Diffusion Micromethod for Nitrogen ROLAND C. HAWES AND EDWIN R. SKAVINSKI’ Laboratories of George Piness, M.D., Los Angeles, Calif.
R
ECENTLY it mas desired to determine the nitrogen content of a number of allergen preparations. Analysis of extractable and “protein” fractions, in duplicate, brought the number of determinations in prospect to about 1800. A micromethod was definitely indicated by the cost of the materials, even if the advantage of economy of reagents, convenience, and speed had not influenced the decision. I n fact, since many pollen extracts contain less than 0.1 per cent (calculated on a dry pollen basis) of nitrogen precipitable with trichloroacetic or tungstic acid, and since it was desired to do a complete analysis on a total sample not exceeding 0.5 gram, a method adapted to the determination of 100 micrograms or less was required. Choice of published methods finally lay between colorimetry using direct nesslerization, and the Borsook and Dubnoff (1) adaptation of the Conway and Byrne (3) diffusion micromethod. The advantages of the former in speed and simplicity were weighed against its more limited precision and the occasional presence of interfering substances from these materials. Experience with the two methods showed that the advantage of the colorimetric method in speed would be overcome to a considerable extent if a satisfactory way could be found of avoiding the sample transfer from the digestion tube into the diffusion vessel, while at the same time one of the chief sources of handling difficulties and variation of results would be avoided. Needham and Boell (6) described apparatus for this purpose, but it has some disadvantages. The setup for large scale use is rather expensive because of the ground joints required, and the diffusion rate is inconveniently slow. While this manuscript was being prepared a paper by Tompkins and Kirk (8) appeared, describing apparatus which attains the same object in a similar way, but which is different in several respects from that shown below, and especially in that the digestion tube cannot so readily be used in the centrifuge and that the diffusion rate is slower. Their paper reviews the need for a n improved method. References in i t and in the Conway book (3) include all the previous work considered except the aeration method of Folin (4).
spection of a table of ionization constants showed that the second hydrogen of hosphoric acid has very nearly the ideal dissociation tendency, wfile the constant for the first hydrogen is sufficiently large that its dissociation is nearly complete at the end point near pH 4.5. The advantage of 1.0 M sodium dihydrogen phosphate monohydrate over boric acid saturated at 20” C. (0.8 M ) may be seen by inspection of the titration curves of Figure 2. They show experimental titrations of 5 ml. of each absorbent with 10.5 N ammonium hydroxide, delivered beneath the surface of the solution from a buret with a capillary tip. The pH values were obtained with a glass electrode pH meter (Beckman laboratory model). Also shown are the pH’s of 1 to 50 dilutions, made by removing 0.02-ml. portions of the solutions at the indicated points during titration with the aid of a set of receivers and diluting each with 1.0 ml. of water for the pH reading. Those at the higher pH values were held only a few moments between removal and dilution, but probably lost appreciable amounts of ammonia in the interval, as suggested by the lines drawn on the plot to indicate various levels of ammonia dissociation in water. Only about 0.08 mole of ammonia per mole of boric acid initially present was required to raise the pH to 6.4, the level of 0.1 per cent ammonia dissociation. The corresponding ratio for phosphate is 0.42. Furthermore, by use of a saturated solution of ammonium dihydrogen phosphate monohydrate the sample size could be increased to about 0.5 mg. of nitrogen with, of course, some loss of end-point precision for smaller samples. In order to determine the time necessary for the diffusion, the rate constant for the apparatus was calculated from measurements on duplicate samples containing 100 micrograms of nitrogen in the form of ammonium sulfate at 0.5, 1, 2, and 3.5 hours. The constant, calculated from the equation
t K = log A / ( A - X) averaged 120 minutes (minimum 103, maximum 142). A is the total amount of ammonia and x is the amount found in the receiver after t minutes of diffusion. The constant shows the time required t o transfer 90 per cent of the ammonia, and may be compared with a constant of about 50 minutes for the standard Conway (9) “unit”, 260 minutes for the device of Tompkins and Kirk (8), and 700 minutes for the Needham and Boell (6) apparatus. (Constants were calculated from data given in the references, corrected for solution composition and t o a temperature of 20’ C.)
