Quantitative Determination of S-35-Labeled Compounds In Complex Mixtures JACKLYN B. MELCHIOR and ARTHUR H. GOLDKAMP Department o f Biochemistry, Graduate schoo/ and Stritch School o f Medicine, L o y o h University, Chicago, ///.
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which weighed about 100 mg. Up to 2 ml. of liquid can be placed in such planchets conveniently.
S THE determination of the radioactivity of solid samples
which emit weak beta particles, it is necessary to consider the absorption of radioactivity by the precipitate itself. Studies of this problem have been carried out with compounds of carbon-14 ( 1 ) and sulfur-35 ( 3 ) . Useful methods for correction of such data for absorption have been presented by Yankwich et al. ( 7 ) . I n general, the final step in the preparation of samples consists of conversion to R single pure substance such as carbonate or sulfate, followed b y precipitation in a standard manner as an insoluble substance. I n many metabolic experiments employing tracer techniques, it is possible to isolate the labeled species from all other labeled suiistances without purifying it. Thus one may have a single labeled amino acid in a solution containing an unknown mixture of tissue component>-as well as inorganic salts. At this point it is tempting to carry out the radioactive determination by evaporating the misture in a planchet and counting directly. Hogncss et al. (4)have described this procedure for use with biological fluids of relatively comtant composition, in which case an absorption curve can be prepared for each set of conditions. The absorption is not necessarily simply a function of the total weight of the sample when this method is used. Serious errors can occur if the curve prepared under one set of conditions is used to interpret data collected under other conditions, such as a change in a buffer or other alteration in the make-up of the medium used. This report concerns an attempt to find a simple procedure for the direct comparison of radioactivity in heterogeneous mixtures containing a single radioactive substance, but avoiding the necessity of preparing a correction curve for each change in variable. The method adopted is a modification of one suggested by Leslie [described by Calvin et al. (1, page 107)], and consists of using a piece of paper in the bottom of the planchet in which the evaporation occurs. With this method it is possible to prepare a master curve for the correction of observed radioactivity as a function of tot,al weight of sample added to the paper, and still stay within the limits of error allowable in many metabolic esperimen t P .
RESULTS
Some preliminary experiments were carried out i n which the solution was evaporated to dryness on a piece of paper hanging in the air. This procedure has been used for carbon-11 determinations by Ruben et al. (6) and has also been used for carbon-14 ( 5 ) . It is dangerous unless partirular precautions are taken to control the rate of evaporation. When a fan or lamp was used to speed evaporation, differences of over 100% were observed in the counting rate of the two sides of the paper. Hence this procedure has heen abandoned as impractical for routine work.
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MILLIGRAMS
SALT
Figure 1. Effect of Inert Additions on Counting Rate of S-35-Labeled Methionine Samples prepared by evaporation on aluminum planchets (Method A). Each point represents average of 10 separate samples
METHODS
All the results reported here were obtained with a sample of S-%-labeled I)x.-methionine, specific activity the order of 19 millicuries per gram, n-hich was obtained from D. L. Tabern of the hbbott Laboratories. About millimole of methionine was used as the labeled addendum in the following measurements, and the activity of this in absence of any other addition wa,s considered to be 100% under the conditions used in each case. Radioactivity determination8 were made with a proportional counter and scaler, using an internal counter tuhe filled with methane, .Ul samples were counted for a sufficient time to record 1000 count6 over the background. Two methods were used in preparing samples for the determination of counting rate. Method A. The samples were evaporated to dryness a t room temperature in aluminum planchets of 3.6-em. diameter. The radioactive amino acid and other substances were added as suitable aliquots of stock solutions, and the final volume was adjusted so that the original dept.h was the same in all planchets. Method B. The samples were prepared as described above. However, the planchet's were constructed of Parafilm (Grade 11, obt,ainablefrom the Marathon Corp., hlenasha, \Vis.) bv pinching the corners to make a plastic dish 3 cm. square. T h e bottom of this dish wae lined with a square of Whatman S o . 1 filter paper,
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Absorption Curves for Method A. With this procedure absorption curves were run using barium chloride and sodium phosphate as inert additions. It is obvious from the curves obtained (Figure 1) that the corrections to be applied to the observed counting rate depend upon the nature of the absorbent as well as the total weight of samples. This is shown also by curve 2 of Figure 2, the self-absorption curve for methionine, which is unlike either of the curves of Figure 1. This serves to emphasize the necessity for preparing a correction curve for each set of conditions if data obtained by direct evaporation of heterogeneous samples are to be interpreted correctly. Self-Absorption. The self-absorption by methionine was found to be differentfor the two methods, as is evident from the curves in Figure 2. Bddition of methionine when the filter paper was present (-Method B) had no effect other than a slight increase in counting rate until over 5 mg. of methionine had been added. Above this point the absorption folloryed a curve similar to that observed when the solution was evaporated in the absence of paper (Method A ) . It is significant that in all the samples containing 10 mg. or more of methionine, crystals of the compound were clearly visible on the top of the paper. Thus it is concluded that the observed counting rate of samples prepared by Method B is essentially independent of the specific activity of the sample as long as conditions are maintained which preclude the formation of crystals on top of the paper.
