concerned, the distribution coefficient decreases montonically, reaching a minimum a t Gd, and then increases slightly with increasing atomic number ( 3 ) . The slope d log K d / d M Mg(NO& decreases with increasing atomic number within the lighter rare earths. Therefore, one can expect the best fractionation of this group to occur in Mg(N03)2solution of the highest molarity, although limited to practically 3M ‘V 1.7). The separation factor of an adjacent pair of the lighter rare earths in 3 to 4M LiN03 was reported by Marcus and Kelson (Y), who gave B : l ~ Y 1.4. In Mg(N03)2 medium, :,CY a t 2M Mg(NOJ2 (equivalent in formality to 4M LiN03) comes out to be -1.5. Therefore, the separation factor of the rare earths in nitrate media appears to be independent of the nature of cations concerned, although the adsorption (Kd) varies considerably with the nature of cation, as demonstrated for Pr by Danon (2). The results given in Figure 1 should
permit the selection of an optimum condition for a given rare earths separation. With the anion exchanger-Mg(NO& system, good separation of the rare earths would be expected in the Mg(N03)Z solution of the highest concentration. I n Figure 2 the results of a typical separation of a mixture of the rare earths are shown. Because of the rather difficult separations involved, elution of the element to be removed first should be as sharp as possible so that the peak spreading is kept minimal. This is possible by decreasing the concentration of eluent a t the obvious sacrifice of the separation factor. The separation of a mixture of the rare earths, which differs in the atomic number by 2 or 3, is satisfactory as shown in the separation of E u and Nd, Pr and La, and Sm and Nd in Figure 2. However, the separation of adjacent pairs of the rare earths-Le., Pm-Nd and Eu-Sm-is less satisfactory using the conditions selected.
LITERATURE CITED
(1) Buchanan, R. F., Faris, J. P., Intern. At. Energy Agency, Vienna, Conf. Proc. 2. 262 f 1962\.
(1964). ( 5 ) Hamaguchi, H., Ohuchi, A., Onuma, Tu’., Kuroda, R., J . Chromatog. 16, 396 (1964). (6j-Ma;cus, Y., Abrahamer, J. Znorg. Nucl. Chem. 22,141 (1961). (7) Marcus, Y., Nelson, F., J . Phys. Chem. 63, 77 (1959). HIROSHI HAMAGUCHI~ KOJIISHIDA IKUKO HIKAWA ROKURO KURODA
Department of Chemistry Tokyo Kyoiku University Bunkyo-ku, Tokyo, Japan 1 Present address, Department of Chemistry, The University of Tokyo, Hongo,
Tokyo, Japan.
Estimation of Vanadium in Biological Material by Neutron Activation Ana lysis SIR: Activation analysis is a rapid and sensitive technique for the detection of vanadium in biological material. The main problem is the completion of an efficient separation within the time limit set by the rapid decay of the radioactive vanadium ( t l / s = 3.8 minutes). The initial destruction of the organic matrix is a step which consumes a considerable amount of time in rapid analytical procedures. This is especially true with hard tissues such as bone or tooth, where a destruction time of 10 to 20 minutes is often required, even when vigorous conditions are used (1, 3 ) . The following method allows counting of the separated vanadium within 8 to 10 minutes of removal from the reactor. Naturally occurring vanadium is 99.76% V51, which on irradiation with thermal neutrons produces V2. The cross-section for thermal neutrons is 4.5 barns and the V52 half-life is 3.8 minutes. Both p and y radiations are emitted, but detection by Geiger counting is preferred because of comparatively low background of this type of counter. There is the possibility of interference by the following first-order reactions: Cr52 (n, p ) V52 Mn55(n, CY) V6* These are only significant in matrices with high chromium or manganese
content and do not require a correction factor in biological materials. The radioactive tissue sample is destroyed in boiling sulfuric acid and the heavily charred solution is cleared by addition of nitrate. After scavenging and solvent extraction with cupferron, the vanadium cupferrate is counted in a Geiger counter accepting liquid samples, diluted to a standard volume, and its absorbance is measured. EXPERIMENTAL A N D RESULTS
