794
ANALYTICAL CHEMISTRY
by the samples was desirable, as it v a s intended to add the standards in the form of solid U308. The grease film was prepared by evaporating one drop of a solution of Apiezon M grease in analytical reagent 60" to 80" C. petroleum ether (0.5 gram in 10 ml.) on the surface. The film was, however, attacked and penetrated with consequent loss in sensitivity for silicon, when solutions containing much nitric acid were evaporated on it. This difficulty was overcome by allowing the electrode with its drop of solution to stand for a few minutes in an atmosphere of ammonia until the yellow ammonium diuranate commenced to form. The ammonia atmosphere was simply obtained by placing a small beaker of 0.880 specific gravity ammonia solution under the crystallizing dish which was used aa a dust cover for the electrodes. To check the sample preparation, several milliliters of a sample were evaporated in a platinum crucible and ignited to U?Os. Quadruplicate exposures were made on Bmg. aliquots of this residue and compared with the results obtained by treatment of the solution on the electrodes as described above a-ith the following results: 1. Ignition in platinum, 93, 100, 98, 100; mean 98 p.p.m. silicon 2. Ignition on thr rlectrodes, 115, 107, 114, 105; mean 110 p.p in silicon The somewhat higher figure arrived at by method 2 was not due to the blank on the grease, as this w a ~ negligible, but might have been due to slight penetration of thr graphite by the nitric acid resulting in the introduction into the arc stream of a small amount of silicate from the electrode. However, a blank run on nitric acid alone showed that no silicon g a s introduced in this way. The difference is probably due to small losses of silicon on the walls of the platinum crucible in method 1; 'no such loss can occur when the solution is processed directly on the electrode. PROCEDURE
A suitable volume of the solution for anal\&, in this case 0.02 ml. which contained 6 mg. of UYOg, was transferred by means of a graduated pipet with plunger control to the prepared electrode, the free acidity was neutralized as described above, and the solution was evaporated to drl-ness under radiant heat lamps. The samples could not be arced in this condition, because copious vapor evolution would have expelled the charge bodily from the electrode cup. They were therefore first ignited for a short time in a Bunsen flame to convert the uranium into UaOs, the base of the electrode being held in platinum-tipped tongs. A little care was necessary in the first stage of the ignition; the surface of the charge had to be heated just outside the flame until the ovide crust became sufficiently porous to allow the passage of vapor
Table I. SaullJlc
Reproducibility of Analysis Silicon P.P.lf. 2 3
1
-
4
Mean
from ivithin the bulk of thcl charge. Aftcr the first two or t,hree ignitions this procedure gave no difficulty. At least two exposures n'ere made on each solution together nith the synthetic standards (6-nig. charge) on the same photographic plate under the following conditions. Haird, grating spectrograph (&meter), 15,000 l i n e per inch. First ordrr. Range 2.5 (2165 to 3580 .A,). Slit 10 microns. Gmting 3-em. aperture, image of source focused on grating. Plate Ilford Ordinary. Exposure 30 seconds at 10 amperes, sample as anode. Plate calibration l.5-st,ep sector at secondary focus, range 8-second order. Hilgcr Littrow spect,rograph. Range 2220 to 2900 h. Slit 10 microns. Plate Ilford Ordinary. Exposure 30 seconds a t 10 amperes, sample as anode. A suitable line pair.for use w t h 1~1thspectrographs was Si 2516.1 and U 2519.0 A , , measurement being made with the Hilger nonrecording microphotometer. .2 calibration curve n-as drawn up of log intensity ratio against log of parts per million of silicon in U308. RESULTS
R.esults were expressed as parts per niillion of silicon in utos and, assuming that the conc,entration of uranium in the original solution was known, could be converted into micrograms per milliliter of solution. Typical results on samples run in quadruplicate are shown in Table I. The standard deviation CAIculated on sixt,y duplicate sample results wm 9.42%. ACKNOWLEDGMENT
Acknowledgment is made to the Director, Atormc Energy Research Establishment, €Tarwell, England, for permission to publish this paper. LITERATURE CITED
(1) Sciibner, A. F., and Mullin, H. R., J . Research Natl. Bur. Statidarda, 37, 379-89 (1946); Research Paper 1753. (2) TTalsh, A , , Spectrochim. Acta, 4 , S o . 1. 47-56 (1950). RECEIVED Jiine 21, 1950
Tiselius-Claesson Interferometric Adsorption Analysis Apparatus Improvements in Design and Use HALF11 T. HOLMAN AND LENNART HAGDAHL' Texas Agriculture Experinen t Station, Texas Agricultural and AMechnicalCollege System, College Station, Tex.
