Adaptation of Beckman Model B Spectrophotometer As Spectrofluorometer Guy Nadeau and 1. P. Joly, Department of Biochemistry, L'H6pital Saint-Michel Archange, anal School of Chemistry, Laval University, Quibec, Can.
of the Beckman M Model DU spectrophotometer for spectrofluorometric determinatioiis have ODIFICATIONS
already been described (1-5). To the authorsJ knowledge, none of these publications has suggested the use of its clinical counterpart, the Model B, for the same purpose.
I n the present adaptation, the sample of fluorescing material, 7, Figure 1, is placed within the solid angle viewed by the entrance mirror, 13. Fluorescence is excited by a beam of ultraviolet radiation originating from an external mercury lamp, 1. The incoming beam is reflected by means of an adjustable mirror, 4, and reaches the sample perpendicular
I
,I I
4
Figure 1. Attachment fluorometry
for
A.
spectro-
Housing for exciting light source Sample eompartment. 1, External mercury lamp 2. Cylindrical mirror 3. Primary filter 4. Adjustable mirror Mechanism for adjusting mirror 4 5. 6. Cell carrier 7. Fluorescing material 8, 9. Plane mirrors 10. Grooved base for cell carrier 1 1. Adjustment screw 12. Brass bor 13. Entrance mirror 14. Brass plate
E.
ao
EJi 6o
3 +
40
PO
0
600
500
WAVE LENGTH, M p
Figure 2. 1. 2.
Uncorrected fluorescence spectra
Quinine sulfate in 0.1N SUIfuric acid solution Fluorescein in pH 8.0 phosphate buffer solution
3.
4.
Phloxine in pH 8.0 phosphate buffer solution Rose Bengal in 95% etbyl alcohol
to the fluorescence light path. This eliminates most of the interference from the ultraviolet source. To allow easier optical alignment, the burner housing attachment used in flame photometry has been converted into a housing for the exciting light source (Figure 1,il). The burner is unscrewed, the mercury lamp (G. E. HlOO A-4) is fitted by a metal collar on the mounting block, and centered on the vertical axis of the overhead chimney. This facilitates cooling of the assembly. On the same mounting block is added an adjustable cylindrical mirror, 2, to reflect toward the sample an additional amount of exciting ultraviolet light. The whole housing is ;slightly shifted to the right (about 6,5 mm.) from its normal position (dotted lines, Figure 1, A) and is secured by screws to the base assembly provided for flame photometry. The back of the spectrophotometer itself is lightproofed by means of a plywood sheet with a window for the ultraviolet light path, a holder for a, primary filter, 3, and an opening for access to the mobile cell carrier, 6. The additional parts necessary to complete the assembly are illustrated in Figure 1, B, and can be described as follows. The tungsten lamp housing is replaced by a brass plate, 14, with a window through which the entrance mirror, 13, can be seen, and which supports three other parts. These are: Brass bar 12, added to carry adjustment screw 11 for the entrance mirror, this screw being left Githout support when the lamp housing is removed; A base, 10, formed of two parallel grooved brass bars on which slides freely a cell carrier, 6, made of blackened hardwood topped with Bakelite. The cell holder has four opposite openings, one on each side, and is supplied with two plane mirrors, 8 and 9, a t right angles in order to increase the intensity of the fluorescence. The cell carrier rests on two guiding brass bars sliding in the grooved base; A plate on which rests variable angle mirror 4 and its adjusting mechanism, 5 . This is intended to reflect the exciting ultraviolet light so that it reaches the sample pcrpendicular to the path of the fluorescent light. The recent addition of an RCA IP28 photomultiplier to the standard Beckman Model B spectrophotometer allows the determination of the fluorescence spectrum of quite dilute solutions of fluclrescent material. As a reference, a solution of 0.25 y of quinine sulfate (USP) per 1x11. of 0.1N sulfuric acid gave a scale reading of 100 against VOL. 29, NO. 4, APRIL 1957
a
583
a blank of the solvent, with the photomultiplier sensitivity set a t maximum, the slit width a t 1.5 mm. using a 3 mm., 18-A. Wratten primary filter, and a 100-watt mercury lamp. A higher sensitivity coald be obtained by using brighter light sources such as the G.E. H250 A-5. Figure 2 illustrates the spectral distribution of fluoresceiice of various substances a t low concentration. These
curves were plotted inerely to appraise the versatility of the apparatus, as there may be som: important shifts of the peaks with variations in concentrations. ACKNO W1IiDGMENT
One of the authors (L.P.J.) is the holder of a studentship from the National Research Coiincil, Canada.