At 20’ C. ammonia diffusion is 99.5 per cent complete in less than 5 hours. Amines ( 2 ) will diffuse more slowly, so that in analyzing digests of most substances of biological interest the recommended overnight diffusion is not unnecessarily long with the present setup. Naturally, the rate can be increased by raising the temperature, and by other means (3, 8) if desired. APPARATUS FIGURE1. SECTIOXAL VI E W O F DIFFUSION It seems worth while, because of the confusion that may result from the method used by Tompkins and Kirk (8) to determine the length of diffusion required for various size The essential feature of the present method is that a plain test samples, to re-emphasize that the above formula has been tube (or conical centrifuge tube, if desired) serves as both digestion shown ( 3 ) to apply generally to the diffusion of substances and diffusion vessel. The diffusion receiver consists of a helix of platinum wire carrying a drop of absorbing solution, held in the having low vapor pressure, such as dilute ammonia. I t s tube by means of a grooved rubber stopper. The apparatus significance is, of course, that for equal relative accuracy of set up for diffusion is shown in Figure 1. determination the same length of time is required for all With this assembly it was apparent that convenience of manipsample sizes. I n unpublished work it was found that the ulation would result if a concentrated solution of a weak acid equation has the same constant and predicts the diffusion were used for the absorbing solution, since then the volume of the droplet need not be measured precisely and the helix could be course as well for 10 as for 100 micrograms of nitrogen. It adjusted so as to pick up the desired volume when dipped in the is, therefore, not clear how Tompkins and Kirk obtained values solution. However, it n-as found that boric acid, which is usually within 1 per cent of theory on various samples concommonly used for the purpose (Z), is too weak to be suitable. A taining less than 10 micrograms of nitrogen, when their 0.02-ml. droplet of saturated solution would provide stoichiometric absorption of ammonia only up to about 20 micrograms recommended diffusion time is sufficient to transfer only of nitrogen. As this was considered an inconveniently small about 90 per cent of the ammonia in such samples, as shown maximum sample size, search was started for a substitute. Inby their own rate curves. S o attempt was made to determine the limits possible with 1 Present address, Southern California Gas Company, Loa Angeles, Calif. 917
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Vol. 14, No. 11
INDUSTRIAL AND ENGINEERING CHEMISTRY
Procedure Samples are pipetted (1)or weighed into ordinary 125 X 15 mm. Pyrex test tubes taking care to kee the upper wall as clean as possible, 0.2 ml. oracid digestion solution is added to each, and excess water is removed overnight in an oven at 100" C. If the sample volume is less than 0.5 ml., the preliminary drying may be omitted if the first heating is done cautiously. Digestion is carried out in a sand bath heated by a Tirrill burner so adjusted as to cause refluxing to about 4 em. from the bottom of the tubes. Ebullition and bumping do not occur. The tubes are covered with glass caps cut from the bottoms of soft-glass vials 21 mm. in diameter, to prevent concentration of the digests and to permit operation outside the fume hood. S o attention is required during digestion as a rule, but if the digests are not nearly colorless in about 4 hours the flame is removed for 10 minutes, and then a small uniform drop of 30 per cent hydrogen peroxide is placed in each tube, including the blanks. Refluxin is continued for at least 30 minutes after &e straw color is gone. When digestion is complete the tubes are placed in a rack and allowed to cool, then 0.6 ml. of water OF ABSORBEKTS WITH STRONG AMMONIA FIGCRE2 . TITRATIOX is added to each, and all are stirred by shaking the rack. After further cooling each in turn is treated as follows: Three-tenths milliliter of 50 per cent sodium hydroxide is pull this diffusion apparatus, but it was found that 10 micrograms down the side of the tube without agitation and so as to avoid can be determined without alteration of the method except wetting the rim and 1 cm. of the uppeFwal1 