V O L U M E 25, NO. 6, J U N E 1 9 5 3
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solutions, probably because of the fairly stable tetraiodocadmate (CdIh--) ion present. Acetic acid in small concentration would be sn interference in very few titration reactions. Retrogradation is presumed t o involve hydrogen bonding between hydroxyl groups on adjacent linear staroh molecules to form insoluble macromolecules. Evidently the undissociated acetic acid in the indicator solution competes successfully with the other starch molecules and the solvent (water) molecules for hydrogen bonding sites (hydroxyl groups) on the linear starch molecule and thus keeps the large linear starch molecule in colloidal solution. Potafisium chloride in 15% solution has been reported (5, 7) as a stabilizing agent for linear starch solutions but such solutions have been found to be stable for only a few weeks. As the blue linear starch-triiodide ion complex is not very water soluble, the indicator solution sbould be added in the usual manner just before the end point is reached. I n this respect, i t is less convenient tbsn the starch glycollate indicator solution reported by Peat, Bourne, and Thrower ( 4 ) in which the starch glycolliLteAriiodideion blue complex is water soluble. These investigators indicate that some iodine-staining capacity may he sacrificed in modifying the starch structure, which in-
volves the replacement of some of the hydroxyl groups on the starch molecule by formation of the starch a-ether derivative of acetic mid ("starchmyacetic" acid). The water solubility of the triiodide complex of this ether derivative of starch is prob ably due to the large number of hydrophilic carboxyl groups in-
LITERATURE CITED
(1) Lambert, J. L.. .~NAL.C a m . , 23, 1247 (1951). (2) I b d . , p. 1251. (3) Lansky, S., Kooi, M., and Sohooh. T. J.. J . Am. C h m . Suc... 7 1., 4066 (1949). (4) Peat. s.. Bourne, E. J., and Thrower, R. D., Nature, 159, 810
,.
,An=,
(5) Pierce. W. C., and Haenisch, E. L., "Quantitative Analysis," 3rd ed.. P.237. New York. John Wiley and Sons. 1948. (6) Rowe. A. W., and Phelps, E. P., J . Am. Chem. Soc., 46, 2078 (1924). (7) Schooh. T. J.. "Advanoea in Carbohvdrate Chemistrv." Vol. I. PP. 247-77, ed. by Pipman, W. W.;and Wolfrom. M. L., I& York. Academic Press. 1945. 8) Sohooh, T .I. J. . Am. Chem. Soc., 64,2957 (1942). t s c ~ r v ~for o review December 24, 1952. Accepted February 26.1953.
Five-Centimeter Absorption Cell for Spectrophotometry with limited Volumes B E R T L. VALLEE y , Cambridge, Mas.S. Department of Biology and the Spectroscopy Laboratory, Massaehz isetts Institute of TechW%olog:
TIE 10: extinFtiOn coefficientof many compounds often sets an undesir~%bl.ble limit to the sensitivity of their spectropbotornetric detection. Optical density readings obtained with such compounds may fall below the range of optimal response of the spectrophotometer used when low concentrations are being measured. In such instances the use of an absorption cell of long path length may materially increase both sensitivity and precision. An optical path len&h of 1 em. is of standard usaee in absarotion cells supplied with widely used instruments such is the Beckman Model D U spectrophotometer. As is apparent from Lambe rt's law, the absorption of light in IS ns+h I s mb".. d h nihinh it +ralrarooo solutions is proportional to t L rill An increase in the length of the absorption cell should, therefore, yield Correspondingimprovement in sensitivity. This is true provided the increase in length is not accompanied by an increase in volume of the coll, necessitating a concomitant dilution of the solute and thus obviating the advantage gained. With an otherwise fixed optical system the theoretical minimal width and height of the cell is defined by the dimensions of the light beam of the spectrophotometer entering and leaving it. Generally, cells have been Constructed to allow for greater than necesmrv clearance between the edges of the light beam and the walls oftbe cell 80 that light scattering would not disturb quantitative spectraphotometrio measurements. The volume acoommodated by such cells becomes much larger than i t need he on the hasis of optical considerations. Long path length cells with appropriate cell holders are supplied with several standard spectrophotometers. Such cells are used to good advantage when adequate quantities of material are available for spectrophotometry. Concentration methods may then be depended upon to lower the limits of deteetion. However, in many instances the final volume consists of but a few milliliters of low optical density. For these conditions optimal results may be obtained by the use of a cell with long path length and diminished capacity of volume. The 5-cm. absorption cells were designed for use with the Beckman Model DU spectrophotometer far which an appropriate attachment was constructed (Figure 1). The light beam passes through these cells without disturbing light scattering from their
.".. ....
