Determination of Carbon and Hydrogen On Micro- or Semimicrosamples with One Compact and Movable Apparatus Sil3IUEL NATELSON, S. STEVEN BRODIE,
R
.&YD EDWIN
B. CO\\NER, J e w i s h Hospital of Brooklyn, Brooklyn, N. Y.
wire to the inch for a space of 12.5 em. (5 inches) to give a temperature of 550" C. 3. The bubble counter has been redesigned and placed behind the scrubber. This makes hydrogen results more constant. 4. Indicating Drierite, instead (of calcium chloride, has been found to have advantages in thescrubber. The internal diameter of the scrubber has been reduced to 9 mm. 5 . A combustion tube 9 mm. in internal diameter and of hard combustion tubing is convenient for analyzing any sample of from 2.5 to 35 mg. If the apparatus is to be used exclusively, for microanalysis, standard tubing 6 to 7 mm. in internal diameter may be substituted. In numerous tests, the use of the narrower tubing has shown no apparent advantage. 6. The method of obtaining a differential in temperature between the end and main part of the heating unit, has been changed. Previously, the turns were xound at greater intervals at the cooler end. This same result may be attained more readily by changing the gage of the R-ire at the cooler end and winding as for the main part of the tube. The total length of the heating coil is 32.5 cm. (13 inches). For the first 23.75 cm. (9.5 inches) No. 26 (B. & S. gage) Sichrome xire is wound. The turns are spaced at 0.6-cm. (0.25-inch) intervals except for the very beginning where, in order to compensate for end cooling, four turns are taken in the first 1.25 cm. (0.5 inch). The end is then spliced to 90.20 wire and the winding is continued for the last 8.75 cm. (3.5 inches). To allow for conduction from the hotter part of the coil, it was found necessary to take only one turn of the No. 20 xire for the first 1.25 cm. (1 inch). The turns for the remaining 6.25 cm. (2.5 inches) are taken at 0.6-cm. (0.25-inch) intervals except for the very end. To alloiv for the end cooling effect, three turns were taken for the last 0.94 cm. (0.375 inch). A simple calculation will indicate why these diameters were chosen. With constant current, the heat developed in a wire is proportional to its resistance and the resistance is inversely proportional to the square of the radii of the wires employed, if the vinding is evenly spaced around the same tube. KO.26 wire at 0.6-cm. (0.25-inch) intervals was found to be suitable for the first 25 cm. (10 inches) (675' to 700" C.). For the next 7.5 cm. (3 inches) a temperature of about 200" C. had to be maintained. Since the same materials (constant specific heat and rate of radiation) were used throughout the combustion coil, it was safe to assume that the heat developed was proportional to the temperature and hence the following proportion was set' up:
ECESTLY (1) the authors described a movable and
easily constructed apparatus for the determination of carbon and hydrogen, designed to analyze a 70-mg. sample. This apparatus has now been successfully scaled down for application to microsamples. The apparatus in its present form (Figures 1 and 2 ) takes up a niaximum of 57.5 em. (23 inches) of desk space when the absorption tubes are not attached. It can be used for sample. of from 2.5 to 35 mg. without increasing the combustion time appreciably beyond that for the standard combustion of microsamples. Kith larger samples, the combustion time is increased beyond a reasonable amount. K h e n a microbalance is available microsamples (3 to 6 mg.) are reighed for analysis; otherwise, a 20- t o 30-mg. sample is weighed using an analytical balance (weighing to 0.1 mg.) or a semimicrobalance (weighing t o 0.01 mg.) for greater accuracy. The time of combustion for a microsample is usually 40 minutes; for a semimicrosample, 55 minutes. A copper oxide spiral is not necessary behind the boat for samples of either size. Although the outfit was designed for microanalysis, with some minor alterations it could be used for samples ranging u p to 3.5 mg., permitting the use of an ordinary analytical balance without losing efficiency as a microcombustion outfit. T h e apparatus is now mounted on a metal frame cast from aluminum Tvitli an iron base.
Apparatus Figure 2 illustrates the present setup of the apparatus, its dimensions, and the filling of the tube. The following improvements may be noted : 1. The capacity of the bulb in the gasometer has been increased to 2 liters. A gasometer of this size can deliver about 650 cc. of oxygen when the gage reads 25 em. of mercury. Less frequent fillings of the gasometer are necessary, and the bubble rate remains constant throughout the combustion. 2. Lead chromate has been eliminated from the preheater. Its presence is unnecessary and is objectionable in that it shortens the life of the Pyrex tube. The heating coil is now in parallel with the main heating coil, making temperature control easier. The preheater coil is prepared by winding nine turns of S o . 28
(Radius of T o . 26 wire)? (Radius of desired wire)*
~
(0.00797 inch)%- 200' C. (Xinoh)? 675O C.