of the tube. Although to use 0.2 M sodium dihydrogen phosphate, if electrometric the solutions become warm, the large difference in specific gravity prevents complete mixing which would cause loss of ammonia. determination of the end point is used. A ground-glass joint, On warm days it may be necessary to cool the tube in a beaker of or possibly a stopper made of a synthetic elastomer in place ice water. It is left undisturbed while the receiver is prepared. of the rubber, and a smaller titration volume would no doubt The latter is removed from the 1.59 X 12.7 cm. ("8 X 5 permit further increases in sensitivity. inch) test tube in which it is kept, and rinsed with distilled water, flamed, and rerinsed. The water is shaken off the helix and wiped off the stopper with cleansing tissue. The helix is dipped Preparation of Receivers in 1.0 M sodium dihydrogen phosphate solution, deeply enough Five turns of 22-ga e platinum wire are close-wound on a rod to cover it, but not so deeply that the platinum- lass junction is 2.4 mm. in diameter ?13-gage), leaving a handle of straight wire wet with the solution. The stopper is lubricate$ with a drop of 1.5 cm. long. The end of the latter is sealed into a short piece of glycerol and then inserted in the tube, taking care that the helix 6-mm. soft-glass tubing inserted through the hole in a No. 0 pure does not touch the wall. If it does it must be rerinsed and regum rubberstopper. charged. Contact may be avoided by placing the rim of the Stop ers, obtained from the Chemical Rubber Company, Clevetube against a notch in the bench edge, holding the receiver in land, Oiio, are treated before making up the receivers by boiling the other hand in a position coaxial with the tube, and sliding the 15 minutes in 1 N sodium hydroxide, then boiling and rinsing in tube up over the receiver, which is held motionless while the tube distilled water. When prepared in this way they contribute the is twisted to seat the stopper. equivalent of less than 0.3 microgram of nitrogen to the value of The digest is now overneutralized by shaking the tube while the blanks. A neat groove in the stopper is most easily made cooling with a stream of tap water. The entire wall of the tube with the aid of an electric drill stock carrying a metal rod to hold is coated with a film of alkali by inverting and rotating, and the the stopper, and a power grinding wheel, but it may also be cut assembly is left overnight on its side on a paper towel or blotter by hand with a sharp knife. The groove is about 1 mm. deep placed on a level surface. and 6 mm. wide, and starts about 4 mm. from the small end of the When ready to titrate, the receiver is carefully removed by restopper. It is effective in preventing the stopper from forcing versing the insertion procedure. The stopper is wiped free of itself out of the tube or moving so as to bring the helix in contact alkali and the helix is immersed in 1ml. of water containing either with the tube wall. indicator or a slight excess of quinhydrone, depending on whether The helix is cleaned either by immersing for a few minutes in the end point is to be detected colorimetrically or electrometrically. hot cleaning solution or by heating to incandescence for a second in a flame. It is then adjusted by pulling with fine forceps or spreading the center turns with a knife blade, until the volume of the drop it carries is within 10 per cent of 0.02 ml. (determined by titration). Uniformity is of more importance than the absolute size of the droplet. The helices are recleaned a t frequent intervals in order to maintain a uniform drop size. Platinum wire was chosen because of the ease of cleaning, although Nichrome wire which waa also tried Kill work, as, no doubt, would other materials, including glass.
Reagents Digestion solution, 18 N sulfuric acid (nitrogen-free) containing 0.1 gram of selenium dioxide and 0.1 gram of copper sulfate per
100 ml. Hydrogen peroxide, 30 per cent, conforming to A. C. S. specifications. Sodium hydroxide, 50 per cent by weight, which has stood or been centrifuged until clear. Absorption solution, 1.0 M monobasic sodium phosphate (Merck reagent). Standardized acid, of strength suited to the microburet and sample size.
0 4 /6 36 M M U ,
,
I
,
I
,
l
,
l
.
225 l
,
,
.
.
625
4#0 I
~
.
~
.
