-to-vacuum coupling of the column to the ion source, is in making this a closed system, taking advantage of the high yield from the column, and providing an opportunity for removal of large quantities of solvent during injection. The control of the solvent peak is very important since the splitless injection techniques employed in our laboratory would send large volumes of solvent directly to the ion source and cause excessively high pressure and contamination. When a sample
is not being run, the interface allows the source to be kept at Torr. The heart of the interface, shown in Figure 1, is a 50-mm piece of 0.3-mm 0.d. and 0.15-mm i.d. platinum capillary (Engelhard Industries) that is silver-soldered into a piece of glass-lined metal capillary tubing, 1.5-mm 0.d. and 0.5-mm i.d. The glass-lined metal tubing is threaded through the line of sight coupling directly into the ion source of the Varian CH-5 mass spectrometer. This CH-5 has been equipped with a 600-l./sec oil diffusion pump on the ion source. Enough glass-lined tubing is used to permit the platinum wire to be positioned inside the gas chromatograph oven. All exposed lines are heated by wrapping with micro heating tape from Clayborn Laboratories. The platinum capillary has been Torr with crimped to give a Pennig gauge reading of 9 X a helium flow rate of 5*ml/min. This is sufficient to handle all of the flow rate from a variety of column types used in our work. The gas chromatographic column is connected directly to the interface by means of a 20-mm piece of glass capillary of 0.31-mm i.d. prepared in quantity on a Hupe glass drawing machine. The platinum is inserted into the end of this glass capillary. The chromatographic column is butted to the glass capillary and sealed with a piece of shrink-type Teflon tubing. All of this is enclosed in a f/l~-inchSwagelok union cross, modified by tapping one side to 0.062-inch i.d. One side of the union cross is used to supply make-up helium gas, the other side of the union cross being connected to a vacuum pump (2-stage, 250 l./min, Edwards) through a fine control needle valve (Whitey, 22R52A). The column is sealed to the union cross through a Ks-inch dead volume fitting using a ferrule machined from high temperature septa material. With the vacuum pump valved off, the ion source is exposed to a constant flow rate determined by the crimping of the platinum wire. This flow rate can be satisfied completely by the flow rate from the column when intermediate i.d. capillaries are used. When small bore capillaries are used, preheated make-up gas from one side of the union cross obtained from a second injection port can be used to maintain constant flow. This is evidenced by the constant source pressure obANALYTICAL CHEMISTRY, VOL. 48, NO. 9, AUGUST 1976
* 1423
