ANALYTICAL CHEMISTRY
1836 in which the (1- X ) volume of toluene in the above formula was replaced with water. No appreciable reaction occurred between the mercury and the sulfur directly, as the wave height was constant for more than 1 hour; the half-wave potential of sulfur was -0.54 volt. The concentration of toluene must not exceed 4% by volume under the above reported conditions; otherwise a part of it is not solubilized, but only emulsified. The sulfur in this emulsified solvent is not recorded by the polarograph. Thus, the wave height decreases on adding toluene. On the other hand, the wave height was slightly increased by ethyl alcohol and considerably by benzyl alcohol. This is shown in Figure 6 which was obtained with a solution containing 1.0 ml. of sulfur (0.5%) in toluene, 12.5 ml. of Aerosol MA 20% in water, 2.5 ml. of 0.33 M acetic acid plus 0.33 M sodium acetate, 0 to 5 ml. of additional solvent, and made up to 25 ml. with water. Determination of Peroxides in Fats. Lard was oxidized by heating a t 90" to 95" C. in an oven for several hours. I t was found that the best solution for polarographic purposes was one containing 5 ml. of a 10% solution of oxidized lard in chloroform, 5 ml. of benzyl alcohol, and made up to 25 ml. with Aerosol AY (20% in water). I n this case, Aerosol .4Y is not only the solubilizing agent, but has to be also the supporting electrolyte, as any additional electrolyte decreases the amount of solubilization. Benzyl alcohol was used because it was found that it improved the shape and height of the wave considerably, probably because it increases the mutual solubility of the soap micelles and the water (Figure 4). Under the above conditions, the peroxides in oxidized lard give a wave a t -0.32 volt (see Figure 5, B ) and a linear relation between wave height and content of peroxide was established; the peroxide numbers of the oxidized fats were determined by the Wheeler method ( l a ) . Determination of tert-Butyl Hydroperoxide. Some liquid organic compounds which are reducible a t the dropping mercury electrode may be solubilized without using an organic solvent. tert-Butyl hydroperoxide is only slightly soluble in water and normally cannot be determined polarographically unless dissolved in a suitable solvent. Aerosol MA can be used to solubilize this material so that it can be determined in aqueous solutions. terf-Butyl hydroperoxide, 0.45 gram, was dissolved in 10 ml. of a 20% aqueous solution of Aerosol MA and a 0.05 M solution was prepared by making up to 100 ml. with water. The solutions electrolyzed contained 0 t o 2.5 ml. of 0.05 M tert-butyl hydroperoxide in 2% aqueous solution of Aerosol MA, 3 ml. of Aerosol MA 20% in water, 2.5 ml. of 0.3 N ammonia plus 0.25 N ammonium chloride, and were made up to 25 ml. with water. Well-shaped polarograms were obtained only in the presence of more than 2% of Aerosol MA as provided in the above formulation. As the peroxide gave a wave a t approximately the
2
4
8
P E R C E N T A G E OF A E R O S O L M A
Figure 6.
Relation between Wave Height and Concentration
A . AerosolMA B . tert-Butyl hydroperoxide (corrected)
same voltage as .4erosol ,Ilh the wave height had to be corrected (Figure 6). Exactly the same relation between the wave height and the concentration of tert-butyl hydroperoxide was obtained when ethyl alcohol was used as solvent for the nonsolubilized peroxide, but the reduction potentials were different, being -1.28 volts in the Aerosol MA solution and - 1.09 volts in ethyl alcohol. ACKNOWLEDGMENT
The author is indebted to the Chief Scientist, Department of Supply, Australia, for permission to publish this paper. LITERATURE CITED
(1) Bachmann, G. B., and Astle, RI. J., J . A m . Chem. Soc., 64, 1303 (1942). (2) Ibid., p. 2177. (3) Bovey, F. A , , and Kolthoff, I. M., Ibid., 69, 2149 (1947). (4) Furman, N. H., and Stone, K. G., Ibid., 70, 3055 (1948). (5) Ibid., p. 3062. (6) Gentry, C. H. R., S a t u z , 157,479 (1946). and de T'ries. Th., ANAL. (7) Lewis, W,R., Quackenbush, F. W,, CHEM.,21,762 (1949). (8) Lingane, J. J., and Laitinen, H. h., ISD. EVG.CHEM.,A N ~ L . ED.,11, 504 (1939). (9) Proske, G. E. O., Angew. Chem., 59, 121 (1947). (10) Proske, G. E. O., Gummi-Ztg. u. Kautschuk, 1, 339 (1948). (11) Sanko, A. M., and JIanussova, F. -$., J . Gen. Chem. (U.S.S.R.), 10,1171 (1946). (12) Wheeler, D. H., Oil and Sonp, 9 , 8 9 (1932). RECEIVED for review Noveniher 19. 1931. Accepted Augiist 20, 1 9 2
New Slit Drive for Beckman IR-2 Infrared Spectrophotometer W. E. TOLBERG AND H. M. BOYD Research Department, General Mills, Znc., Minneapolis 13, M i n n . OR routine spectral scanning with a Beckman IR-2 infrared Fspectrophotometer, it is desirable to compensate for the exponential decrease in the intensity of the energy from the source over the wave-length range from 2 to 15 microns in order to maintain a constant level of background energy. Since the intensity of the energy decreases, an increase in the area of the beamthat is, a widening of the entrance slits as the wave length is increased-compensates for the decrease. Ordinarily a complete spectrum taken from the IR-2 or similar instrument having no device for continuously widening the en-