I
I
900 I
1
FIGURE 3. pH OF DILUTEDABSORBENTAND ABSORPTION EFFICIENCY FOR VARIOUS AXOCKTSO F AMhlONIUM I O N
November 15, 1942
ANALYTICAL EDITION
The solution is titrated with standardized acid from a microburet t o the pH given by 0.02 ml. of the buffer solution diluted in the same way. The end point lies between pH 4.3 and 4.7. Blanks are, of course, included with each set of determinations. With electrometric titration the end point can be determined to 0.02 pH, or 0.2 per cent of the 100 microgram “optimal” sample. Indicator end oint error is about three times as great, but could be decreased f y decreasing the titration volume (for a discussion of limitations see Conway, 3). Drying and digestion introduce greater variation, but duplicate determinations on protein solutions almost always agree within 1 per cent and agree with larger samples run by the semimicromethod ( 7 ) t o within the same limit. Bn important advantage of electrometric titration, in addition to greater recision, more warning of end-point approach, and no need to gack-titrate if the end point is overrun (small excesses can be read from a previously prepared titration curve), is that the size of the droplet of absorbent solution can be checked by comparing the pH before titration with the amount of acid required to reach the end point. [A pH meter with continuous (nonballistic) indication of balance is more convenient and rapid than the ballistic t y e, especially for titration purposes.] In this way it is easy t o &e sure that sufficient buffer was present t o ensure stoichiometric absorption of the ammonia. I n Figure 3 is shown the normal relation between p H and sample nitrogen content, also the completeness of absorption, for a considerable range of amounts of ammonia added as ammonium sulfate solution. Table I gives the results of a series of determinations done on an ammonium sulfate solution contairiing 0.500 gram of nitrogen per liter (2.358 grams of the salt, recrystallized from water, and dried in vacuo over fresh calcium chloride.) A semimicro-Kjeldahl (7) determination done in triplicate on the same ammonium sulfate solution and referred to the same independent standardization of acid and alkali gave a value of 0.4982 * 0.0005 mg. per ml. Since the main point of novelty in the method and consequently of interest in these determinations was the diffusion procedure, the samples were not carried through the digestion steps prior to the diffusion or distillation. The actual titration volumes are given and the analytical results which were calculated from the averages, as well as the standard errors of the individual determinations and of the averages and results. All the data obtained under the described conditions for this solution of ammonium sulfate are reported, except for a single determination obtained with pipet B, which was 163.8 microliter and was discarded as probably in error through a fault in pipetting technique. The difference between the value obtained with pipet E and the semimicro-Kjeldahl value, 0.0016 * 0.0008 mg. per ml., is of doubtful significance, but the differences between the larger sample and the smaller, 0.0031 * 0.0010, and between the larger sample and the semimicrodetermination, 0.0057 * 0.0010, are significant. All the differences are in the direction to be expected if transfer of the ammonia is incomplete, and they probably may be taken as a measure of this incompleteness for correction of other analyses, if the highest accuracy is wanted. The diffusion time was ample to ensure the establishment of an effective equilibrium between the evolving and absorbing solutions.
digestion rack and is considerably easier to obtain a t the present time.
It consists of a 20-em. (%inch) hemispherical iron bath with 18 notches cut with tin snips in the periphery. Enough fine sand to make a layer about 10 em. (4inches) in diameter is placed in it and the tubes are leaned into the notches with their bottoms around the outer edge immersed t o about the level of the digest, of the sand layer. The sand bath also makes a very satisfactory digestion rack for six 30-ml. Kieldahl flasks as used in the semimicro or micromethod, but m&t then be placed in the fume hood and heated with a hEker-type burner.
A titration stand is an even greater convenience for microthan for macroanalytical work. That used in this laboratory may be of interest because of its simple construction. It is shown in Figure 4.
TABLEI.
