Microdetermination of Nitrogen in Seed Protein Extracts with the Salicylate-Dichloroisocyanurate Color Reaction Peter Felker Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan 48824
With the advent of new protein and amino acid microtechniques such as fluorimetry, GLC, and electrophoresis, it has become desirable to develop a total nitrogen assay of comparable sensitivity. A number of micromethods for total nitrogen employing the salicylate-dichloroisocyanurate-nitroprusside color reaction with NH3 have been described (1-5). The previously described methods determine the color from an aliquot of the diluted digest either manually or with a “Technicon Autoanalyzer”. A manual method is here reported, for those laboratories not having an autoanalyzer, in which the color development is performed on the entire digest in the digestion flask, thus avoiding dilutions, washings, and transfers. The useful range of this assay is from 0.5 to 5 pg total N in the sample.
EXPERIMENTAL Reagents. The salicylate reagent contains 2.12 g of sodium salicylate and 15 mg of nitroprusside in 25 mL of doubly distilled HzO. The dichloroisocyanuratereagent contains 50 mg of sodium dichloroisocyanurate (Eastman Chemical Co.) in 10 mL of 1 m sodium phosphate buffer pH 12.5. This pH, previously considered optimal ( 4 ) , is close to the optimum of 12.8 to 13.0 reported subsequent to the completion of this study (5). The phosphate buffer is prepared by mixing 84 g of Na2HPO4with 152 g of Na3P04.12H 2 0 per liter of water and adjusting to pH 12.5 with NaOH. Indicator paper (SpezialindikatorpH 11.&13.0, E. Merck, Des Plaines, Ill.) is used to check the pH of the phosphate buffer to avoid electrode error at pH 12 with 1 M sodium ion. Procedure. Up to 100 WLof nitrogen containing solution and 100 WLof concentrated H2S04are pipetted into the digestion flasks (5136422 freeze drying ampule, A. H. Thomas Co., Philadelphia, Pa.). The contents are mixed on a vortex mixer and the digestion flasks placed on a cold micro-Kjeldahl digestion apparatus (Aminco 4-1851) and heating is begun. Bumping may occur on a preheated apparatus. Digestion is for only 10 min from the time fumes first appear in the flasks, since longer heating times with the above described flasks cause loss of “3. The flasks are allowed to cool and 1.0 mL of water is added followed by 2.0 mL of 1.0 M phosphate buffer pH 12.5. The flasks are then stoppered with serum cap stoppers (8753-D22, A. H. Thomas Co., Philadelphia, Pa.) as the next step will cause the pH to rise above the pK, of NH3, thus converting nonvolatile (NH&S04 into volatile NH3. A 0.5-mL solution of 30% NaOH is then added through the serum cap with a 1.0-mL plastic tuberculin syringe equipped with a 3-inch, 22-gauge needle. After allowing the flask to cool, 0.4 mL of the salicylate reagent, followed by 0.3 mL of the dichloroisocyanuricacid reagent, is added with minimal mixing in quick succession with the aid of two prefilled tuberculin syringes. The flasks are rotated by hand to mix the contents and are then placed in a reciprocating metabolic shaker in water at 37 OC for 20 min. After cooling, the absorbances are determined with a Gilford 240 Spectrophotometer at 660 nm against a reagent blank. RESULTS A N D DISCUSSION The method presented here has several advantageous features. First, the analysis can be accomplished directly on the entire protein digest, thus requiring less sample; secondly, the use of a buffer permits pH control even in a manual micromethod; and last, a manual method is better adapted
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to those laboratories needing only an occasional nitrogen assay on a microgram scale. Standard curves should be prepared daily. With glycine standards, containing 0 to 5 pg of nitrogen, we obtained a straight line plot of Am vs. pg of nitrogen with a slope of 0.255 and an intercept of zero. There was an average of f 4 % error for seven duplicates of seed protein extracts. Phosphate buffer (pK, = 12.7) was used to maintain the p H in this assay. As color development has been shown to be pH dependent ( 4 , 5 ) ,care should be taken in pipetting the NaOH, H2S04,and phosphate buffer, as the volumes and concentrations have been chosen to stoichiometrically neutralize and buffer the H2S04acid digest. It was necessary to make the manual assay a closed system, as is the automated system, with the use of serum caps in order to minimize losses of NH3,and to prevent absorption of COzwhich would lower the pH. Different concentrations and proportions of the salicylate and dichloroisocyanurate reagents were tried before the present procedure was adopted. The salicylate and dichloroisocyanurate reagents should be made fresh daily and this is especially true for the dichloroisocyanurate reagent. Because of the small size of the digestion flasks and the small volume of sulfuric acid (0.1 mL) used, vigorous boiling was obtained in less than 30 s and clearing was often obtained in 5-7 min with seed proteins. A digestion time curve of glycine had a maximum a t 5 and 10 min with lower values at 15,30, and 60 min using the procedure described here, and therefore a digestion time of 10 min was adopted. Longer digestion times might give higher results for some proteins and heterocyclic nitrogen compounds. For 30 pg or less of seed protein (which yields the maximum useful color development), a 10-min digestion time is satisfactory. The color development was identical at 45 and 60 min at 37 O C with 84%, 95%, and 99% of color development being reached a t 11,20, and 30 min. A heating time of 20 rnin was used for routine analysis. More recent work (5), suggests the ,A, is 666 nm rather than 660 nm as previously reported ( 4 ) , and used here, but little loss of sensitivity would be incurred because of the flat broad absorption maxima. As the upper limit of the assay is approximately 30 pg of protein, an amount which is difficult to weigh, the assay is most useful in analyzing the nitrogen content of fluids. ACKNOWLEDGMENT The author is indebted to Robert S. Bandurski for his council and advice and to Brenda Goucher for her help. LITERATURE CITED (1) J. Reardon, J. A. Foreman, and R. L. Searcy. Clin. Chim. Acta, 14, 403-405 (1966). (2) A. R. Fraser and J. D. Russell, Clay Miner., 8, 229-230 (1969). (3) W. M. Crooke and W. E. Slmpson, J. Sci. FoodAgric., 22, 9-10 (1971). (4) J. A. Bletz, Anal. Chem., 48, 1617-1618 (1974). (5) R. V. E. Pym and P. J. Milham, Anal. Chern., 48, 1413-1415 (1976).
RECEIVED for review December 27,1976. Accepted February 22, 1977. This work was supported by a National Science Foundation Grant (GB-40821-X) to Robert S. Bandurski.
