Low Temperature Apparatus for Cleanup of Biological Sample Extracts H. A. McLea Research Laboratories, Food a M Drug urrectoraie, Ottawa, Canada
Low
TEMPERATURE equipment for the precipitation of fats, oils, and waxes in foods and other biological sample extracts had been described by Anglin and McKinley (I), McKinley and Savary (Z), McCuUy and McKinley (3,Bates (4). Latham and Blumer (3, and Grussendorf et a[. (6). Each apparatus was custom made and had distinguishing features that were optimum for certain parameters relating to efficient cleanup. Some have provision for mechanical stirring of the extract during the precipitation step, but filtering was carried out external to the bath (Z-3,s). The filtering apparatus (2,3,5) was cooled by methanol circulating from the bath through jacketed funnels and return. Bates (4) and Grussendorf e f al. (6) used all glass precipitation and filtering apparatus that enabled these analytical steps to be carried out in the bath. However, no provision was made to stir the mixture during precipitation or washing of the precipitate. McCully and McKinley (7) have demonstrated that there is a need for constant stirring during the precipitation step to obtain quantitative recovery of some compounds. This note describes the specifications and construction details for a low temperature bath and its ancillary equipment developed in this laboratory for a cleanup procedure of pesticide residues. Precipitation, stirring, filtering, and washing of the precipitate are all performed in the bath. One feature is novel, others are derived from equipment previously reported (3,4,6). The novel feature is the use of nitrogen gas, instead of mechanical stirrers, to agitate the sample extract during the precipitation step. Six samples may be handled at one time.
Figure 1. A right side riew of the assembled low temperature bath and ancillary equipment
CONSTRUCTION
The equipment consists of the following major components: A stainless steel bath, with a stainless steel top cut to hold 6 sets of 3 glassware units (Figure 1). A nitrogen tank and a two-stage regulator (Figure 1). A vacuum motor with adjustable vacuum controls (Figure 1). A set of three glassware units (Figure 2) consisting of two SO-ml test tubes with lips (20 X 2.5 cm) and one filter tube. Also shown in this figure is the nitrogen bubbling capillary ball and socket tubes, a 125-1111 vacuum flask, and glass stoppers used to cover the reaction and filtering tubes. A filtering tube and a test tube rack (Figure 5). (1) Constance Anglin and W. P. McKinley, J. Agr. Food Chem., 8,
186 (1960).
(2) W. P. McKinley and G. Savary, ibid., 10, 229 (1962). (3) K. A. McCully and W.P. McKinley, J. Ass. Oflc. Agr. Chem., 47. a59 (1964). (4) i. A. R. Bates, ~ n o l y ~90,453 t, (1965). ( 5 ) S. b. Latham and T. N. Blumer, 3. Ass. Oflc.Anal. Ckem.,
53,789 (1970). (6) 0. W. Grussendorf, A. J. McGinnis, and J. Solomon, ibid.,p lW ._.I.
(7) K. A. McCully and W. P. 47, 652 (1964).
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McKinley, J. Ass: OJfic. Agr. Ckem.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972
Figure 2. An end view of the stainless steel top holding a glassware set used for each sample extract Reading from left to right; (1) a capillary tube with ball and socket joint leading into (2) a 5 0-ml test tube in which the precipitation reaction is carried out. (3) a 50-ml test tube to hold the wash solution. (4) a filtrationtube with a capillary tube leading out of the bath and into a vacuum flask with B tight fittingcork Figure 3 gives the dimensions of the bath and its components. Part A is the cover, constructed from stainless steel and having l'/% to 2 inches of styrofoam insulation. It is used to cover the bath when not in use. Part B is constructed of sheet, stainless steel with openings cut as illustrated and with the dimensions given in the blown up portion at the right hand edge of the illustration. This part is the working sur-
Figure 3. Dimensions and construction details for the low temperature bath
7’
I
L TEST TUBE RACK
FILTERING APPARATUS
FILTERING TUBE RACK
Figure 4. Dimensions and construction details for test tube rack, filtering tube rack, and filtering apparatus ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, J U N E 1972
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Figure 5. A photograph of the filtrationand 50-1111 test tubes face of the bath. Part C, right siae view iiiusrrares me glass vacuum manifold, rubber tubing and “C” clamp mounts. Part C, left side view illustrates the glass manifold for distributing nitrogen gas to 6 rubber tubings, each connected to a 6-cm glass tubing with a socket joint size 12/1, Pyrex No, 6162. Part D is a stainless steel bracket mounted on the side of the bath to hold the glass sockets. Also shown in this illustration are the external and internal (one) dimensions of the bath. The diagram labelled “section thru Part C” gives the height and width of the stainless steel interior dimensions of the bath, the Styrofoam insulation and veneer plywood outside walls. The material can be of any convenient thickness.