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ANALYTICAL CHEMISTRY
Absorption Curves for Method B. Substances other than the radioactive substance itself exerted a definite effect on the counting rate, which appeared to decrease logarithmically as the total weight increased. An absorption curve for a number of samples prepared with varying amounts of monosodium phosphate is shown in Figure 3. I n Figure 4 the effect of a wide variety of materials on the counting rate is shown. The mean deviation of these points from the phosphate curve is 3.7%. It is evident that the effect of a given weight of these substances on the counting rate of samples prepared by Method B is relatively constant.
Table I.
Table 11. Effect of Area of Paper on Counting Rate of Samples Prepared by Method B (Each value represents an average of ten determinations) .4rea, Square Inches Counts per Minute 1.56 1051 0.56 1356 0.14 1825
Effect of Cupric Ion on Counting Rate of Samples Prepared by Method B
(Each value represents average of 10 determinations) Sdditions Mg. % Total Activity CuClr.2HzO 9 82.0 6 Glycine 87.3 9 CuClz.2HoO Glycine 6 Calculated" 69.3 Observed 83.0 CuClz.2H10 9 90.0 HC1 0 . 1 meq. Calculated on assumption that effects of cupric chloride and glycine are additive.
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Effect of Complex Formation. The effect of cupric ion, known to form complexes with amino acids (8), was investigated. The data presented in Figure 5 indicate anomalous behavior in the presence of copper. It s e e m likely that the complex formation alters the distribution of the methionine in the paper. This is further emphasized by the data in Table I, where the effect of the cupric ion is reversed by adding an excess of glycine to compete for the copper ion, or by making the solution acid, which reduces complex formation. Both treatments brought the counting rate up to that expected on the basis of Figure 4.
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Figure 3.
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Effect of Sodium Phosphate on Counting Rate of S-35 ,Methionine
Samples prepared by evaporation in plastic planchets lined with filter paper (Method B)
the counting rate by less than a factor of 2. Thus the limits of paper area are wide for all practical purposes. Effect of Total Activity. The observed counting rate of samples prepared by Method B is linear with respect to the actual number of disintegrations, This is shown in Table 111, where the amount of labeled methionine was varied over a factor of 10. DISCUSSION
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The reason for the logarithmic relationship shown in Figure 3 is not clear, as there is evidence that the distribution in the paper is not uniform. In general, if the papers are lifted out of the planchets and counted, the upper side is about 10% more active. In one experiment five identical sheets of paper were stacked in a planchet in place of the usual single sheet and counted separately I
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Figure 2.
METHIONINE
Self-Absorption Curves for Methionine
1. Samples prepared by evaporation in plastic planchets lined with filter paper (Method B) 2. Samples evaporated i n aluminum plancheta (Method A). Each point represents average of 10 samples
Effect of Area of Paper. In these experiments samples were prepared by Method B, but the area of the plastic planchet was altered. In each case an identical amount of methionine was added. Water was added to the larger planchets so that the depth of the liquid was the same in each planchet a t the outset. The data presented in Table I1 show the observed counting rate to increase a.s the planchet size is diminished. However, a decrease of over tenfold in the size of the paper is seen to increase
M ILL1G RAMS
Figure 4. Effect of Additions on Counting Rate of Samples Prepared by Method B Smooth curve taken from data on NaHaPO4.HzO. Each point is average of 10 samples. Milligram axis refers to total weight of solids. Forms present were: BaClg.2HzO CaUi 3HzO MnClt.SHz0 SrCl2.6H2OO, CdCiz, MgCit.bHid, and NiClz.b$@
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V O L U M E 2 6 , N O . 1, J A N U A R Y 1 9 5 4 Table 111. Effect of Total Activity S-35-Labeled Methionine, Micromole 0.0176 0.00878 0.00351 0.00176
Observed Specific Activity Counts/ Min./Mioromole 166 142 165 156
Counts/Min. 2925 1243 580 275
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MILLIGRAMS
cUC12 2H20
Figure 5. Effect of Copper on Counting Rate of Samples Prepared by Method B
on planchets. The standard error for a mean calculated from triplicate determinations is less than 4% by either method. This compares favorably with the precision obtained after the more tedious procedure involving conversion to barium or benzidine sulfate ( S ) , particularly as such methods require at least two additional transfers of small amounts of materials. The less desirable features of this method of sample preparation include the absorption of radiation by the paper. The counting rate of samples prepared by Method B was approximately 20% of that observed with identical samples prepared by Method .4. The availability of compounds of high specific activity makes this loss permissible in many procedures. It is also obvious that the independence of counting rate and specific activity can introduce errors if large amounts of the labeled species are present and the total weight of sample is not suitably corrected. Finally, any factor, such as complex formation, which alters the movement of the labeled species through the paper, can lead to erroneous results. I n practice, these sources of error can be generally avoided in designing the experiment. The chief advantage is the relatively small and constant alteration in counting rate introduced by other components of the mixture in which the radioactivity is to be determined. The changes in absorbers tested represent more drastic changes than would normally occur in practice. This would seem to make this a satisfactory method for the direct comparison of radioactivity in heterogeneous mixtures.