-..~
."
i. ,.J6 c. c ml. y-u ."A Lo .."A" walls. Tho +n+ol nanrni+.r '~.~'J -Yen smaller. No changes in the optical system of the instrument are required. y l l
\I.yJ
-".?
_L.Ur
n
CONSTRUCTION AND DESIGN OF CELL
One type of cell W ~ Rmanufactured from Vycor glass (Corning Glass Works) for use in the visible part of th e spectrum (Figure 2).
Y.U.II"IU.
.$
. .
Figure 1. Adapter to Receive 5-Cm. Path Length Cell for Attachment to Model DU Beckman Spectrophotometer
A piece of opaque Vycor glass of appropriate dimensions was bent to U-shape, and optically flat Vycor end plates were fused to the body of the cell by means of Laminae (American Cyanamid Co.), a polymerizing resin, which was applied to each surface to be cemented. Polymerization was brought about by irradiation with ultraviolet light for a period of 100 hours. Another type of cell was constructed of fused quartz throughout for use in the ultraviolet region of the spectrum (Figure 2). Fusion of quartz was accomplished in two separate steps using
ANALYTICAL CHEMISTRY
986 stainless steel jigs. The body and end faces were fashioned and polished first to the requisite dimensions, parallelism, and optical flatness. The end plates were then fused onto the body in a separate operation. The light beams of four Beckman Model DU spectrophotometers were measured and found to be 2 mm. wide a t the plane of entrance and 5 mm. wide a t the plane of exit from the cell. The beam was 12 mm. high a t both planes. The dimensions of the absorption cells (obtainable from the Jarrell-Ash Co., Boston Mass.) are as follows (Figure 2):
0:5
0.4 t
t VI z
#
hZm.
Length External height External width Internal height Internal width
0.6
0.3
-I
50+01 24 + 0.1 28 zt 0.1 14 0.1 8 i 0.1
a 2
: 0.2 I-
+
The inner tetrahedron, having a volume of 5.60 ml., is based on a semicylinder (the bottom of the U), of 4-mm. radius, 50-mm. length and a resulting volume of 1.26'ml. The total capacity of the cell is therefore 6.86 ml. The over-all parallelism of the cell is 3 minutes, the end plates are 1 minute parallel, and the flatness of the end plates is 0.5 fringe measured with the 5460.740 A. mercury green line.
0 .I
pg OF COPPER IN SEVEN mi OF SOLUTION
Figure 3.
G
L
h
Figure 2.