Preparation of Samples. Tissue samples are vacuum-dried and receive no treatment before irradiation so t h a t contamination is kept t o a minimum. With hard tissue such as tooth, sections are used as obtained from a cutter, after rinsing with distilled water. The cutter is carefully protected against contamination. I t is necessary to investigate the cutting wheels to ascertain that they are not sources of vanadium contamination. The standard sample is prepared by evaporating a known weight (about 1 pg.) of vanadium from solution onto a piece of polyethylene sheet which has been washed thoroughly with concentrated nitric acid and distilled water. The evaporation is effected by heating under an infrared lamp so that the polyethylene is not damaged. The standard solution is prepared by dissolving “Specpure” vanadium pentoxide in distilled water. The samples and standard are packed in individual small poly-
ethylene envelopes which are heatsealed and irradiated together in l/2inch diameter polyethylene containers for 4 minutes a t a thermal neutron flux of 10l2neutrons/sq. cm./sec. Reagents. Where possible high purity reagents are used. Cupferron solutions (5% w./v.) are unstable, so only small amounts are prepared a t a time, and these can be kept in dark bottles in a refrigerator. Destruction of the Organic Matrix. The standard may be digested in the normal manner or, if preferred, it may be shaken with the vanadium carrier/ sulfuric acid mixture in the cold, when complete exchange is obtained. The technique chosen for digestion of the sample is as follows. A 1.5-mg. sample of vanadium carrier (0.5 ml. of a 0.05M ammonium vanadate solution) and one drop of molybdenum carrier (10 mg./ml.) are added t o 3 ml. of 1811.1 sulfuric acid, and the mixture is heated in a 125-ml. tall form silica beaker until the acid is refluxing on its walls about 1 inch above the surface. The piece of tooth or tissue, which weighs up to 0.5 gram, is dropped into the acid and digests in 60 to 90 seconds with heavy charring. The charring is removed by the addition of 20 or 30 mg. of sodium nitrate. The solution, which is green to yellow and sometimes contains a white precipitate of calcium sulfate, is cooled as follows: swirl the solution in the flask in air for 30 seconds; cool further by immersing the flask in boiling water for a few seconds; bring to normal temperature by immersion in an ice bath. VOL 37, NO. 10, SEPTEMBER 1965
1285
When the solution is cool, 30 ml. of water a t 0' C. are added, often resulting in an even larger precipitation of calcium sulfate if bone or tooth has been destroyed. It is not profitable to hold the calcium sulfate in solution a t this stage by the addition of sulfuric acid, because dilution to the required 10% acid concentration (4)for extraction immediately precipitates it. The acid volume chosen for digestion is the one which gives no loss in digestion time and results in the most reasonable volume for further handling. Separation. After digestion and during cooling in the ice bath, the solution is scavenged by adding two drops of 2M hydrochloric acid followed by five drops of silver nitrate solution (4%. w./v.). As the precipitation is carried out in the presence of excess chloride, the precipitate acts as a cation scavenger, removing elements which might be extracted in the separation. The AgCl precipitate is lost with the aqueous layer and wash water during the first cupferron extraction. The solution and precipitate are transferred to a 100-ml. separating funnel and extracted with 10 ml. of an cybenzoin oxime solution (1 mg./ml.) in chloroform. Decay curves of the activity separated from different biological
Table 1. Volume of Cupferron Required under Hard Tissue Digestion Conditions
Volume of cupferron (5% w./v.), ml.
Extraction color 1st extract 2nd extract Yellow-brown Red-brown Wine-red Yellow-brown Wine-red Pale yellow
1
2 3
Table 11. Variation in Recovery with Time of Standing in Chloroform
Time (minutes)
Absorbance
2
0.275
21 30 41
0.015
ii
Table 111.