Figure 4 gives the dimensions of the test tube and filtering tube racks and filtering apparatus. The racks provide convenience in handling the test tubes and filter tubes. They can be constructed of sheet aluminum or staidless steel. The filtering apparatus consists of (A) a glass stopper size J 19; (B) a specially constructed filtering tube with a capillary tube lead out; (E) a 125-ml vacuum flask with cork stopper (D). The filter tube is a Pyrex No. 36060, 15 ml ASTM, 40-50C fritted glass filter, modified by fusing the top with an open end tube to give the dimension shown at “B”. The stem is made of a Pyrex capillary tube 5.5-mm 0.d. and approximately 1-mm i.d. The socket portion of the nitrogen capillary tube (6.5 mm 0.d. X 1.0 mm. id.) shown in the left hand test tube of Figure 2 must be extended to have a total length of 24-25 cm. A right angle bend is made 5 cm from the ball end. This tube is also used to mix the precipitate with the wash solution before filtration. The equipment has been used successfully in multipesticide screening procedures for foods and biological samples. Evaluation data on its efficiency have been presented elsewhere (8). ACKNOWLEDGMENT
The author thanks A. Moore for construction of the glass components, J. L. Kelly and staff for the sheet metal and cabinet assembly, G. Morris for drawings, and the National Health and Welfare Information Services for the photographs. RECEIVZD for review June 11,1971. Accepted November 19, 1911. H. A. McLeod and Patricia J. Wales, J. Agr. Food Chem., in press.
(8)
Versatile Colorimetric Coulometer William D. Ellis and Daniel T. Baker Honeywell Corporate Research Center, 500 Washington Avenue South, Hopkins, Minn. 55343
A VARIETY of chemical coulometers have been devised in the past. Lingane has reviewed the more common ones in detail (I). In this note, we describe a colorimetric coulometer based on the oxidation of the iodide ion to iodine, which is simpler, more sensitive, and more versatile than those previously reported.
place in the cuvette during the absorbance measurement and were later reconnected to the current source for further reaction. For the experiments on the timer version of the coulometer, a controlled constant 0.5 mA at 0.5 volt was passed through 2.6 ml of solution which was 0.23M in KI, 0.12M in NaHCOa, and 2.9 X lO-’Min As20a.
EXPERIMENTAL
Electrochemicalparameters were controlled with a National Instrument Laboratories Electrolab unit. Optical measurements were made using a Cary 14 spectrophotometer with 1-cm cuvettes. All chemicals used were reagent grade. The procedure for obtaining the colorimetric coulometer data was to pipet 2.5 ml of 0.24M KI solution, pH 6.5, into a cuvette containing a 2.1 cm2platinum working electrode and a nickel wire auxiliary electrode. The electrodes were placed so that they were out of the optical path in the cuvette. A controlled constant current of 0.1 mA at 0.5 volt was then passed through the cell for a specified period of time. The solution was stirred and the absorbance measured at 287 nm, the peak maximum for iodine. The electrodes were left in (1) J. J. Lingane, “Electroanalytical Chemistry,” 2nd Ed., Interscience Publishers, New York, N.Y., 1958, pp 452-459. 1330
ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972
RESULTS AND DISCUSSION
Iodine was chosen for this work because of its high molar absorptivity and because it can be generated with 99.99+ Z efficiency (2). The linearity of the resulting colorimetric coulometer is indicated by the results of a typical experiment io which fourteen data points were obtained over a coulombic input ranging up to 2.4 x 1W2coulomb, resulting in absorbance readings up to a value of 1.8. The computer fit linear least-squaresregression line for the data is: coulombs = (-2.4
+ 0.8) X
+
r(1.351 f 0.008) X 10-q absorbance (1) (2) G. Marinenko and J. K. Taylor, ANAL. CHEM.,39,1568 (1967).