LITERATURE CITED
(1) Burdett, R. A., Jones, L. C., J. Opt. SOC.Amer. 37, 554 (1947). (2) Gornall, A. G., Ilalant, H., ANAL. CHIXI.27, 474 (1955). (3) H u h , F. B., Heidel, R. H., Fassel, V. A.. J . Opt. SOC.Anier. 43, 400 (1953j. (4) . . Lauer. J. L., Rosenbaum, E. J., Ibid. 41;450 (1951). (5) Lowry, 0. H., J . B i d . Chem. 180, 389 (1949).
Inert-Atmosphere Dry Box Thomas R. P. Gibb, Jr., Tufts University, Medford, Mass.
increasing use of air-sensitive or materials in research laboratories has resulted in the design of a variety of devices for handling such materials (1,8,4,5,11-14). Several types of dry boxes are available commercially (11). This paper describes a large, relatively inexpensive, and versatile apparatus which may be employed for a vide variety of analytical manipulations, such as weighing, grinding, and transferring, in an atmosphere of inert gas, particularly nitrogen. HE
Tmoisture-sensitive
level and wooden parts of a balance must be removed before evacuation and replaced by suitable substitutes.) The windows of th‘e box are safety glass set by means of plastic compound into angle irons brazed to the $ides of the aperture. DETAILS OF CONSTRUCTION
The back, front, top, bottom, and right side are 1/8-mh hobrolled steel sheet. The left side, which supports the vacuuni lock, is 1/&xh hot-rolled steel.
The vacuum lock is steel tubing 14 inches in diameter with a wall thickness of inch. The shoulders are trued and slightly rounded on a lathe to reduce wear on the rubber-gasketed doors. The inner door fittings are silver-soldered in place; the tube is pushed through a tight-fitting circular hole in the box and brazed in place. The remaining fittings are then silver-soldered. The hinges are 1/2-inch steel, melded to the lock, and slotted so that the hinge pin is floating. This avoids unsymmetricai er
GENERAL DESCRIPTION
The design and operation of the dry box may be made clear by the follov-ing description and by reference to Figures 1to 3. The main part of the all-welded steel box contains dry nitrogen or argon, which is continually leaked into the box through a drying train a t the rate of about 0.1 cc. per minute. The gas.in the box is constantly purified by circulating a t the rate of several cubic feet per minute through a high voltage sodium arc (Figure 4). A twin centrifugal blower in the box accomplishes this circulation and also very rapid movement of gas in the entire box. The box is illuminated by a fluorescent lamp, and contains several electric outlets, gas cocks, and large pipe connections through the exterior wall and through the mall of the vacuum lock. The lock has two hinged and gasketed doors of the “porthole” type, which may be made vacuum-tight by the wing bolts shown. The vacuum lock is connected by’ largecapacity stopcocks (1and 4) to a largecapacity mechanical pump, by small cocks (2) to a supply of purified nitrogen or argon, and directly (cock 5 ) to the dry box. It has a fitting a t the bottom through which leads for an electric heater may be brought, permitting its use as a vacuum oven. The lock is large enough so that a small ball mill or partly dismantled analytical balance may be brought through it, (The spirit 584
ANALYTICAL CHEMISTRY
Figure 1 .
Front view of box
Vacuum lock at left, portion of spark purifier at extreme right. Tubing connections indicated by standard symbols. Vacuum and inert gas are piped to lock, left side of box, front of box (for coiinection to port covers), and rear of box (not shown). Blower, shown in dotted lines, is lccated within box at right
I v2‘ 1/4”
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4.”
............-....... ,/
............................
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,y ............................ ........
.......
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Figure 2. Top view of box Blower and spark purifier at right. Outlined rectangle near center is a fluorescent lamp. Cocks 9 and 10 arc: within box. A port cover is shown in position at lower right