HE adsorption analysis apparatus developed by Tiselius in the
has proved to bc a Chesson ( I , chromatographic separation of sugars (9,121, amino acids and peptides ('O), fatty acids ( 4 , maCromolecules (', 8)' '), Experience gained through building two instruments for this laboratory has led to some modifications in design that have increased the versatility of the apparatus and altered and simplified its manipulation. The ~ k ~ l iapparatus ~ ~ coIlsists - ~ ofl tu-o ~ main ~ ~ parts, ~ the vertical chromatographic column and the horizontal interferometerby of Tvhich observations are made upon the effluent. The model described is shown in Figure 1. 1 Permanent address, Biocheniical Institute, University of Gppsala, Uppsala. Sweden.
COUPLED FILTER SYSTEM
The chromatographic colunln, A , is segmented, consecutive segments decreasing in internal diameter from the top to the l,ottom of the column. The filters (segments) are joined by couplings having capillary bores through which the effluent passes. This arrangement, first used by Hagdahl (S'), thoroughly mixes the effluent from one filter and passes it on to a fresh filter of diameter. The inevitable irregular front! are thus ~smaller ~ "ironed out," and, as the fronts pass to successively smaller filters, these irregularities are expressed in smaller and smaller volumes of effluent. Frequently fronts are observed enconipassing only 1 or 2 ml. Without this coupled filter system and the mixer, observation of the separated zones is difficult.
V O L U M E 2 3 , N O . 5, M A Y 1 9 5 1
Figure 1.
795
Modified Tiaeliur-Claeslioii Adsorptiou . h u l y s i s Apparntxis
A.
I..
C. I). E.
P.
6.
Ii.
x.
iM.
N. 0.
ene gaskets, D,are held in place on the ends of the cuvette by brass end plates, F. The outlet tubes, G, made of stainless steel needle tubing can be oonnected conveniently to capillary plastic tubing to conduct the effluent wherever desired. An extra opening to the flowing channel is provided t o allow the Hushing out of any interfering bubbles which may collect in the cuvette and prevent observation. The needle tubings are protected by larger jacket tubings, H , which terminate in the cuvette cover. I. By Dravidinr the cuvette v-ith its own cove,, the G&ument-ean be used in a windy place without any noticeahle disturbance of the interference fringes. By using interchangeable cuvettes of differing lengths, one e m quickly select different ranges of refractive index without making adjustments of the instmment itself. When the "crau$ng-over" technique is used ( 5 ) , x spool, J , carrymg a few meters of capillary plastic tubing, is placed around the foot of the column on the ouvette, and the ends of the spool are connected t o the Hawing tube and one inlet of the eomps;rison tube. EWuent is then collected from the other comparison tube inlet. An auxiliary cuvette (Figure 1, I ) has been eonstrueted to allow simultaneous observations to be made in the Beckman spectrophotometer. This cuvette consists of a quartz cell having a 0.2-mm. light path and is provided with an inlet a t the bottom and an outlet a t the top. The entire unit replaces the cuvette carrier of the Beckman spectrouhotometer. The interferometer cuvette is codnected with the Beckman cuvette by the capillary plastic tubing.
Q.
F.
OPTICAL SYSTEM
S. T.
11. 1.
.I.
The optical system, similar to that of Claesson
11.
The set of coupled filters pictured in Figure 1 is constructed of R-0. 303 stainless steel and is resistant to corrosion. The outer diameter of the filters has been increased to 1.25 inches (3.1 em.) t o allow larger internal diameters for greater capacity, to permit stronger threads, and to lend sturdiness t o the system. Nine s t a n d a d filters of camcities varvine from0.5 to20 cc. are shown in Fimue 1, €3. Thk ratios of I e n h h to diameter are approxi-
o a n y polyethylene gasket8 in both faces.
.