DETERMINdTIONS OF A STANDARD,
SULFATESOLUTION
(0.5mg. of N per ml.) Titrations Standard Analytical with 0.0495 Error” N Acid Result M g . N/ml. Pl. 0.4 0.12 0.3 0.6 0.4 0.5 0.7 0.5 0.5 0.6 0.6 0.3 0.4 0.5 0.4 0.4 Av. 0.47 0.031
Sample Blanks
Pipet E , 0.1645 ml (82 r e , )
Av.
118.0 117.6 117.8 118.3 117.4 117.4 118.7 118.7 118.1 118.7 118.6 118.9 118.8 118.8 118.27
Av.
162.3 160.7 161.8 160.7 160.1 161.5 160.3 162.5 161.7 162.1 161.37
Av.
3.7b 1.5 1.0 3.0 2.30
Av.
165.8b 165.9 165.5 164.8 166.1 166.2 165.72
Pipet B. 0.2261 ml (113 rg.)
Blanks b
Notes The oven used a t b s t for removing excess water previous to digestion had been employed in organic preparation work for several years. Despite repeated cleanings, including sluicing out with water, and baking out a t high temperature, large and variable blanks which were easily traced to the drying operation were obtained until a new oven was constructed. I n the latter the heat is radiated downward from an element in the ceiling, resulting in rapid evaporation without danger of loss by bumping. The sand bath is a flexible and inexpensive substitute for the drilled aluminum bar first used in this laboratory as a
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Pipet B (11.3 p g . )
b
-
AMMONIUM Standard Errora
0.55 (0
0.147
0.4966
0.0006
0.4s35
0.0008
0.86 (0.53%)
0.272 1.3
0.63 1.62 (0.98%)
0.66
0.5012
-
0.0028
-
aStandard error of individual observation, 8 , dZdV(n l), and standard error of average 8 / d / n , where d is deviation of each observation from average and n is number of observations. bIn these,analyses 0.2 3 N P H I P O ~was used as absorbent. Ammonium sulfate solution and titration acid were 1 to 10 dilutions of standards, each made with same pipet and volumetric flask.
920
INDUSTRIAL AND ENGINEERING CHEMISTRY
t - / u cm, _i STAND AXD CALOMEL HALF-CELL FIGURE 4. TITRATION
The stirring motor is a 1400 r. p. m. shaded-poleinduction motor, with a chuck from a small hand drill attached to hold the stirring paddle. The latter is an old platinum foil electrode, 3 mm. wide by 5 mm. high, sealed into a piece of soft-glass tubing. The motor is mounted on two shelf brackets fastened to a block of wood which also carries a rod to support the electrode terminal block and a piece of Bakelite in which holes for the electrodes and stirrer and a slotted hole to carry the receiver have been cut as shown. The titration vessel, consisting of a beaker 10 mm. high and 15 mm. in diameter, is sup orted on a counterweighted hinged shelf of Masonite. Electrogs are held in the holes in the Bakelite sheet by means of small soft wood wedges. The construction of the calomel half-cell and agar bridge is also illustrated in Figure 4. It is made of Pyrex glass, except for the internal platinum electrode, which is sealed into a small softglass tube. A fiber of acid-washed asbestos sealed into the tip may be substituted for the agar bridge, if desired. When not in use the electrode is stored with the agar bridge immersed in saturated potassium chloride in a squat bottle closed with a onehole rubber stopper. The quinhydrone electrode consists of a short piece of 22-gage platinum wire sealed into a piece of 8-mm. soft-glass tubin drawn down at the tip t o about 2-mm. inside fiameter. It was substituted for the small glass electrode used by Borsook and Dubnoff because of the fragility of the latter. For these titrations pure quinhydrone provides an entirely satisfactory half-cell.
