Figure 1. Fluorescence attachment
A . Cuvette holder clamped B.
C. D. E.
G.
being measured, the shutter is closed, to minimize photochemical decomposition of the sample. The optical arrangement is such t h a t the angle betxeen the incident light upon the cuvette and the emergent light viewed by the condensing mirror is approximately 20”. Because the sample is viewed on the illuminated side, errors due to overlap of absorption and emission spectra are diminished ( 2 ) . However, in a cuvette of large diameter, concentration effects shift the position of maximum intensity of emitted light within the cuvette and may make i t necessary to adjust the horizontal focus of the mirror. T o permit easy adjustment of the horizontal mirror focus after the instrument is assembled, a wheel (13/4 inches in diameter) with a knurled edge is attached to the end of the horizontal adjustment screw. Once the vertical focus of the mirror and position of the cuvette holder have been adjusted, it is rarely necessary to change them.
on lamp bracket of backplate Beckman Model 3240 hydrogen lamp backplate Horizontal focus screw Filter house with side cut away to show filter in place Shutter assembly displaced vertically from position between filter house and lamp house, F Supporting rods screwed into backplate
The modified backplate is attached to the lamp house of the spectrophotometer and the mercury arc lamp is turned on. A cuvette containing the sample is placed in the cuvette holder and the lid of the lamp house is replaced. The sensitivity control of the spectrophotometer is turned to maximum sensitivity (fully counterclockwise). The sensitivity switch of the photomultiplier attachment is turned to full. The slit is opened to 2 mm., or less if intensity of fluorescence permits. The wave length selector is rotated to the desired position and the transmittance is set a t zero. The selector switch is set at 1.0 or 0.1, depending on the intensity of the fluorescence. The shutter switch is opened, and the dark current control is adjusted to bring the galvanometer to zero. The shutter on the backplate attachment is then lifted to illuminate the sample. The intensity of the emitted light is read on the transmittance scale in arbitrary units. K h e n an emission spectrum has been obtained, the wave length selector may be set at a position of peak intensity and the mirror focus adjusted to achieve a
450
500 mJJ
Figure 2. Fluorescence spectrum of quinine sulfate
maximum reading on the transmittance scale. -4 solution of quinine sulfate containing 0.3 y per nil. in 0.1N sulfuric acid, a t 460 mp and slit n-idth of 2 mm., pare a reading of 52.9 ==I 0.47 (standard deviation) on the transmittance scale in 10 measurements made by removing and replacing the cuvette. The rmission spectrum of fluorescence of quinine sulfate shown in Figure 2 was obtained by using a solution containing 0.06 y per ml. in 0 . 0 2 s sulfuric acid. The slit of the spectrophotometrr was set a t 2 mm. A modified tungsten lamp backplate has been described by Cardon, Alvord, Rand, and Hitchcock ( 4 ) . ACKNOWLEDGMENT
The author is indebted to the Kational Cancer Institute of Canada and the Dalhousie Medical Research Fund for grants. LITERATURE CITED
Beckman Instruments, Inc., Fullerton, Calif., Bull. 19 (1957);( Bowen, E. J., Wokes, F., Fluorescence of Solutions,” Longmans, Green Co., Toronto, 1953. Burdett, R. A., Jones, L. C., J. Opt. SOC.Amer. 37, 554 (1947). Cardon, S. Z., Alvord, E. T., Rand, H. J., Hitchcock, R., Brit. J Cancer 10, 485-96 (1956). Gornall, A. G., Kalant, H., ANAL. CHEN.27, 474 (1955). Huke, F. B., Heidel, R. H., Fassel, V. A., Ibid., 43, 400 (1953). Lauer, J. L., Rosenbaum, E. J., Ibid., 4 1, 450 (1951).