",
plates, N , and a second plano-convex le&, 6, whioh bhngs the two rays to cross at thc focal point of the cylindrical lens in the eyepieoe, P. The compensator, N , is the only part of the optical system that has been significantly modified. A view of the compensator system with shade tube removed is shown in Figure 3. In the compensator, the glass plates, A , ape mounted in rods, B , which rotate ahout axes which intercept the light beams. With this arrangement the same spot of the compensator glass intercepts t h e light mi in all angular positions of tho plate, and the glass does not move out of the light path. The arms of the eompensar tor, C, nhich are activated by the micrometers, D,are not permanently fixed t o the rods hearing the cornpensator glasses, but are fixed to sleeves, E , which can be adjusted t o positions selected to provide for any desired angular ranre of the compensator
The column head is fitted with Luer-Lok syringe adapter, D, making possible convenient and quantitative charging of the column with the sample for analysis. The bottom of the oolumn carries a mixer, E (S),the function of which is to homogenize the effluent and prevent any layering of the solution in the ohservation cuvette, R. The syringe, G? which drives the column is a stainless, steel hollowhoned oylmder which contains a close-fitting piston. The cap of the syringe is provided with a detachable fitting eonneeted t o a source of compressed gas, H , by pressure tubing. The nose of the syringe is threaded to fit the column head, D. Recently i t has been found that for use with solutions that do not attack rubher, B piston carrying t w rubber O-rings makes a more leakproof fit and no special care is required to prevent scoring of the honed surface. With the steel piston removed, small Byringes c&n conveniently be placed within the cylinder for delivery of small volumes of liquid into the column.
eters below the optical path j n d by shortening t.Ke focal length of the plano-convex lens, 0, the operator e m more comfortably reach and read the micrometers. The eyepiece, P , is provided with v e r t i d and horiaontal slits which allow the operator to exclude stray or reflected light and to select only the rays involved in the interference pattern. The eyepiece is also provided with a curved eye shade, exoluding side light from the operator's eye. An adjustable fine glass hairline, Q,is mounted a t the focal point of the oylindrical lens of the eyepic
THE CUVETTE
THERMOSTATED BATH
For use with fatty acid separations it was necessary to abandon the conventional gold-plated brass cuvette to prevent leanhing of copper by the fatty aeid solutions.
'lhe aoume-walled bath of the original instruments ( I , 1 1 ) has been retained. An attempt was made to use 5 simple bath, hut the disturbanom caused by sbirring and heating were too great.
The cuvette, shown in Figure 2, is made of stainless steel and represents a new type of construction. It consists of two blocks of stainless steel with channels milled into their surfaces. The observation channels were milled into the upper surface of the lower block, B, and channels to conduct the liquid to the needle tubing outlets, G, were milled into the lower surface of the upper black, A . The two blocks are bolted together with ia polyethylene gasket, C , separating them. Glass faceplates, E, over polyethyl-
The outer bath, R, containing water is provided with a level indicator, and the cover of bhe hitth carries a motor stirrer, a thermoregulator (37" + 0.2"), two electric heaters, m d a thermometer. When the cuvette is in place, its cover completely cover8 the opening of the inner bath, minimizing cooling by the atmosphere. The heaters axe Connected in such a mannef that they can be used either in parallel for rapid heating, or In sene8 for
C~~~~
~~
796
ANALYTICAL CHEMISTR
sustained heating free from surges in temperrtture. Controls for motor, heaters, micrometer lamps, S , and band lamp are grouped in a control box, T. JSE OF THE APPARATUS
A u ion of the manipulations of the adsorption a d the coupled filters is not available in the andysA. -,,yeAeyuo literature. To give a olertr pioture of how the apparatus is used, the manipulations involved in a simple displacement experiment are described here.
:ure 3. Compensator with Shade Tube Removed
. -
Figure 2.
Exph,ded View of Cuvette
E.
E".F'atep
.
The number and sizes of filters chosen for a desired separation depend upon the quantity of the sample, retention of displacer, and difficulty of separation of components. Columns should always be built with lowest filter the smallest (0.5 cc.), increasing the capacity of the oolumn by adding larger filters a t the top. Neighboring filters should preferably not differ in capacity in steps greater than a factor of 2.5.
into the largest filter of the column, and the'salvent is drained from below by slight suction. When the filter is full of packed, yet moist, adsorbent, the absorbent is pressed down with a spatula, and the excess is removed. A filter aper oirole is laid over the adsorbent. The filter is now invertea and sorewed into the column head, D, which is previously flooded with solvent.