trance slits consists of a number of sections of the spectral range. Each section is recorded a t a constant slit width and in such a way as to overlap part of the range covered by the preceding section. I n order to obtain a complete spectrum in terms of per cent transmittance, the background absorption, the sample absorption, and the zero transmittance curves are recorded. The per cent transmittance of the sample is calculated from the ratio of the distance from the zero line to the sample curve and from the zero line to the background curve a t the particular wave length. Obviously this method is tedious and leaves a great deal to be desired when
1832
V O L U M E 24, N O . 11, N O V E M B E R 1 9 5 2 it is compared with the relative ease with which spectra are recorded on instruments h a ~ n ag slit drive. Two slit drivm have recently been described in the literature. One of these, the ladder chain-sprocket gear arrangement of Shreve and Heether (3), was too limited for the purpose. The other, the memory-controlled servomechanism of Madsen et al. (t), was much too complex to attempt to build, The mechanism designed and built in this laboratory consisted of a spiral cam mounted on the wave-length drive and connected by a drive cable to a threaded cvlinder mounted on the slit pinion. The details of the completed k i t drives for both the rock salt and lithium fluoride monochromators are shown in Figure 1.
f2 is equal t o f t when the sverage radius of the cam is substituted for the variable (r cam).
8.05 f, I cylinder = 18.5 f, (cam) i
This expression shows that the ratio, 7 cam17 cylinder, is the k e d ratio. Ohviously, the first derivative, dr cam/dr cylinder, is also equal to the fixed ratio. The point on the cam a t which its radius eqnals its average radius, the index point, was located by finding on the curve a slope equal to 8.05/18.5. The radius of the cam a t its narrowest point wm made large enough to ensure the proper strength. The remaining radii of the cam were oalculated by using the proportionality of the radii to the slope of the ourve. The radius of the cylinder was calculated from the fixed CONTINUOUS SLIT DRIVE M)R ROCK SALT ratio and the radius of the cam a t the index p i n t . MONOCHROMATOI I Construction. The machine tools available were net capable of duplicating the profile as laid out. Therefore, the profile was Design. Examination of the gear assembly disclosed that the broken down into conical segments (frusta), the sides of which a p proposed cam could be mounted on the idler gear of the waveproximated the outline. The segments were threaded and aslength drive. The relationship between the wave length and slit sembled, and the cam assembly was mounted on the monochrowidth was defined in terms of the revolutions of the idler versus mator. The drive cable was wound on the cam with one loop the revolutions of the slit pinion. The number of turns of the extending to the cylinder on the slit pinion. Tension on the cable slit pinion required to give a reading of 100% transmittance mas maintained by means of a threaded cylinder on a swinging (mplification set a t lox gain plus 1 / 1 thecapacity of the variable rack. The spring which provides the tension was wound on the attenuator) on the null potentiometer were plotted a8 a function axis of rotationof the rack. The threaded cylinder also kept the of integral turns of the idler. cable in a horizontal plane (see Figure 1). The shape of the cam was checked by rnnning a background spectrum from 2 to 15 microns. Part of the CUNe showed increasing energy, indicating that the slits were being opened too fast. The corresponding part of the cam was cut down to give a linear energy response. The part of the cam which gave a decreasing energy level was built np with solder. I n this way the cam was given a find shaping so that the background curve was satisfactorily linear except from 14 to 15 microns where the energy level is very I O N . Figure 2 shows 8 background curve. For efficient operation, an electrical system was installed to provide automatic stops a t 8 and 15 microns and to return to the %micron position, a t which point there is also a stop. The Figure 1. Monochromators with Slit Drives stop a t 8 microns permits change of drive speeds Left. Rockaaltmonochromatorwithcam and rave-length drivein plaee and of the shutter. RiEht. Lithium fluoride monochmmntor6 t h G e m in place The instrument was calibrated ( I ) , and the equation derived from the calibration d a t a was solved for 200 frequencies between 2.5 and 15 microns. The The slope of the curve was the rate a t which the cam must turn solutions were expressed in terms of inches of chart paper. The the slit pinion, The radius of the cam a t any point was, therefore, proportional