Material NaV08 VOSOl
VOSOl SnCh
1286
+
0.115 0,040
0.015
Colors of Various Vanadium Cupferrate Extracts
Color of chloroColor of form cupferrate solution on Aq: in solution chloro- stzndcolor form ing Yellow Wine-red Yellow Wine-red Yellow Blue Yellow Blue Yellow
ANALYTICAL CHEMISTRY
samples without this molybdenum extraction step indicate the presence of Moiol (t1,2 = 15 minutes.) Tracer experiments prove that up to %yo of added molybdenum goes through the separation. The solvent extraction technique is introduced to remove molybdenum and does this efficiently, so that no molybdenum is detectable after extraction. Subsequent decay curve measurements on the activity separated from the same biological samples as above, with the molybdenum extraction included, show removal of that section of the decay curve attributable to RfolO*. The extract is discarded. A 3-ml. portion of ammonium cupferrate solution (5y0 w./v.) and 15 ml. of chloroform are added. The funnel is shaken vigorously to extract the vanadium as the cupferrate into the chloroform layer, which turns deep wine-red in color. I t is possible to tell if the extraction is incomplete by the color of the cupferrate/chloroform extract. If it is light brown the recovery is low; and if red-brown, the recovery is high. A colorimeter with a filter of maximum transmittance a t 430 mp showed that the minimum volume of cupferron solution (5y0w./v.) required to extract all the vanadium is 0.5 ml. The extraction of the 1.5 mg. of vanadium carrier is into chloroform from 10 ml. of 10% sulfuric acid. The extracts are made up to 100 ml. with chloroform. The volume of cupferron sufficient for complete extraction of vanadium in the absence of a piece of hard tissuee.g., tooth-is not sufficient when this tissue is present. This suggests a change in the state of the vanadium to a form which is much less extractable. This is demonstrated, when the cupferron is added, by the formation of a yellow precipitate rather than a red one. Table I shows the results obtained by extracting 1.5 mg. vanadium carrier from the solution obtained by the digestion of 0.2-gram tooth samples, using varying amounts of cupferron. Two complete extractions, including the addition of cupferron, are made of each sample with 15-ml. portions of chloroform. The color of the extract is an indication of the completeness of the extraction-the more red the solution, the more complete the extraction. The excess of cupferron converts the much less soluble yellow form to the more soluble brown form which extracts to give the wine-red solution. Hence, in the separation, 3 ml. of cupferron solution are used. The chloroform layer is washed in a separating funnel containing 15 ml. of water and is then run into a clean separating funnel containing 10 ml. of l M sodium hydroxide and two drops of (ethylenedinitri1o)tetraacetic acid (disodium salt) solution (1% w./v.). The EDTA is used to remove the last or CuG6which may have traces of been extracted by the cupferron. Willard (6) has used this technique in the determination of vanadium with cupferron in the presence of copper.
The funnel is shaken vigorously until the chloroform layer is pale olive in color. This layer is discarded and the aqueous layer washed with a further volume of chloroform, which is discarded. A further 10 ml. of fresh chloroform are added and the aqueous layer is made acid with 2df hydrochloric acid (6-7 ml.) ; the vanadium cupferrate is re-extracted into the chloroform layer and washed as before. At this stage sufficient radiochemical purity has been attained, so the solution is used directly for counting and colorimetry. The resulting chloroform layer is run into a Geiger tube accepting liquid samples and counted. The chemical yield is estimated by making the chloroform solution from the Geiger tube up t o 100 ml. and measuring its absorbance a t 400 mp on a spectrophotometer, using 1-cm. quartz cells. The figures obtained are corrected for recovery and decay and the results obtained by comparison of samples and standard. The recovery may be poor if the chloroform solution resulting from the first cupferron extraction is not treated with sodium hydroxide immediately. It has been suggested that the color change of the vanadium cupferrate in chloroform solution (from M ine-red to yellow) is due to reduction of 5-valent vanadium by the chloroform (2). I t is found that standing for various times so that a change from the wine-red takes place results in lon-er recoveries, as shown in Table 11, where the recoveries are proportional to the absorbance. This is due to the nonextraction into sodium hydroxide solution of the yellow vanadium complex. To examine this point further the following experiments were made : Five-valent vanadium (NaVOa) was extracted as the cupferrate into chloroform. Four-valent vanadium (VOSO,) was extracted as the cupferrate into chloroform. Four-valent vanadium (VOSO,) was extracted as the cupferrate into chloroform in the presence of stannous chloride. The results shown in Table I11 indicate that vanadium(1V) is readily oxidized to the five-valent state and extracted to give a wine-red color. On standing the wine-red color changes to yellow fairly rapidly as shown in Table 11, or immediately if a small amount of stannous cupferrate in chloroform is added. The absorbance measurements for the recovery correction are made on the yellow-colored solution. DISCUSSION
The application of activation analysis to vanadium microestimations allows gram to amounts of the order of be measured. As the half-life is relatively short, this sensitivity is that obtainable when activity mensuremcnt s are made after removal from the reactor. A series of trials were made on
.
solutions of known vanadium content, covering the range 10-9 gram to 10-7 gram* The error found was within the range &5%. In similar trials on biological material the range proved to be the same. The method eliminates blanks other than the standard sample which gives the specific activity of the vanadium. Microseparations are avoided by adding inactive vanadium carrier in convenient amounts. Samples of hard and soft tissue may be analyzed within a reasonable time after irradiation SO that the sensitivity is not lost by decay.