On solving for the unknon-n one obtains 0.041 cm. (0.0164 inch), which is closest to the radius of KO.20 5%-ire. That the
FIGURE1. APPARATUS SETUPFOR MICROLVALYSIS 609
INDUSTRIAL AND ENGINEERING CHEMISTRY
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VOL. 10, KO.10
tubing is subjected to temperatures just below the boiling point of water. 8. The combustion tube is now filled as shown in Figure 2. All the asbestos plugs need not be wider -.-..-..-..-.-.-..- .-.-,than 1 to 2 mm. PrecipiE tated silver may be used rather than the usual silver E i wire. I t was observed that compounds with a high percentage of chlorine (more A than 40 per cent) consistently gave slightly high results for carbon \Then silver wool, wire, or foil was used. ASCARITE DRIERITE The silver filling as used in this tube absorbed 300 mg. of chlorine (calculated from the weights and percentage of chlorine of the compounds used) before any indication of breakdown was observable. The silver could then be regenerated in the usual way. For the usual run of analyses silver wire may serve as well. The precipitated silver may be prepared by treating silver nitrate with zinc dust (less than theoretical) FIGURE 2. SCHEYdTIC DIAGRAhf O F COUBUSTION .kPP.&R.&TcS and heating on a water bath. The precipitated silver is filtered off, boiled with dilute sulfuric acid t o remove traces of zinc, filtered, &shed, reasoning is correct was evidenced by the fact that KO.20 nire and dried in an oven. The finely divided silver is then heated at for the last 6.25 cm. (2.5 inches) gave the required temperature 675" C. (in the combustion train) for 1 hour, in order to avoid (200" C.) within 10' when spaced at the same interval 0.6 cm. (0.25 inch) as the KO.26 wire. A smoothly uniform temperaany shrinkage which may occur in the tube later. It is then alture is thus readily obtainable in both parts of the tube. To imlowed to cool and transferred to a bottle for further use. prove its appearance, the insulation around the heating coils is 9. For analysis of semimicrosamples, absorption tubes patterned after the usual microabsorption tubes were used (Figure now cast in a split metal mold. 7. I n order to avoid the condensation of moisture at the 3). For microdeterminations, tubes of standard size were used. 10. The design of the support for both micro- andsemimicromouth of the combustion tube, a split metal tube hinged on one absorption tubes on one small ring stand is worthy of note side is attached to the heating coil. When attaching the absorp(Figure 1). tion tubes, the upper part of this metal tube is moved back on its hinges and replaced after connection has been made. This split tube, kept warm by conduction from the heating coil, is long Procedure and Results enough to force the water directly into the absorption tube. An auxiliary metal tube, cut from a cork borer of the roper dimenFor the combustion of the microsamples the standard prosions, may be used by sliding part of it in and out orthe split tube cedure is used, 10 minutes for the first combustion and 10 if any additional length is needed. The connection between the combustion and absorption tubes minutes for reheating in order to assure complete combusis made by means of the usual heavy red nitrometer tubing, tion. The washing time is approximately 20 minutes, makpreviously heated to 110" C. and carefully cleaned inside and ing the complete combustion time about 40 minutes. On out. After more than 6 months' continuous use, no apparent samples of about 25 mg. the two combustion times combined deterioration had taken place. During the combustion, this
-
I
GI.
Cast aluminum disk B . Heat conductor (split hinged tube) C. Auxiliarv heat conductor D . Absorpdon tube (Hz0) E Ahsor tion tube (Cot) F: Guaratube (Cot and Hz0) A.