to the slope of the curve at a corresponding corresponding frequencies were engraved on transparent scales point, From the ourve, i t was seen that the desired cam should by means of whioh the frequencies of absorption bands were read decrease gradually in diameter from 2 to 11 microns and increase directly from the spectrograms. Performance. The slit drive, the automatic stops, and the rapidly from 11to 15 microns. calibrated scales permit efficient production and use of spectra. Empirical equations were derived from the data and differentiated. The derivatives were solved for integral turns of tbe The slit drive provides a nearly constant slit schedule, and spectra cam, and a profile of the cam was laid out. The dimensions of are easily compared. the cam and of the cylinder were calculated on the basis of The background curve varies with thermocouple sensitivity two mechanical considerations. The total length of a cable and with the Condition of the glower. Comparison of backtaken up by the cam was necessarily equal to the total length ground curves over a period of time provides a check on their of cable given up by the cylinder. The ratio of the number condition. This also shows the condition of the glass shutter and of revolutions made by the cam to the number of revolutions of the rock salt window nearest the glower. made by the slit pinion is a k e d ratio, since the length The gain control shifts the entire cume linearly on the transof cable taken up in 18.5 turns of the cam must be given mittance scale, while a change in the initial slit setting a t 2 miup in 8.05 turns of the cylinder. From 1.83 to 15 microns the crons changes the shape of the curve. number of turns of the cam is 18.5. The number of turns of the CONTINUOUS S U T DRIVE FOR LITHIUM FLUORIDE slit pinion for the same wavelength interval is 8.05. MONOCHROMATOR The length of cable per turn is a function, f ~of, the radius of the cylinder and another function, fi, of the radii of the cam. The The black body radiation for the rock salt optics had a maxinumber of turns of the cylinder times the length per turn is eqnal mum at about 1.8 microns, Since the spectrum is observed from to the number of turns of the cam times the length per turn: 2 to 15 microns, the radiation is a continuously decreasing function. The black body cume of the lithium fluoride optics has a 8.05f, (T oylinder) = 18.5f 2 (1 cam)
.
1838
ANALYTICAL CHEMISTRY maximum a t about 1.3 microns, and the radiation is not a continuously decreasing function since the spectrum is observed from 1.0 to 6.3 microns. In order to give a linear background, the slits must close to a minimum at 1.3 microns before opening continuously from 1.3 microns to the endof the lithium fluoride range. It was observed that the Beckman slit jaws reached a minimum closure and reopened when the slit pinionwas rotated past the dial stop a t zero. The slit jaws were reset so that the minimum closure was equal to the slit width required to give 100% transmittance a t 1.3 microns, with a gain factor of 10 plus half the capacity of the variable attenuator. The slit scale was set arbitrarily to read zero a t the slit width required at 1.0 micron. The slit width versus &aye length curve was determined, and the design was calculated according to the method outlined. The radius at 1.0 micron Isas very large and decreased to the normal radius in two turns of the cam. The radius at 1.0 micron was too large for the space available. An alternative was to maintain the change in cam iadius for the final two turns the same as for the preceding five turns and calculate the changes in radius of the cylinder Where the cam
V O L U M E 24, N O . 1 1 , N O V E M B E R 1 9 5 2
1839
would have been very large, the cylinder N&S very small. The cam and cylinder were then cut and assembled according to these plans. The performance of the slit drive was satidactmy. No final shaping necessary. The background curve is a function of the condition of the glower and the thermocouple. The slit drive has increased the value of the lithium fluoride monochromatar by permitting convenient operation of the unit and by presenting a nearly linear transmittance sesle. Figure 3 shows the lithium fluoride background curve. ACKNOWLEDGMENT
The authors wish to express their thanks to George Long, Takuzo Tsuchiys, and Peter Smiruov of the Mechanical Engi-
neering Department for preparing drawings of the design, and to E. G . Buday, Hilding Lindquist, and R. B. Hilton far building . .. the c:ams ana electrical systems.
.
.
LITERATURE CITED
(1) IcKimey, D. 5..and Friedel, R. A,, J . O p l i d Sac. Am., 38,22 (1948). (2)
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-.,
-.,
1 1 1 1 ,
A. O., presented before conference of Instrument Society of Amerioa. St. Louis, Mo.. September 12 to 16. 1949. (3) Shrew, 0. D.,and meether, M. R., ANAL. CHEM.,22, 836-7 D s y 18, 1951. Amepted August 19, 195.2. 1~ E C E ~ V EM tleries. Research Depsrtment, General Mills, Inc.