ACKNOWLEDGMENT
The authors thank the Scottish Research Reactor Center staff for the use of their laboratories and irradiation facilities. They further thank professor Gilbert Forbes and Dr. George Nixon for support and encouragement during the of the work.
LITERATURE CITED
& ? ~ ~ ~ $(I;&&’ ,
( ‘ ? ~ ~ ~ . A H ~ e ~ ~ . (2) Crowther, P., Kemp, D. M., Anal. Chim. Acta 29, 97 (1963).
(3) Fukai, R., Illeinke, W. W., Limn. Oceanogr. 4, 398 (1959). (4) Furman, N. H., Mason, W. B., Pekola, J. S.,ANAL.CHEM.21, 1325
(1949). ( 5 ) Willard, H. H., Martin, E. L., Feltham, R. Ibid., 25, 1863 (1953).
H. D. LIVINGSTON HAMILTON SMITH Department of Forensic Medicine Glasgow University Glasgow W.2, Scotland H. D. Livingston received financial support from the Scottish Hospitals Endowment Research Trust.
Modified Kjeldahl Determination of Nitrogen in Potassium Metal the method may be an indication that nitrogen has been removed from the metallic material. No matter whether this is due to KIN or interstitial nitrides originally in the metallic materials, the ammonia test would result and confirm some mass transfer mechanism. The procedure, applicable only to reducible nitrogen, involves transfer of the potassium from a special extrusion sampling system to a unique distillation apparatus under argon and hexane. The alkali metal is then reacted with phosphoric acid under the hexane, converting potassium to the phosphate and any nitride nitrogen as well as some other possible compounds of nitrogen to ammonium phosphate because of the
SIR: A modified Kjeldahl method has been developed for the determination of nitrogen in potassium. While stable potassium nitride seems unlikely to be found in potassium, other metallic nitrides or nitrogenous compounds may be expected. Such nitrides may arise from the containment material during purification steps or after exposure to them a t elevated temperatures. If only non-nitrogen bearing containment materials are used and ammonia is detected, KIN is assumed to be present (assuming the absence of lithium impurity). If nitrogen-bearing metallic materials for containment were used, the detection of ammonia when the potassium is dissolved and treated by
.t-MLCRO BURET
CONNECTIOU TO TUBING
nascent hydrogen. The hexane is then distilled off and discarded. Next, a known excess of sodium hydroxide solution is added. This converts the nitrogen present to free ammonia which is then steam distilled from the sample solution into a receiver flask containing excess boric acid solution to neutralize the ammonia. Nessler’s reagent is added to the flask to form the characteristic yellow-brown colored solution. Final determination of the amount of nitrogen is made spectrophotometrically. To determine the weight of potassium sample used, a method based on flame photometric measurement of the element in the spent solution was developed. ilccuracy within 5y0 and excellent reproducibility were demonstrated by adding known amounts of nitrogen in the form of ferrous ammonium sulfate a t levels of 100, 50, 25, 12.5, and 7.5 p.p.m. Reagent blanks were in the order of 12 to 15 p.p.m. of nitrogen. Nitrogen was determined in four samples of potassium with results in the range of 3 p.p.m. One procedure is recorded (2) for the direct addition of water to lithium to form the corresponding alkali hydroxide and to liberate ammonia directly. Obviously, only nitride nitrogen is involved. EXPERIMENTAL
+3,8C+
+ 6.4cm t+ Figure 1.
Distillation assembly for nitrogen in potassium
The apparatus simulates that used for the standard method for analysis of nitrogen in titanium (1). In this procedure only a small volume of solution needs to be distilled to effect complete separation of the ammonia. The main difference is in the original design of the reaction flask which is shown in Figure 1. The purpose of the large No. 50 “ 0 1 7 ring is for the attachment to a special sampling apparatus beyond the scope of this letter. Chemically pure ammonium chloride, NH4C1, was used for all standardization VOL. 37, NO. 10, SEPTEMBER 1965
1287