Glass Pens for Beckman Model DK-2 Spectrophotometer A. C. Arcus, Nutrition Research Department, Medical School, Dunedin, New Zealand
of spectra in different colors with the Beckman Model DK-2 recording spectrophotometer has been facilitated in this laboratory by the use of readily interchangeable glass pens requiring almost no attention. ECORDIKG
A capillary 2 em. long with a bore of about 0.05 em. is drawn out from the end of a n 8-cm. length of borosilicate glass tubing (external diameter, 0.3 em.; bore, 0.2 em.). The tube is then clamped vertically, capillary end downward, and t h e capillary flamed evenly with a
microburner held directly underneath (Figure l),until the bore has narrowed sufficiently near its upper end. The surplus capillary is cut off a t the narrow place and the tip of the pen is ground t o a point of the size wanted. The finished tip, which is thick walled and strong, should be about 1 em. long and have a bore narrowing t o about 0.006 cm. The correct bore is best gaged by comparing its size visually through a magnifying glass with that of a pen found t o work well. On the recorder the pens are pushed into a stout spring clip made from brass
strip (24-gage, 0.9 cm. wide), which is permanently and rigidly screwed onto the pen carrier, as shown in Figure 2. This arrangement ensures that the pen does not vibrate on the pen carrier and that the position of the pen point on the chart is accurately reproduced &-hen pens are changed. K i t h the five pens being used a t present in this laboratory, the points all fall on the chart within a circle of 0.025-em. radius, corresponding to a maximal variation of =kO.l% on the transmittance scale. The reproduciVOL. 30, NO. 1, JANUARY 1958
159
bility on the Kave length scale is not affected because the wave length setting can be checked before each run. The pens are stored partly submerged in colored writing ink. For this purpose 0.8-em. holes are drilled centrally in the screw caps of the ink bottles; under each cap is fitted a rubber washer with a 0.2-cm. central hole for the pen and another small hole for an air leak. I n this way the pens are prevented from drying out and kept loaded with ink. Unlike the pen supplied with the instrument, the glass pen described seldom blocks. Khen it does, the blockage is removed by flushing with water. Although it has been possible to test these pens only n i t h the Model DK-2 spectrophotometer, no doubt they could be used with any similar instrument.
1
PEN
r ?
PEN
-
SPECTRO PHOTOMETER
POINT OF NARWING
MICRO BURNER
Figure 1.
PEN CARRIAGE
1
\ /
'-/
,
-
Formation Figure 2. Glass pen ottached to pen carriage
of capillary tip on glass pen
Device for Measuring Rf Values Robert
v
L.
Clements', Department of Agricultural Biochemistry, The Ohio State University, Columbus, Ohio
have been used for the rapid determination of Rfvalues on paper chromatograms. The following instrument is particularly useful for small chromatograms, but larger versions can be constructed to accommodate papers of any size. The device does not cover the paper during measurements; thus notations can be made directly on the chromatogram. It is easily constructed, and a high degree of precision is possible without special machining or engraving. ARIOCS DEVICES
The instrument consists of a rigid right angle with a straight pointer pivoted a t the end of one leg and a decimal scale laid out along the other. The scale, A, is positioned along the vertical leg, so that the zero point lies on the upper edge of the horizontal leg. The pointer, B , is pivoted from a point, C, which lies on the upper edge of the horizontal leg, and is located one scale length from the vertical scale-i.e., 2 = y. A small sliding indicator, D, is attached to the horizontal leg, and a reference line, E, is engraved through the pivot parallel to the scale. I n use, the slide indicator is first set so that the distance, f, from the slide to the pivot is equal to the frontal distance. This may be done by measuring off the distance by superimposition, or by placing the upper edge of the horizontal leg on the starting line of the Chromatogram, setting the pointer t o R , = 1, and setting the slide t o correspond to the point a t which thp pointer crosses the front. Readings are then made with the upper edge of Present address, Department of Plant Biochemistry, University of California, Riverside, Calif. 160
ANALYTICAL CHEMISTRY
the horizontal leg on the starting line of the chromatogram, and with the slide indicator at the point of application of the sample. The pointer is positioned over each spot (resulting from that particular application) and the corresponding R f values are read from the points at which the pointer crosses the scale. When there is a slope in the solvent front, Rfvalues may be determined by adjusting the slide indicator for each application (as though it were an individual chromatogram). The instrument can be modified for measurement
of distances on chromatograms on which the solvent front has been allowed to run off the paper and a value based on a reference compound is used -e.g., Rglucose. For this application the decimal scale should extend t o 2.0 (or greater, as dictated by the particular case). The slide indicator distance, f, would then be set with the pointer a t 1.0 on the scale for the particular reference compound. The existing models were constructed of aluminum, but any rigid material should suffice. The pointer consists of
A. Decimal scale
-
Chromatogram
-
Front
-
Starting line