The pipet is filled with water and placed in a horizontal position with the tip downward. The orifice is adjusted by breaking off ver short pieces of the tip until the delivery rate is between 0.1 an80.2 microliter per second, with the tip just immersed. I t is now placed on the titration stand as shown. The height of the water column in the test tube, which is held in place by a spring brass clamp so that it can be moved up or down, is adjusted with the tube in its uppermost position until the delivery rate is about 100 microliters per minute. A mark is made on the tube at the level of the water surface. In this way two constant delivery speeds which can be used as needed are available: a rapid “approach” speed, and a slow “end- oint” speed. The former will determine to some extent the cayibration of the buret, and should therefore be maintained reasonably constant. The buret is adjusted in height until it is horizontal when the tip is immersed in 1 ml. of water in the titration beaker. It is filled by immersing the tip, lacing a finger firmly on the rubber connecting tubing, and rolying to the left. The meniscus is brought to the zero point by immersing the buret tip in a beaker of water, then the buret rack is moved to the titration stand, and the titration is started by immersing the tip and raising the test tube. When the end point is nearly reached the test tube is lowered (if a drop hangs across the inner tube a dro of oil may be floated on the water) and the end point is approacBed by alternately immersing and withdrawing the tip. The tip height adjustment lever is of sufficiently thin metal that it.can be sprung out of the way for reading if the meniscus is near it. The buret is calibrated by weight of water or by titration. Four or more points should be taken along its length, as these pipets are seldom found to have perfectly uniform bore. A reservoir for the standard acid may be attached to the buret by a stopcock, one-way mercury valve, or other device, just behind the graduations, if greater convenience in filling is wanted.
A convenient indicator is 0.0015 per cent of bromocresol green plus 0.0002 per cent of methyl red. The combination, which has been in use in this laboratory for several years, was recently recommended (6) for the increased sharpness of end point i t affords (in the p H range within mrhich both indicators change color-i. e., 4.4 to 5.4), but no mention was made of another equally important advantage it has over the commonly used methylene blue-methyl red combination (Tashthat i t gives considerably more iro’s reagent)-namely, warning of end-point approach. Optimal proportions of the two dyes vary with the endpoint pH and should be adjusted, with the aid of a solution buffered at the desired end point, so as to give a gray or neutral tint. Suitable proportions for the usual macro- or microKjeldahl procedure with the end point at about p H 5.4 are 0.0001 per cent methyl red and 0.002 per cent bromocresoI green. The indicator is made u p in alcohol to 100 times the specified final concentrations.
An easily constructed microburet is illustrated in Figure 5. Its outflow is started and stopped by immersion and withdrawal of the tip from the liquid being titrated, eliminating the need for a stopcock or any kind of positive displacement device. It is made from a Kahn serological pipet, 0.2 ml. graduated in microliters. A constriction 4 cm. long and 2 mm. in diameter is drawn, as near the ti as possible, pulling the tip with a handle or Ebrceps. The farthest point of the constriction is further drawn until it has a diameter of about 0.2 mm., and it is broken at the narrowest pointio The entire tip is bent at an angle of about 70 away from the side carrying the graduations.
Vol. 14, No. 11
FIGURE 5. MICROBURET AND STAND
ANALYTICAL EDITION
November 15, 1942
Summary A micro-Kjeldahl method for nitrogen in 10- to 1 ~ h n i c r o gram amounts is based on the diffusion principle developed by Conway. In terms of the time required for manipulations ‘it is more rapid than a n y other of like precision. The apparatus required is inexpensive and is easily made from readily available materials. Two pieces of accessory apparatus described are a simple microburet and an easily made titration stand. The advantages of an indicator t o replace the commonly used blue-methyl red combination are pointed out.
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Literature Cited (1) Borsook, H., and Dubnoff, J. W., J . Bioi. Chem., 131,163 (1939). (2) Bradstreet, R. B., Chem. Reo., 27, 231 (1940). (3) Conway, E. J., “Micro-Diffusion Analysis and Volumetric Error”, New York, D. Van Nostrand Co., 1940. (4) Folin, O.,J . B i d . Chem., 97,141 (1932). (5) Ma, T. S., and Zuazaga, G., 1x11.ENG.CHEM.,ANAL. ED., 14, 280 (1942). (6) Needham, J., and Boell, E. J., Bwchem. J.,33, 149 (1939). (7) Shriner, R. L., “Quantitative Analysis of Organic Compounds”, Ann Arbor, Mich., Edwards Brothers, 1940. (8) Tompkins, E. R.,and Kirk, P. L., J. Biol. Chem., 142, 477 (1942).