TUBESFOR SEMIMICROSAMPLES FIGURE 3. ABSORPTION Designed for weighing on balance pana
OCTOBER 15, 1938
ANALYTlCAL EDITIOK
611
FIGURE 4, DESIGNOF ASPIRATORFOR COSDITIOSISG .~BSORPTIOS TUBES
are about 30 minutes and the mashing time is about 25 minutes, making a complete combustion time of about 55 minutes. In the combustion of the microsample 175 cc. of oxygen are used, and 325 cc. of oxygen are used for the conibustion of the semimicrosample. I n the case of the semimicrosample the oxygen has to be passed at the relatively rapid rate of about 5.5 cc. per minute. This can easily be achieved because the resistance of the semimicrotubes is much less than that of the microtubes. I n the authors’ apparatus the bubble rate mas maintained a t about 40 bubbles per minute. When the microtubes were attached the rate was maintained a t about 4 cc. per minute or a little less than 2 bubbles per second. The bubble rate is set a t the start of the combustion. The position of the adjusting stopcock is not changed until the combustion is finished, even though a t times i t may vary temporarily when burned gases gather in the tube. Where combustion is difficult-i. e., with samples of high chlorine or sulfur content-the bubble rate must be slower and the time of combustion longer. For best results ( 2 ) i t is good practice, before and after the combustion, to aspirate air (dried and free of carbon dioxide) through the absorption tubes, in order to weigh the tubes filled with air rather than oxygen. For this purpose many experimenters have used the RIariotte bottle. The authors have designed an apparatus which avoids some of the disadvantages of the Mariotte bottle-the need to be present when the required amount of water has gone into the graduated cylinder, the inconrenience encountered in t h e refilling of the bottle, and the change in bubble rate and hence in time for the required 50 cc. as the level is loir.ered in the bottle. T h e apparatus recommended is automatic, drawing through the required amount and stopping of its own accord. There is no pouring of water and the time to deliver the required volume is always the same. A glance a t Figure 4 will indicate the design. When the reservoir, A , is lowered to the required level, the amount of air desired will be drawn through and movement will cease. The two-way stopcock may then be turned so that when
t h e reservoir i y raised, the aspirated air is expelled. The stopcock is then returned to its original position and the apparatus is ready for use again. Instead of the 50-cc. bulbs, a 100-cc. graduated cylinder tube may be used. The bubble rate is regulated by means of a glass stopper, pinchclamp, or capillary located behind the aspirator. One filling of the tube as shown or the graduated cylinder will aspirate two sets of microtubes or one set of semimicrotubes. For microdeterniinations, 50 cc. of air are passed through in 10 minutes. The tubes may then be placed directly on the balance and weighed n-ithout further delay. For the semimicrotubes 100 cc. of air are passed through in the same time (10 minutes) and the tubes are weighed directly. Both semimicro- and microtubes should be wiped carefully before weighing.
K i t h two sets of tubes about six determinations of either micro- or semimicrosamples may be readily performed in one day. Table I s h o m representative results obtained on the same outfit by alternating from micro- to semimicrosamples of compounds n-ith a variety of characteristics. A Kuhlman microbalance was used for t’he microsamples. The semimicrosamples were weighed to 0.1 mg. by means of an analytical balance fit,ted with a magnetic damper. It is apparent that for the semimicrosamples the accuracy of the results is limited by the accuracy of the weighing.
Discussion
It is apparent that with minor differences the procedure and time are almost the same for micro- and larger samples using this apparatus. I n the authors’ experience both sizes of sample h a r e given consistent results. T h e choice therefore depends upon the amount of material available and whether one is to use the micro- or semimicrobalance. For instructional purposes this apparatus has the advantage that one may learn the technic of analysis on semimicrosamples with the aid of an ordinary balance and then transfer the same procedure t o the determination of microsamples; the only new procedure to be learned is the operation of amicrobalance. The apparatus retains the advantages of being compact and movable. It may be stored without dismantling and may be set in operation on little notice.
ITDUSTRIAL AND ENGINEERING CHEMISTRY
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Precipitated silver is used in the combustion tube to hold back large amounts of halogen.
TABLEI. REPRESESTATIVE RESULTS Substance
COS Sample Found MQ. AVQ.
Formula
Resorcinol
CeH+O:
Benzoic acid
C~HBO?
p-Xtrobenzoyl chloride Cystine
c~H~~o~
I
a .is
C:HIOJSCI
5,040 12.112 20.2 48.5 2.938 7.446 19.0 48.1 3.171 .272 38,1a 5 63.6 N3.801 ~ s ~ 4.176 23.5 25.8
H20 C H C H Found Found Found Calod. Calcd. I?‘Q. % % % % 2.497 10.0 1.249 8.7 0.548 6.6
65.54 63.48 69.12 69.04 45.35 45.52 1.845 29.97 10.8 29.94
5 54 S.55
VOL. 10, NO. 10
65.43 5 4 9
4 76 GS:85 4:91 5.12 1.93 4;:ZS 2:iS 1.94 5,43 30:00 ~ : O O 5.14 .. ..
