darker pink solutions as the iron content >\-asincreased. Apparatus. Automatic pipets [available fiom Corning Glass Works, Catalog Eo. 96126, in 2-ml. (special order), 5-mi., and 10-ml. capacities] are used to dispense the reagent solutions and the alcohol; 125-mi. separatory funnels and a Burrell rvrist-action shaker are used for thc alcohol extractions. The modifird technique employed small flat-bottoinerl vials (Fisher Scientific Co., Catalog No. 3-330, 4-ml. size) for color comparisons and estimations. The sides of the rials are painted black to inch from the mouth opening. Plastic
'!.
vial stoppers and vinyl filmfor covering the working surface are used to minimize contamination. Procedure. A 100-ml. sample of water (or a standard with a known iron content) is treated in a separatory funnel with 2 ml. of 10% hydroxylamine hydrochloride and 10 ml. of Bathovhenanthroline solution. After these a r e thoroughly mixed, j ,,I, of n-hexyl alcohol is addec{, . . ~are~ and the funnel and. rnnt,mt,s shaken for 10 min utes. (severnl samples and/or standa.rds may he rnii simultaneously.) The aqueous layer is drawn off and the ailcohol extract is uoured into the viewirtg vial bo within ~~~~
inch of the top.
Samples can bc ""2 yillu ,olor intensities visually evaluated by viewing the solutions against a white SUI' face from above-Le., axially. Color intensity of an unknown can he readily matched with standards in increments of 1 up to 10 p.p.b. and 2 up to 20 p.p.1~. .l+:,..n l Y l l
",.-.""*A
L'v.LEp~Lcu
-+""A""A"
oyLIIIucLIuI)
n
The range Can be extended U P ~ ~ ~ ~ r ~ sample dill:~ reduction ~ ~Of ~ ~quantity, . by tion of the alcolio1 extraction, or use I::t a spectrophoton reter at 533 mp. . - A - - L L "I/Llr WORKperformed .jlllUrl aUnpAGrr U. 8. Atomi2 Energy Commission.
ulic
^C / I
Improved Cutting De!{ice for Stanch Block Ellectrophswesis
..
Hans Bloemendal and Leendert Bosch, Department at Biochemistry Antoni van Leeuwenhoek-Huis, .I ., , , r , Nernerianas Cancer insrirure, Amsterdam, The Netherlands
... .
OTARCH block
electronhoresis is of
3 gron-ingimportancc 6 proteiii clicm-
istry for analytical studies as well ns isolatiou on a preparative scale. The supporting medium, pota.to starch, is mixed with buffer and poured int,o a Perspex trough or box. Excess fluid is withdrawn with blotting paper. The st,a,rch block is connrctrd to clectrode vessels by paper strips, plastic s])onges, moist, cloth, or agar bridges. According to Kunkel and Slater ( 5 ) ,the starch block is sliced into equal segments after the electrophoretic run, with a thin spatula (3, 4) or a knife. Paigen (6) marked the starch bed with a comb having teeth spaced 0.5 cm.; then sliced off the sections and removed them in serial order, using a plastic skip with beveled edge. Carlson (2) noted that it is difficult to cut t.he starch with precision. Errors of 10 to 20% may arise when 0.5-em.
I,
seunents are cut as orimnanv clescrlDea b; Kunkel and Srater." Paigen's method is insufficiently exact when proteins, the mobilities of which differ ouly a little, are under investigation. To circumvent these troubles a calibrated box (Figure 1,A) was used to permit more exact cutting (1). Over it iyas placed a guillotinelike frame, B, ryhich holds a stainless steel kiiife of cross section equal to the starch block, C. Although this considerably improves the slicing technique, small distortions of the starch block may still occur, resulting in poorly reproducible patterns after protein estimation. Furthermore, this procedure takes much time, particularly when long boxes are usrd. Substantial improvement is obtained with the apparatus shown in Figure 2. A Perspex box (Figure 3,A) with removable side walls, B, fits in a cutting device illustrated in Figure 2. The latter consists of a brass frame, (Figure 3, D) joined to a metal plate, C, by hinges. Thin stainless steel threads are spanned on the frame a t 1-cm. intervals. After an electrophoretic run the two side walls of the box are removed and the bottom with the starch hlock i s
placea m a nxea ~ O S I Z I U I I UII LIK I I I ~ : L I U I plate. The frame is turned down Kith the aid of the handles, E. The bottom of the box is supplied with notches a t 1-em. intervals, int.0 which the threads fit. Cutting is performed in a single manipulation and exactly equal starch sogments are obtained, which are easilrtransferred to centrifuge tubes. ACKNOWLEDGMENl
Thanks are due to Xi. J. BergstrCiii and N. A. van Zwol for constructing the apparatus. LITERATURE CITED
(1) Bloemendal, H., Proc. Koninkl. Acucl-
Wetenschap. C 59, 22 (1958). (2) Carlson, L. A,, Acta Chenz. Scand. 8,
510 (1954). (3) Kunkel, H. G., "Methods of Biochemical Analysis," Vol. 1, p. 157, Interscience, New Yark, 1954. (4) Kunkel, H. G., Slater, R. J.: J . C& Invest. 31,677 (1952). (5) Kunkel, H. G., Slater, R. J., Proe. Soe. Ezptl. R i d M c d . 80,42 (1952). (6) l'aigen, K . , A v ~ r . .CtimI. 28, 284 (1956).
Figure 3. Cutting device Figure 1 .
Cutting device
A. Calibrated box. 4-mm. Persoex. 20 X 40 X
400 mm. B . Holder. 12-mm. Perrpex C. Stainlen steel knife [knife 20 X 40 X 0.4 m m , handle 20 X 6 0 X 4 mm.)
1446
. I
ANALYTICAL CHEMISTRY
Figure 2. spex box
Cutting device with Per-
A. Bottom of 12-mm. Perrpex box 8. Removable ride w~III,6-mm. Perrpex C. Dura! ground plate of cutting device, 20 mm. D . Bmrr frome with rtoinlers ,tee1 threads, 0.3
E.
mm. Handles,