Spectrographic Analysis of Rat Tissues for Ingested Vanadium ESTHER PETERSON DANIEL’, ELIZABETH 31. HEWSTON, AND MARIAN W. KIES’ Bureau of Home Economics, U. S. Department of Agriculture, Washington, D. C., and Johns Hopkins University, Baltimore, Md.
A spectrographic method for the quantitative estimation of vanadium in limited amounts of biological material is presented. Chromium is employed as an internal standard in the arc spectrum and use is made of standard reference curves established by plotting the ratios of line blackening produced by standard solutions of vanadium and chromium against the con-
D
URIKG the course of a study of the distribution of in-
gested vanadium in r a t tissues, the following method for determining very small amounts of this element in limited amounts of biological material was developed.
Preparation of Rat Tissues for Analysis Tissues from rats fed sodium metavanadate were dissected out under ether anesthesia, washed thoroughly in physiological saline to remove the blood, and wet-ashed, after combining with Eimilar organs of other rats on a diet containing the same level of vanadium. Ashing was begun in micro-Kjeldahl flasks over microburners, and after complete digestion the samples were transferred to weighed silica dishes and brought to dryness. A iittle perchloric acid was used in the final stages of digestion of fatty tissues.which were difficult to ash with nitric acid alone.
Method of Analysis Analyses were made by means of a Bausch & Lomb high-dispersion, Littrow-type quartz spectrograph. The arc was operated on a 220-volt direct current line with a large 24-step resistor jn series, thereby affording a much steadier arc than is possible on a 110-volt line. A voltmeter connected across the electrode terminals made it ossible to determine the potential of the arc gap at any time. condensing lens was used in concentrating the Sight from the sample. Although this practice is not generally used in quantitative spectroscopy, it seemed desirable in the present case because the smallest detectable quantities of vanadium were sought. A Bausch &- Lomb nonrecording densitom-
1
1 Present address, Food and Drug Administration, Federal Security Agency, Washington, D. C . Present address, Bureau of Agricultural Chemistry and Engineering, U.S.Department of Agriculture, Washington, D. C. f
centrations of vanadium. The spectrum lines, vanadium 3185.406 A. and chromium 3188.0 A,, were measured by means of a nonrecording densitometer. From a number of ashed tissues analyzed for vanadium, blood samples taken from rats fed three different levels of this element were chosen to illustrate the applicability of the method. eter modified according to Scribner (4) n’as used to read the plates. The most sensitive group of vanadium lines in the arc spectrum, 3185.406, 3183.99, and 3183.415 A., was selected for measurement. Consideration was given to such factors as current, slit aperture, type of electrodes, interelectrode distance, size of sample, depth of electrode well, creeping of the sample, internal standard, burning time, and background. Duffendack, Wolfe, and Smith (3) noted that fluctuations in the relative intensities of line pairs are insignificant above an arc current of approximately 12 amperes. I n the present study i t was observed that amperages of 12 to 15, while producing a somewhat steadier arc than lesser currents, caused too much background on the photographic plate for densitometer readings. At 10 amperes this background could be cut down sufficiently t o read the spectrograms and at the same time remain in a range which did not exceed a tolerable change in the relative intensities with variation in current, The voltage across the arc with this current was 50. In practically every case, background was the factor which in the end greatly influenced the choice of procedure. A slit width of 30 microns was selected as a compromise between one which allowed lines of s a c i e n t width for measurement and one which did not cause excessive background. A slit length of 1 mm. was found satisfactory and practical in that it permitted taking 60 spectrograms on one 10 X 25 cm. (4 X 10 inch) plate. By simple shielding, the range of light from the densitometer could be readily narrowed to accommodate spectrograms of this width. The vanadium impurity in ordinary carbon electrodes excluded their use. Purified “Hilger spectro” vanadium-free copper electrodes were found unsatisfactory, since, a t the current used,
,