Each point is average of 10 samples
after drying in the usual manner. In this case the counting rate for the upper side of the sheets, stdrting with the top one, was 7 4 i , 192, 121, 131, and 380 counts per minute, respectively. Although this procedure introduced the additional variable of a series of interfaces between the sheets, the results serve to indicate that the active species moves both up and down through the paper as evaporation proceeds. I t aould seem that the equations derived by Henriques et al. for solid precipitates do not apply to this process (S). Presumably the actual rate of movement depends on a variety of factors, including temperature. I t is significant that when water was added to samples and permitted to re-evaporate under the same conditions, no marked effect on the counting rate was observed. I n spite of the complexity of the process leading to the distribution of the labeled substance in the paper, the precision obtained is a t least as great as that observed with direct evaporation
LITERATURE CITED
(1) Calvin, &I., Heidelberger, C., Ried, J., Tolbert, B., and Yankwich, P., “Isotopic Carbon,” New York, John Wiley & Sons, 1949.
(2) Greenberg, D. ll., “Amino .kcids and Proteins,” Springfield,
Charles C Thomas, 1951. (3) Henriques, F. C., Kistiakowsky, G. B., Nargoretti, C., and Schneider, W. G., IND.ESG. CHEM.,ANAL.ED.,18, 349 (1946). (4) Hogness, J. R., Roth, L. J., Leifer, E., and Langham, W. H., J . Ant. Chem. S ~ C 70, . , 3840 (1948). (5) Le Page, G. A., and Heidelberger, C., J . Bid. Chern., 188, 593 (1951). (6) Ruben, S., Hassid, W. Z., and Kamen, 31.D., J . Am. Chena. SOC. 61, 661 (1939). ( 7 ) Yankwich, P., Sorris, T., and Huston, J., -4v.k~.CHEM.,19, 439 (1947). RECEIVED for review May 15, 1953. .4ccepted August 17, 1953. Supported by a grant-in-aid from The American Cancer Society upon recommendation of the Committee on Growth of the National Research Council.
Determination of Nitro Nitrogen by the Kjeldahl Method R. B. BRADSTREET The Bradstreet ~!aboratorks, lnc., 7356 North Broad St., Hillside,
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H E Kjeldahl method ( 4 , is not applicable to every form of organic nitrogen. However, owing to the comparative ease of operation, many attempts have been made to generalize the method. Treatment of nitro conipound.5 with phenols to convert them into a more easily reducible form was first suggested by Jodlbauer ( 3 ) . Later, Cope ( 2 ) substituted salicylic acid for phenol. Complete recovery of the nitrogen in nitro and other so-called refractory compounds is not always accomplished by the use of these, even in conjunction with sodium thiosulfate. The generally accepted Procedure for nitro compounds uses 5 grams of sodium thiosulfate and 10 grams of potassium sulfate a boiling point raiser. The severity of this reaction is increased by using larger amounts of potassium sulfate, which has been shown by other investigators (5,6). Using p-dinitrobenzene as a typical compound whose nitrogen is not readily available, it
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was determined that 18 grams of potassium sulfate was the optimum (see Table I). EFFECT OF INCREASED DIGESTION TERIPER.4TURES
T~ demonstrate the difference between the use of the conventional 10 grams of potassium sulfate and 18 grams, a number of compounds were using the follon+ng procedure: Weigh into a Kjeldahl flask 0.1 to 0.15 gram of sample and 35 ml. of concentrated sulfuric acid containing 1 gram of salicylic acid. Let stand for 0.5 hour on the steam bath, or until the sample has completely dissolved. Transfer to the digestion rack and add 5 grams of sodium thiosulfate. Let stand for 0.5 hour and then heat gently until the mixture carbonizes. Cool and add potassium (I), and 0.25 gram Of mixed (FeSOa.iHzO-Se). Heat strongly until the mixture clears, and boil gently for 1 hour, Coo], dilute wit,h distilled water, determine the nitrogen in the usual manner.