Calibration Curves for Copper with Sodium Diethyldithiocarbamate at 450 Mp
0, I-cm. path
l e n g t h cell; 0, 5-cm. p a t h l e n g t h c e l l
losses do not occw as a result of reflections from the walls of the cell. The marked gain in optical density for identical quantities is apparent. Table I shows optical density and copper values obtained on replicate samples of plasma. Sixteen 1-ml. aliquots from the same lot of stored plasma were analyzed. The Beckman Model DC spectrophotometer with the 50-mm. Vycor cell was used a t 450 mp] where the complex of copper with sodium diethyldithiocarbamate absorbs maximally. Plasma-freed of protein-absorbs some radiation at this wave length, presumably due to the presence of bile pigments. Column I shows the absorption of radiation at 450 mp in the protein-free supernatant of trichloroacetic acid-precipitated plasma prior to the addition of sodium diethyldithiocarbamate. Column 2 shows the absorption observed following the addition of the dye. Column 3 $ h o w the absorption due to the presence of copper, and Column 4 s h o w the calculated amount of copper in 1 ml. of plasma. These values are higher than encountered in normal human sera (B), presumably as a result of the extraction of copper from the glass bottles used for prolonged
Five-Centimeter Cells
Left, Vycor cell; r i g h t , q u a r t z cell C,o u t e r cell wall; L, light beam; H , L u c i t e holder
While the internal dimensions of the quartz cells are identical with those of the Vycor cells, the external diameters are markedly decreased to assist fusion without distortion. A Lucite bottom plate was found suitable to hold the quartz cells in the same adapter used for the Vycor cells. APPLlCATlON IN QUALITATIVE AND QUANTITATIVE WORK
The 5-cm. cells have been used for the quantitative determination of copper with diethyldithiocarbamate in human sera ( 1 ) . Figure 3 shows calibration curves obtained with the 1-cm. and the 5-cm. cells on aliquots of the same solution. By direct measurement with a micrometer the 1-cm. cell measured 0.999 cm., the 5-cm cell measured 5.017 cm. The theoretical ratio of path length of the 5-cm. and I-cm. cells, therefore, is 5.02 cm. The calibration factors, derived from the density values, were 43.3 for the 1cm. cell and 8.64 for the 5-em. cell; the actual gain in optical path is therefore 5.02, in agreement with the expected value. The direct correspondence of these ratios demonstrates that light
Table I.
Copper Content of 1-M1. .4liquots of Pooled and Stored Human Plasma
(Beckman Model D U spectrophotometer; 5-cm. Vycor cell) Copper MicroAbsorption a t 450 mp gramd/,Ml. of Sampleb Difference PlasmaC 0.139 1.77 0.156 0.138 1.76 0.154 0.141 1.80 0.155 1.79 0.140 0.154 0.142 1.81 0.157 1.81 0.142 0.159 1.76 0.152 0.138 1.79 0.140 0.153 0.140 1.79 0.154 1.82 0.143 0.156 1.82 0.143 0.158 1.85 0.145 0.162 1.89 0.148 0.015 0.163 1.81 0.142 0.015 0.157 1.85 0.145 0.015 0.160 1.89 0.021 0.148 0.169 0.142 1.81 0.157 Mean 0.015 f0.0032 AO.04 AO.0026 10.0024 S.D. a Reagents and plasma. b Reagents and plasma plus sodium diethyldithiocarbamate. C Column 3 X calibration constant X dilution factor. Controla 0.017 0.016 0.014 0.014 0.015 0.017 0.014 0.013 0.014 0.013 0.015 0.017
V O L U M E 25, NO. 6, J U N E 1 9 5 3
987
storage. The standard deviations indicate very good reproducibility. The coefficient of variation of this series is 2.2%. -4previous communication ( 2 ) demonstrated the precision and sensitivity attained when these cells are used for copper drterminations of human sera. SUMMARY
Absorption cells with a path length of 5 cm. but accommodating much smaller volunies than conventional cella have been constructed for the Beckman Model DU spectrophotometer. S o changes in the optical system of the instrument are required. With these cells marked gain in sensitivity and precision can be, achieved in analytical spectrophotometry. This is illustrated by data on the application of these cells to the determination of copper in biological material which could be ineasured with a co-
efficient of variation of 2.2Fo n hell thc total quantity to be determined was 1.8 micrograms. ACKNOW LEDGRIE\T
The author is indebted to Frederick Brech for help with the construction of the cells. and Thomas L. Coombs for technical assistance. This work \vas supported by the Charles F. Kettering Fountlation, Dayton, Ohio, and the Howard Hughes Foundation of Hollyv-ood, Calif. LlTERATURE ClTED
AI. E., .ishenbrucker, H., C:artwvright, G. E.. a n d \Tintrobe, AI. XI., J . Biol. Chem., 196,200 (1962;. (2) Yallee, B. L., Metabolism, I, 420 (1962). (1) G u b l e r , C. J., L a h e y ,
R E C E I V Efor D review November 17, 1952.
Acceiited February 12, 19>3.
