that no significant amount of COz is being lost. RESULTS
When the same procedure and compounds were used as for the sulfur determination ( I ) , the results shown in Figure 2 were obtained wit,h samples ranging from 2 to 5 mg. The straight line drawn through the points is the predicted value using diphenyl sulfone as a standard. LITERATURE CITED
Figure 2.
Calibration chart
(1) Beuerman, D. R., Meloan, C. E., ANAL. CHEM. 34,319 (1962).
DONALD R. BEUERhlANl E. hfELOAN CLIFTON Dept. of Chemistry Kansas State University Manhattan, Kan.
Abstracted from a thesis by Donald R. Beuerman in partial fulfillment for the degree of Master of Science a t Kansas State University. Financial support from the Kansas State University Bureau of
General Research knowledged.
is
gratefully
ac-
Present Address: Monsanto Chemical Go., East St. Louis, Mo.
Attachment for Making low Temperature Spectra in a Recording Spectrophotometer Willard B. Elliott and William Tanski, Department of Biochemistry and Medical Shop, University of Buffalo School of Medicine, Buffalo 14, N. Y.
T
HE history of low temperature spectrophotometry in the study of cytochromes has recently been reviewed by Estabrook ( 2 ) . The usual apparatus used in recent years h a r e been composed of separate monochromator and detector systems ($, spectrophotometers such as the Reckman D U with extensive modifications including a large. lightproof dry bo.; between the monochromator and the phototube compartment, or special spectrophotometers such as those described by Estabrook (8)' By designing a special Dewar flask (Figure I), fabricated by H. S. hIartin Co., Eranston, Ill., a-ith a flattened oval cross section, of a size to fit inside the cell compartment of the Cary Model 11 spectrophotometer, and a suitable sample holder based on designs used by Yocum (4) and Bonner ( I ) , it has been possible to obtain liquid nitrogen spectra of cytochrome and cell particulate preparations without rebuilding the spectrophotometer. The apparatus can be constructed a t a cost of approxi-
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ANALYTICAL CHEMISTRY
mately 3125 per unit (two units would be required for difference spectra). The design of the Dewar, with only the curvature of the wall necessary to prevent implosion, and the acrylic plastic Dewar holder, B, place the sample as close to the exit opening of the cell compartment as possible to
Figure 1.
Dewar flask
reduce the light loss from scattering b y the turbid sample. The Dewar flask holds sufficient liquid nitrogen to freeze the sample and to maintain it at liquid nitrogen temperature for about 45 minutes. The liquid nitrogen level is below the optical path at the end of the freezing period and during the scan. The optical path passes through the Dewar in an area of minimum curvature of the walls of the Dewar flask. The cuvette, D ,is 5 em. rride by 4 em. high, with 2.5 x 2.5 cm. opening aluminum spacer, and acrylic plastic windows 1i16-inchthick and 3 cm. high. A thin film of Apiezon N ib used to seal the plates to the spacer. The cuvette and its support (Figure 2 ) are designed to permit ease in positioning in the optical path of the spectrophotometer and in lowering the cell into the narrow opening of the Dewar. The spline on the brass support rod, J , and adjustable ring stop, F,make the positioning of the cuvettes in the Dewar reproducible. The positioning screw> H , on the aluminum base plate of the holder positions the holder in the cell compartment. where it rests against
a
Figure 2.
Cuvette and cuvette support
Blueprint showing details of construction available on request
the rear wall of the compartment on the rods t h a t normally support the sample cuvette carrier base plate. Cuvettes with light paths of 1 to 5 mm., depending on the thickness of the aluminum spacer, are readily inserted into the cuvette support arm.
.Isthe only alterations to the spectrophotometer are removal of the cuvette carrier base plate and the use of a simple metal box to extend the height of the sample compartment, the change-over to do low temperature spectra requires approximately 5 minutes. -1modified cuvette holder for use in the reference cuvette compartment has a shorter post to support the cuvette as a result of the height limitations imposed by the compartment design. Frosting of the upper outer surface of the Dewar in the spectrophotometer is eliminated by the normal operating temperature of the cuvette compartment and the passage of dry nitrogen through the curette Compartment, except when used for more than 1 to 2 hours during periods of high relative humidity. The spectra obtained a t room and liquid nitrogen temperatures are compared in Figure 3. The liquid nitrogen
Figure 3.
- 192' C.
Comparison of spectra
y x c t r u m of the 01 and p regions of cysteine-reduced cytochrome c in phosphate buffer compares favorably with that obtained by Estabrook in the Chance spectrophotometer ( 2 ) . The 25" C. spectrum (thick line) was made with high gain (dynode 1, slit control 10) n-ith the cytochrome and solution in place in the 5-mm. lorn temperature cuvette in the Dewar n i t h air in the reference beam. The line thickness indicates the peak to peak noise level. After the cytochrome solution had been frozen in the cuvette b y lowering it into liquid nitrogen, the cuvette was placed in the Dewar along with sufficient liquid nitrogen to cover the cuvette up t o the windom-. The light-scattering loss caused by the ice crystals was compensated for b y placing filter paper in the reference beam. The balance control n a s adjusted to 0.00 4. a t 600 mp. The dynode and slit control settings were not changed. The recording pen was reset t o 0.00 -4,when i t reached 0.80 A., so that the upper portion of the peak would be continuous,
made
at
25" and
Slight modification of the basic design would permit its use in other spectrophotometers, where the distance from the cell compartment exit opening to the light detector is minimal. ACKNOWLEDGMENT
G. F. Doebbler, Linde Co., generously supplied the liquid nitrogen used in the development of this apparatus. LITERATURE CITED
(1) Bonner, IT, D., Jr., "Haematin Enzymes," Part 2, J. E. Falk, R. Lemberg, R. K. Morton. eds.. International Union of Biochemistrv Symposium, Vol. 19, p. 479, Pergamon, Oxford, 1961. (2) Estabrook, R. W., Ibid., p. 436. (3) . . Estabrook, R. W., J . Bid. Chem. 223, 781 (1956). ' (4). Yocum, Conrad, personal communication.
WORKsupported by P. H. S. Research Grant (RG 6241) from the Division of General Medical Sciences. The senior author is a P. H. S. Research Career Development Awardee (GM-K3-lS,514).
VOL. 34, NO. 12, NOVEMBER 1962
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