.
a safeguard I5 minutes’ extra combustion time was allowed for this larger sample.
iicknowledgmen t The authors are indebted to hlr. Weiskopf of the Empire Laboratory Supply Co., 559 West’ 132nd St., Yew York, N. Y., for his continued cooperation in the development of this apparatus, t o A. Elek of the Rockefeller Institute for reviewing the manuscriut. and t o B. Kramer of the JemiLh Hospital for his support. I
,
Summary
Literature Cited
A compact and movable outfit is described for the determination of carbon and hydrogen on samples ranging from 2.s to 35 mg. -in analytical or microbalance may be used, depending upon the amount of material that is available. Little more time iS required for the determination Of the seniimicrosample than for the microsample. An aspirator is described for conditioning the absorption tubes.
(1) Natelson and C o m e r , IKD. ESG.CHEM.,Anal. E d . . 10, 276 (1938). (2) Niederl and Kiederl, “hlicro Methods of Quantitative Organic Elementary Analysis,” New York, J o h n Wiley & Sons, 1938. (3) Pregl, “Quantitative Organic hlicroanalysis,“ Philadelphia, P. Blakiston’s Sons, 1930. T h e literature in this field is
SO
voluminous t h a t i t has been decided
to refer only t o two textbooks and t o the authors’ previous paper on the subject. RECEIVED July 20, 1938.
Determination of Iodine in Biological Materials Refinements of the Chromium Trioxide Oxidation Method NORMAN L. XATTHEB‘S, GEORGE AI. CLTRTIS, 4 n B’.ALLACE ~ R. BRODE The Ohio State University, Columbus. Ohio
Q
UASTITATIVE studies of iodine metabolism require adequate analytical procedures, adapted to the determination of the minute amounts of iodine present in biological materials. Owing to the complexity of former methods ( I O ) , difficulties have been experienced in obtaining consistent results, while comparative studies from different laboratories have shown considerable variation ( 2 ) . Most procedures for destroying the organic matter, preparatory to actual extraction and determination of the iodine, have involved combustion in a closed system or basic ashing. 4 procedure developed by Leipert (8) in 1933, however, involved oxidizing the organic matter, and all the contained iodine to iodine pentoxide, by means of chromium trioxide in a sulfuric acid medium and in the presence of ceric sulfate as a catalyst. Leipert’s method also employed steam distillation in partial vacuum to separate the small amounts of iodine from the sulfuric acid solution, after the iodine pentoxide and excess chromium trioxide had been reduced by arsenious oxide. Trevorrow and Fashena (12) employed potassium dichromate instead of chromium trioxide as the oxidizing agent, since chromium trioxide usually contains iodine and is difficult to purify. They also found that the use of arsenious oxide resulted in unwarranted high iodine values, and consequently substituted phosphorous acid in its place as the reducing agent. Later ( 3 ) , moreover, they found that their former method was not quantitative and replaced the vacuumsteam distillation by a combination of aeration and distillation. The authors have devised a simple method for preparing
chromium trioxide of low iodine content.
Ceric sulfate as
a catalyst may be omitted in analyses of blood, urine, feces, thyroid gland, and milk of lower iodine content than approximately 1 mg. The chromium trioxide method is not directly applicable to biological specimens which contain very minute concentrations of iodine. Thus, for the accurate analysis of mixed dried food, combustion in oxygen by the von Kolnitz and Remington modification (‘7) of the Karns procedure (6) precedes chromium trioxide oxidation (9). This makes convenient the oxidation of several hundred grams of material which is subsequently completed in the chromium trioxide procedure. Iodine may be separated from the chromic sulfate and ~ . ~ l f ~ i r i c acid solution by a simple distillation procedure. [C. D. Stevens of the DeCourcey Clinic of Cincinnati visited the authors’ laboratory to inrestigate their procedures ftir the microdetermination of iodine. He later published A procedure employing the simple distillation (14.1 The apparatus, designed for making this distillation (Figure I), is easier to manipulate and more compact than either that of Leipert or of Fashena and Trevorrow. Phosphorous acid is most effective as a reducing agent in the quantitative recovery by simple distillation of the smaller, more .‘biological” amounts of iodine. Larger amounts ( 2 mg. in 100 ml. of sulfuric acid) are only 90 to 95 per cent recovered when this reagent is employed (9). The use of oxalic acid (1) may result in the distillation of reducing substances. A permanganate procedure, proposed by Groak ( 4 ) t o