Micromethod for Measurement of Carbon-14-Labeled Material JOHK H. P E T E R S
AND HELMUT
R . GUT3IANK
Radioisotope Unit, Veterans ’ddministration Hospital, and Department of Physiological Chemistry, L-niversity of Minnesota, Minneapolis, Minn. TOR
the routine assay of radioactive cat bon in biological
E materials or organic compounds the method of Lindenbaum, (1, 4 ) or a recent modification (8) has
Armstrong, and Schubert been shonn to be accurate and rrpioducilJk. .liter wet combustion of the sample, the radioactive carbon dioxide is absorbed in saturated barium hydroxide (4)or in 1% sodium hydroxide, followed by precipitation with barium chloride (8),and the radioactivity of the solid carbonate is meawred. In boih procedures the carbon content of the material is of such a magnitude that the weight of the radioactive barium carbonate falls within the range of “infinite thickness.” However, when only microquantities of material are available, as is often the case in the isolation of metabolites or in the tissue assay of small organs, it is advantageous to be able to measuie the iadioactivities of smaller samples than were employed in the original method (I, 4 ) . The present paper describes a simple and rapid modification of the procedure of Lindenbaum el al. ( 4 ) to samples which may range in carbon content from 0.5 to 3.0 mg. The barium cai honate precipitates which are counted have a thickness of 3 to 10 mg. per square centimeter. T o facilitate the handling of small amounts the size of the original apparatus has been reduced (Figure 1). The saturated barium hydroxide (4)has been re-
placed by a measured volume of approximately 0.25 aV IJarium hydroxide u-hich contains 2 grams of barium chloride for each 100 ml. of solution (6). This eliminates both the filtration of the saturated barium hydroside as it is introduced into the receiver and the centrifugation of the final mixture (4). I n addition, the use of barium chloride-barium h? droside solution minimizes the coprecipitation of barium hydroxide which is troublesome with saturated barium hydroxide ( 5 ) . Following combustion, the excess of barium hydroxide is titrated with standard acid to the phenolphthalein end point ( 2 ) . The weight, of the barium carbonate is calculated from the t,itration data, which obviates the necessity for the tedious quantitative collectioii of the radioactive precipitate (1, 8). .%iter collecting and xveighing, the radioact.ivity of the carlionate is measured. The radioactivity of the sample is calculated a s follo\vs: n e t couiits,’niiii. X Radioactivitj- of the sample = -vli-al sorption factor mg. of carhonatr titrated mg. of carbonate wighed ~
JVith the size of sample employed, R combustion time of 2 instead of 3 minut,es ( 4 ) has been found adequat,e. Sweeping of the apparatus following combustion with air freed of carbon dioxide (8) has been found unnc~essary. Combustion of the sample, absorption of carbon dioxide, and titration require only 20 minutes, whereas the improved macroprocedure takes 35 to 40 minutes (8). PROCEDURE:
Figure 1.
Conihiistiori Apparatus
The coml)ustion apparatus is show1 in Figure 1. The combustion tube A has a capacity of 12 to 13 nil. The receiver D is a 40-ml. borosilicate glass centrifuge tube with conical hottom, fitted with a two-hole rubber stopper through which pas.< both t,he delivery tube for the carbon dioxide and a one-way stopcock n-hich connects the apparatus to a vacuum pump. The rubber stopper in D which gives rise to leaks after some use may l)e eliminated by use of the ground glass unit E . The sample, which should contain 0.5 to 3.0 mg. of carbon, is weighed into the combustion tube if it is a solid. In the case of solutions, the sample is pipetted into the combustion tube with a micropipet, and the solvent is evaporated. Three hundred milligrams of a mixture of potassium iodate ( 2 arts by weight) and potassium dichromate (1 part) is added. !tandard barium hydroxide solution (4.00 to 5.00 ml.) is delivered into the receiver from a 10-ml. buret. The buret and the barium hydroxide reservoir are protected from atmospheric carbon dioxide by soda lime tubes. The apparatus is assembled, and all ground surfaces in direct contact with the combustion mixture are lubricated with phosphoric acid. Two milliliters of modified Van Slyke-Folch reagent ( 7 )is placed into reservoir B and run into the combustion
