Coordinate System for Electrophoresis VERNON L. FRAJMPTON AND KENNETH E. HARWELL Basic Cotton Research Laboratory, University of Texas, Austin, Tez. The imposition of an orthogonal coordinate system on the scanning negatives obtained in electmphoresis studies permits a more convenient and acetirate determination of the boundary positions and of the areas occupied by the shadows. The orthogonal coordinate system is produced on the negative by a comhination of a millimeter scale etched on the face of the electrophoresis eel1 and one, normal to that on the cell, etched on a glass plate that is placed immediately before the unexposed plate in the plate holder. The advantage gained is the elimination of the microcornparator and of the planimeter measurements.
T .
HE advantages experienced by the superposition of a
co-
ordinate system upon the manning pattern obtained in electrophoretic work include a more convenient method than tho
Figure 1. Millimeter Scale I Engraved on 11-MI. Tiselius$isrnn
Figure 2.
one currently used for the determination of the position of tlm boundary, and a more precise and convenient method fur t h e determination of the a m s occupied by the shadows than the one involving the'use of % planimeter. Certain errors are also reduced by the scheme presented in this paper, inasmuch as it is: independent of optical defects in the camera, and the inaccuracies in determining the magnification factor are not important. T h e errors introduced because of mechanical movements bctn-ccn t h e camera and cell are also reduced. A millimeter scale was engraved on thc face of each Icg of the center section of the Tiselius-type, 11-ml. electrophoresis cell, as shown in Figure 1. The method of engraving was cntirelg conventional. Molten pwsffin IV&S brushed over lhe face of each leg of the cell to give a smooth coating. The cell was then clamped to the table of a dividing engine and aligned with a machinist's square. The knife edge of the engine was drawn through the parafi, making a mark on each leg of the cell. The cell was then advanced 1 mm. and an additional set of marks u-a8 made, This was continued throughout the entire length of the cell, except that every tenth line was omitted. The cell was then removed from the engine and inspected with a lens. A cotton swab, moistened with a 48% solution of hydrofluoric acid, tvas then brushcd over the division'marks, and aftcr half a
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.
Pattern Obtained with Graduated Cell Ur tg Longsworth Scanning ' .chnique
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ANALYTICAL CHEMISTRY
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Y
Figure 3.
Superposition of Scanning Pattern upon Coordinate Sjstern Using Longsworth Technique
minute, the cell was thoroughly washed with water. The paraffin was subsequently removed with benzene. Finer gcaduittions m y be ruled with a diamond (a). The scale engraved on the face of the cell was checked with a microcompsrator and was found t o be accurate to +0.003 mm. The scale lines appearing in the photographs taken during the normal course of an electrophoresis experiment extend over the
Figure 4.
.
length of the plate. Became of the groove nhape of the scale l i e s , the light passing through the loner portion of the groove is refracted upward, whereas the light passing through the upper portion is refracted downward. The effect is that in the schlieren method the dark part of the image of the scale lines is emphasized in the light areas of the photograph and the light part of the image is emphasized in the dark part of the photograph. 'One
Philpot Scanning Pattern
Mask st focal plane bsfore cemua lens above, rather than bslor,
light image wduated cell
sod millimeter plate vaad
V O L U M E 2 3 , NO. 5, M A Y 1 9 5 1 obtains an illusion that the linrs are dinplaced a t the junction of the light and dark parts of the photograph. Actually, there is no displacement of the lines except in the schlieren shadow, as may be seen if the inner edges of the light and dark portions of the line images are considered Measurements should be made from these inner edges. In Figure 2 the scale lines appearing in the schlieren shadows are displaced in the direction of increasing concentration gradient in the cell, but the displacement of the lines is reduced to a minimum by virtue of the nearness of the scale to the solution in the cell. The displacement of the scale lines in the photograph, however, is of no importance in connection with the determination of the rate of boundary movement. Khen a particular boundary has moved five divisions, it has moved 5 mm. In the actual operation, the cell is placed in the bath with the scale lines toward the camera. A photograph is taken a t zero time after the boundaries have been moved into the center section, and the knife edge has been placed in a position to give the narrowest shadow possible. This operation records the original position of the boundary. The electrophoresis is then continued in the normal manner, and all measurements of the boundary positions observed in the photographs taken subsequently are referred to the original photograph. The number of divi*ions through which the boundary has moved is a measure of the distance moved, and it is independent of the camera system. The accuracy of any determination ma\ be improved by intrrpolation with the aid of a microcomparator. The scale lines may be used in determining the area occupied by the shadows in the electrophoresis pattern. The sum of the lengths of the lines in any given shadow, multiplied by the