Paper 118. Journal
Determination of Cholesterol in Microgram Quantities of Tissue DANIEL J. CAVANAUGH'L AND DAVID GLICK Department of Physiological Chemistry, Medical Scho01, University of Minnesota, Minneapolis, Minn.
T I S the purpose of this paper to describe a quantitative method
I for the determination of both free and total cholesterol in microgram quantities of tissue--e.g., microtome sections-w+th an accuracy equal to that of macroprocedures. Microtome sections are used so that a correlation of the histology with the cholesterol analyses can he made. The original method of Schoenheimer and Sperry (7), as modified by Sperry and Webb (a),was adapted to the micro scale required, and the solvent mixture of Nieft and Deuel(6) was employed for extraction of the cholesterol since it permitted extraction without heating.
stanaard aointion. n .024 o n9 . miP1.~O-PRm I_____._.. .... ~ ~ , t~. ...... .. phnieqt+Troi r__ microliter of glacial acetic acid. All steps were carried out in glass reaction tubes of about 25 mm. in length and 0.2 ml. in volume, which could he fitted with ~
tion (& electric mkcrocentrifuge, Microchemical Specialties 50.~ Berkeley, Calif., %-as used). Stirring --as carried out .by .me-
EXPERIMENTAL
.
. "..
.
Preparation of Tissue Sections. The tissue was rapidly re. ,. .. 1 moveu nom %neanimal, neea 01 oiooa, connective ussue, ana fat, and then frozen either an a block of dry ice or in the freezing compartment of a refrigerator. No evidence of a difference in cholesterol distribution dependent on the freezing method was found: however, the former method was preferred since it should 1ead to less tissue change than the slower freezing procedure.
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Using stainless steel cylindrical tissue borers with diameter? of > I I"
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-p."'L'"Y .-"I."
F.""I ,..".."FL l Y U C l l
+:D^..n y.oo"I
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ramn...4 -"A .m. III.I".b.u Y.." ~
mediately frozen to the head of a freezing attachment on a rotary microtome. Careful alignment of the cylinders N&S necessary t o ensure that the plane of sectioning was perpendicular t o the axis of the cylinder. The cylinder diameters chosen depended on the cholesterol concentration and the section thickness. Sections were usually cut a t 30 microns. The sections to be analyzed far cholesterol were placed on illurninurn foil diRkR (4mm. in diameter) and dried t o constant
tended to stick to porcelain or glass'surfaces if dried on these miterials. Cholesterol Determination. REAGENTS A N D APPAR4TUS. Alcohol-ether solution, 3 to 2, volume for volume, made with ah8olute ethyl alcohol and anhydrous ethyl ether. Alcohol-acetone solution, 1 to 1 volume for volume, absolute ethyl alcohol and reagent grade acetone. Acetic acid, glacial, reagent grade. Thirty per cent acetic acid in distilled water. Digitonin, 0.5%, in 50% ethyl alcohol. Phenolphthalein, 0.2%, in 1t o 1alcohol-acetone solution. Potassium hydroxide, 50%, in distilled water. Color reagent, made by adding concentrated, reagent grade, sulfuric acid dropwise to ice-cold, reagent grade,,aceta anhydride in the proportion of 1 to 9, volume for volume, lust before using. This reagent must he freshly prepared, and should not he used aftera few hours. ~
1 Present address, Chemioal Department. Nuclear Instrument and Chemical Gorp., 223 West Erie St., Chicago 10. Ill.
F i g u r e 1.
Electric Heater for Small
Tuhes
ohanieal vibration or "buzzing," by touching the bottom of the vessel to a rapidly rotating flattened nail (I). Colorimetry N= carried out using a microscope colorimeter ( 5 ) with capillary glass cuvets having a length of 7 mm. and lumen of 1 mm. in diameter. A tung8ten ribbon filament light source was used with a Farrand interference filter having maximum transmission at 619 mN. The reaction vessels were heated electrically in an appazatus which was especially designed for this purpose, and which was capshle of maintaining temperatures constant within 1' C., Figures 1 and 2. The double pole, double throw, toggle switch of the heating apparatus permitted use of either one or both electric heating coils. A variable transformer (750 VA.) was used to regulate the temperature hy controlling the power Supply from the 115-volt house line t o the heater. Temperatures up to 290" C. (far a room temperature of 26" C.) could be obtained with one heating coil, and >360" C. with both coils. For the