to the compressed gas, and solvent is forced through the filter to wash it. The next filter is packed and inverted, and its top is joined to a coupling previously flooded with solvent. The other side of the coupling is then filled with solvent and joined to the bottom of the first filter. The remaining filters are packed and assembled in the Same way, and the mixer, E, is joined t o the
bottom filter. This method of building and washing a oolumn saves considerable time, for when the column is built, it is already nearly washed. The sample to be separated is dissolved in a convenient volume ( 5 t o 10ml. j of the solvent, and placed in a Lner-Lak syringe. The stainless steel syringe is removed from the column and thc glass syringe is fixed in its adapter. The contents of the syringe are discharged into the filter column by hand. It is often convenient to loosen B joint in the center of the column before pressing in the sample, thereby reducing the back pressure. The column is then retightened, the syringe removed from its socket, and the oolumn replaced on the large syringe whioh has been filled with displacer solution. The oolumn with syringe is then screwed into its place in the cuvette and the cuvette is placed in the bath. ' The contents of the compssison tube are next washed out with solvent from a glass syringe aud the needle tubings are joined by a short length of plastic c%pillarytubing. The flowing channel is also flushed with solvent and the u-ash outlet plugged. The driving syringe is conneoted to the source of gas presrmre and the development of the column is begun. When the apparatus comes to bath temperature, the fringes are located at or near the em (base 1ine)valueon themicrometer. If fringes cannot he found, the cuvette chnnels are inspected by means of a mirror. Should the light be obscured, the channels are washed again until the light emerges from both channels. The usual source of trouble is the lodging of bubbles in the channels. If bubbles appear in the Rowing channel during an experiment, they usually can be dislodged by pinching the plastic outflow tube. This develops internal pressure enough to dissolve the bubble partially, and when the pressure is released the bubble is flushed out. Pinching the tube s few timm usually removes the bubble from the channel; if not, it can always be washed away by a stream of solvent injected into the wash tube. Observations are made at convenient intervals of volume during the course of the experiment by turning tho micrometer screws to bring the zone of interference fringes across the hairline, approximately the same number of fringes lying on either side. A convenient flow rate is 20 to 30 ml. per hour. When the micrometer reading reaches the value previously determined for the displacer solution, the experiment is terminated, far a t that time the column is saturated a i t h displacer and the sample has been displaced. When high concentrat,ions of displitcer are to be used, or when the components have high refractive indexes, so that measurements would normally exceed the range of the two micrometer screws, the crossing-over technique ( 5 ) is of value. T h e effluent is conducted from the outflow tuhe, through a plastic ca.pillary
797
V O L U M E 23, NO. 5, M A Y 1 9 5 1 tube spacer wound on a spool, to the inlet of the comparison tube, and from thence to the receiver, U. Thus, refractive index gradients across a volume interval equal to the spacer are measured. As long as the concentration of substance in the effluent is constant, the refractive index gradient is zero. When a zone of substance arrives in the cuvette, the refractive index rises to a new level, and this is then observed as a spike. Thus, rather than a stepwise rise in refractive index, one obtains a series of maxima rising from the base line, corresponding to the steps of the ordinary displacement diagram. In the discussion here, displacement separation has been used as an example. The apparatus is well adapted also to elution, frontal, and carrier displacement separation. ACKNOWLEDGMEhT
Opportunity is taken to commend the workmanship of D. Milton Kvanbeck of Minneapolii, Minn., who constructed the custom-built model discussed in this paper. This work ww
supported by a granbin-aid from the Williams-Waterman Fund of the Research Gorp. LITERATURE CITED
(1) (2) (3) (4)
Claesson, S., Arkiv Kemi Mineral. Geol., 23A, 1 (1946). Ibid., 26A,24 (1949). Hagdahl, L., Acta Chem. Scaad., 2, 574 (1948). Hagdahl, L., and Holman, R. T., J . Am. Chem. Soc., 72, 701
(1950). (5) Holman, R. T., AN.4L. C H E Y . , 22, 832 (1950).
(6) Holman, R. T., and Hagdahl, L., Arch. Biochm., 17,301 (1945). (7) Holman, R. T., and Hagdahl, L., J . B i d . C h m . , 182,421 (1950). (8) Moring-Claesson, I., Biochim. et Biophys. Acta, 2,389 (1948). (9) Tiselius, A,, Arkiv Kemi Mineral. Geol., 16A, 18 (1943). (10) Tiselius, A., “The Svedberg,” Stockholm,4lmquistandT~iksells, 1944. (11) Tiselius, A., and Claesson, S., Arkiv Kemi Mineral. Geol., 15B,18 (1942). (12) Tiselius, A , , and Hahn, L., KoZloid Z . , 105,177 (1943). RECEIVEDAugust 28, 19.50. Presented before the Division of $nalyticsl Chemistry at the 117th hfeeting of the AZ~IERICAN C x ~ n a r c . 4 ~SOCIETY. Houston. Tex.