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magnification factor of the camera system, is equal to the area. The determination of the lengths of the lines is greatly facilitated by superimposing the pattern upon a coordinate system. The engravings on the face of the cell effect the production of a scale in one direction in the photograph. .4 scale, normal to the first, is produced by placing a millimeter scale immediately before the unexposed plate in the plate holder. The scale used in this work was etched on a 9 X 12 cm. glass plate with hydrofluoric acid by the procedure employed with the cell. Thus, a coordinate system, as well as the scanning pattern, appears on the negative, as illustrated in Figure 3. The patterns presented in Figure 3 were obtained using the Longsworth scanning technique ( I ) , but similar results are ohtained with the technique for obtaining electrophoresis patterns described by Philpot (S), and by Svensson (4). The pattern shown in Figure 4 was obtained by the Philpot technique. The graduated cell, together with the millimeter scale in the plate holder, were used in the manner indicated in the Longsworth scanning procedure. In this instance, however, the mask was set a t 45"with the horizontal, and was placed above, rather than below, the light image immediately before the camera lens. Thus the pattern was reversed-that is, the pattern was light and the background dark. LITERATURE CITED (1) Longsworth, L. G., Chem. Revs., 30,323 (1942). (2) Longsworth, L.G., J . Am. Chem. SOC.,66,449(1944) (3) Philpot, J. S.L., Nature, 141, 283 (1938). (1) Svensson, H.,Kolloid-Z., 87, 181 (1940). RECEIVED .2ugust 19, 1949.
Fluorometric Determination of 2-Nitronaphthalene F. L. ENGLISH AND J . W. EPPERT Chambers Works, E. 1. d u Pont d e .Vemours & Co., Znc., Deepwater, S. J . This work was undertaken to establish a rapid method for the estimation of small amounts of 2nitronaphthalene in crude and refined l-nitronaphthalene. The method depends upon sulfonation, reduction, and measurement of the blue fluorescent light intensity produced by- the beta isomer when irradiated with ultraviolet light of 365 mp wave
THE
3 RE are many references in the literature to the fluorescent properties of naphthalene derivatives ( I ) , but none of them appeared t o apply directly t o the estimation of small amounts of Znitronaphthalene (beta) in refined and crude l-nitronaphthalene (alpha). Preliminary tests showed that neither of these compounds fluoresces appreciably under light of 365 mp wave length. It is well known, however, that under these condition. the corresponding amines do fluoresce, the beta much more strongly than the alpha. The investigation was, therefore, conducted along this line.
APPARATUS
A Coleman Model 14 Universal spectrophotometer with fluorometer attachment, operated through a constant voltage transformer, was used in the ex rimental work. It was provided with a Coleman UV-1 li h t K e r for the mercury lamp and Corning Nos. 5433 and 3850 alters for the photoelectric cell. Fluorescence measurements were taken from a 20 X 40 mm. cuvette. T o minimize undesired reflection of stray light, the entire surface of the cuvette carrier was roughened with emery cloth and it, as well as the inside of the light compartment of the spectrophotometer, was painted with a flat black paint.
length, using suitable filters to exclude the dull green light produced by the alpha. The procedure will detect 0.05% beta and covers the range up to 5 % with a precision of about *3% of the amount present. Naphthalene and dinitronaphthalene normally present in crude material do not interfere. 4 single determination can be run in an hour. REAGENTS
Monohydrate, 99 to 100% sulfuric acid. Oleum, 58 to 6070 free sulfur trioxide. Charcoal, Nuchar W. Darco D-51 was tried and found unsatipfactory. Rash xater, 2 ml. of 9570 sulfuric acid diluted to 1 liter. Sodium acetate, 100 grams of sodium acetate trihydrate dissolved in 100 ml. of water. STANDARD REFERENCE SOLUTION
Sulfonate 0.100 gram of purified 2-naphthylamine as directed below for the analysis of samples and dilute to 500 ml. This solution should be clear and colorless without charcoal treatment or filtration. Dilute a 5-ml. aliquot to 500 ml. Both solutions are stable for a t least a month. Prepare the final reference solution daily as needed by diluting 5 ml. of the second solution plus 1 ml. of the sodium acetate buffer to 100 ml. PROCEDURE
Weigh 0.100 gram of sample into a 6-inch test tube, add 1 ml. of monohydrate, heat for about a half minute in a water bath at 50' C. to effect complete solution, cool to 25' in a water bath, and while swirling the tube vigorously add rapidly 2 ml. of oleum.