Microdetermination of Chromium in Catgut Sutures HERBERT Y, WEISS, \-ISTON E. SILER,
AYD
PETER R . BUECHLER
Department of Surgery, t-niversily of Cincinnati, Cincinnati, Ohio
TITRIMETRIC method for the determination of chromium
A in catgut mtures, using 1-to %gram samples, has been re-
ported ( 5 ) . However, studies in progress a t this laboratory involve the determination of chromium in small strips of catgut suture, weighing 3 to 4 mg. As the chroniium content of these sutures ranges between 0.15 and 0.2575, a more senfiitive method of analysis is required. The high sensitivity of the diphenylcarbazide method for determining chromium (3) has been adapted to the determination of chromium in leather ( 2 ) . The procedure described is not within the working range for the minute quantities of chromium to be determined in catgut sutures. AIodifications of this procedure have, however, afforded the accurate determination of chromium in 3- to 4-nig. samples of catgut suture. The method is simple, rapid, and should he generally applicable to the determination of minute amounts of chromium in biological materials-e.g., in the stratoassessographic analysis of chrome-tanned leather ( 1 ) . Iron, vanadium, molybdenum, and mercury interfere with color development in the diphenylcarbazide method. Of these, iron is the only interfering metal that may possibly be encountered in catgut sutures. (Phenyl mercuric benzoate is added to certain tubing fluids. However, the amount employed is insufficient to produce interference in the diphenylcarbazide reaction.) The addition of phosphoric acid, to complex the iron possibly present, effectively minimizes this interference ( 2 ) . A 108s of chromium in the perchloric acid oxidation, resulting from the formation of ehromyl chloride, may occur ( 4 ) . This is obviated by adjusting the temperature below the boiling point of constant boiling perchloric acid, and yet maintaining a sufficiently high temperature to permit complete conversion of chromium t o the hexavalent state. PREPARATION OF SAMPLE
The sutures, following removal from the tubes, were cut into 1-inch (2.5cm.) strips, allowed to come to equilibrium in a room which remained relatively constant with regard to humidity and temperature, and then weighed, For determining absolute values of chromium content, 1-inch strips of euture were transferred to micro weighing bottles, oven-dried under vacuum a t 70” C. for 18 hours, and weighed. REAGENTS
Buffer solution, pH 1.6. Take 250 ml. of 0.2.Y potassium chloride (14.9 gramF per liter), mix with 150 nil. of 0.2 -Irhydro.
chloric acid (17.1 grams of conctntrated hydrochloric acid per liter), and dilute to 1 liter. Diphenylcarbazide, 0.25% in 1 to 1 acetone-water. This solution should be prepared daily and kept in the dark while not in use. EXPERI?vlENTAL PROCEDURE
The sample is transferred to a 12 X 75 mm. test tube. Two drops of concentrated sulfuric acid and 4 drops of concentrated nitric acid are added, and the mixture is boiled gentlyon a hot plate until carbonization occurs. The sample is cooled, and one drop of hydrogen peroxide (3070),isadded. The mixture is heated again to charring, removed from the hot plate, and cooled, and the treatment with a drop of hydrogen peroxide is repeated. Usually 3 drops of hydrogen peroxide are required to effect complete digestion of the sample in this manner. Following clearing, the sample is allowed to remain on the hot plate for 0.5 hour to remove traces of unreacted hydrogen peroxide. .-ifter cooling, 2 drops of constant boiling perchloric acid (70 t o 72%) are added to the digestion tube. The test tube iz inserted about. 15 mm. below surface of a constant temperature bath adjusted at 200” * 2 C. and oxidized for 15 minutes. After oxidation, the tube is immediately inserted into a cold water bath and diluted with distilled water to a volume of approximately 3 ml. The solution is transferred into a 25-ml. volumetric flask. About 10 ml. of water are used for the transfer. The volumetric flask is placed on a hot plate and the solution is boiled for several minutes to remove liberated chlorine. After cooling, one drop of concentrated phos horic acid and 10 ml. of the buffer solution are added. One milliher of diphenylcarbazide is transferred to the flask, water is added to the mark, and the solution is shaken. The color is allowed to develop for 1.5 minutes. Readings are made in an Evelyn colorimeter using a S o . 540 Evelyn filter.
tp
PREPARATION OF ST.AYDARD GRAPH
Known samples of hexavalent chromium a t five concentrations, ranging from 3.6 to 10.8 micrograms, were transferred to the digestion tubes, reduced with an excess of sodium thiosulfate, digested, and oxidized in the manner described. Six samples at each concentration were determined. The color developed was read against a blank solution treated similarly. Per cent transmittance for the concentrations indicated ranged from 25 to 65. The optical density of the mean reading a t each concentration was plotted; Beer’s law was followed. The average deviation from the mean values of opt’ical density at the particular concentrations was 1.i’70 and the maximum deviation from the mean (at 3.6 micropr:tnis) was 6.2% RECOVERY EXPERIMEIITS
Solutions of potassium dichromate were added to 1-inch (2.5cm.) strips of ordinary nonchromicized catgut. Four samples a t
