ACKNOWLEDGMENT
We gratefully acknowledge the assistance of S.P. Rogovin, who supplied the samples of polysaccharides recovered by pilot-plant-scale precipitation with QN salts. +
LITERATURE CITED
(1) Albrecht, W. J., Rogovin, S. P., Griffin, E. L., Jr., n’alure 194, 1279
(1962). (2) Albrecht, W. J., Sohns, V. E., Rogovin, S. P., Biotechnol. Bioeng. 5, 91 (1963). (3) Anderson, E. K., Acta Chem. Scand. 8 , 157 (1954). (4) Auerback, M. E., IND.ENG.CHEM., ANAL.ED. 16, 739 (1944). ( 5 ) Cadmus, M. C., Lagoda, A . A,, Anderson, R. F., A p p l . Microbiol. IO, 153 (1962).
(6) Cunningham, K. G., Dawson, IT., Spring, F. S., J . Chem. SOC.1951, 2305. (7) Fogh, J., Rasniussen, P. 0. H., Skadhauge, K., A i i . 4 ~ . CHEM. 26, (j 19,541. -__-,.
(8) Jasinski, T., A c t a Polon. Phnrm. 14,
45 (1957). (9) Jeanes, Allene, Pittsley, J. E., Senti, F. R., J . A p p l . Polymer S c i . 5, 519 (1961). (10) Jeanes, Allene, Pittsley, J. E., Watson, P. R., Ibid., 8, 2775 (1864). (11) hletcalfe, L. D., ANAL.CHEx 32, 70 (1960). (12) Mukerjee, P., Zhid., 28, 870 (1956). (13) Rogovin, S. P., Anderson, R. F., Cadmus. 31. C.. J. Biochem. Microbiol. Technol.’Enn. 3: 51 11963). (14) Siele, \-. I., Picard, J. P., A p p l . S p e c t r y . 12, 8 (1958). (15) Sheinker, Yu. N.,Golovrier, B. hl., Izvest. Akad. .Vauk S.S.S.R. Ser. Fiz. 17, 681 (1953). Y
Z
(16) Scott, J. E., Chem. I d . (London) 1955. 1568. (17) -SCott,J. E., “FIethods in Biochemical Analysis,” David Glick, ed., Vol. 8, p. 14.5, Interscience, Nerv York, 1960. (18) Sloneker, J. H., Jeanes, Allene, Can. J . Chem. 40, 2066 (1962). (19) Zj.ka, J., Phnrmazie 10, 170 (1955). RECEIVEDfor review July 23, 1964. Accepted December 14, 1964. Division pf Carbohydrate Chemistry 148th ?\leeting, ACS, Chicago, Ill., August 1964. The Northern Laboratory is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture. The mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over other firms or similar products not mentioned.
Spec t rophoto metric Determina ti o n of Pyra z o Ie with Trisodium Penta cya no a mino ferra te T. A. LARUE Prairie Regional laboratory, National Research Council, Saskatoon, Saskatchewan, Canada
b A rapid spectrophotometric determination of pyrazole is described. It is based on the absorbance a t 458 mp of an acid-stable, yellow complex of pyrazole, pentacyanoaminoferrate and nitrite as an oxidant. lndazole and some substituted pyrazoles give similar reactions. P(pyrazoly1-N)alanine does not form a colored complex. This amino acid and the constituents of several nutrient media do not interfere with the determination of pyrazole in bacterial broths. The procedure has a lower limit of sensitivity of 0.04 pmole of pyrazole.
P
is a precursor of @ (pyrazolyl-x)-alanine in melon seeds (3) and is a product of the microbiological degradation of this amino acid (16). Pyrazolyl-alanine is the only known naturally occurring derivative of pyrazole, and these compounds have only been reported in melon seeds. The study of the distribution and metabolism of these compounds has been hampered by the lack of a sensitive and specific assay for pyrazole. Preliminary to studies on the microbiological degradation of the amino acid, we required a method of analysis for pyrazole which would not suffer interference from pyrazolyl-alanine or the complex mixtures added to media to provide microbial growth factors. Since pyrazole contains a nitrogen-nitrogen linkage, we tested a reagent which has been used for hydrazine and its derivatives. Feigl, Anger, and Frehden (6) reported that trisodium pentacyanoaminofrrrate (TPF) formed a red comYRAZOLE
246
ANALYTICAL CHEMISTRY
plex with hydrazines. This reaction has since been used in the analysis of isonicotinic acid hydrazide (14) and unsymmetrical dimethyl hydrazine ( I S ) . Fearon (4, 6) found that the reaction required the oxidized form of the reagent. He reported that the ferrate (111) could be obtained by exposing the reagent to air or treating with an oxidant, and that the product with hydrazines was stable in dilute acetic acid. Fearon noted that similar colors were produced by amidines and guanidines, and suggested that these reactions were due to the presence of a hydraziring formed by the closure of the nitrogens in these compounds :
/“, h
--
----+ R\/:H
R--c H
c l
H/\NH
However, he did not test the specificity of the reagent with heterocyclic compounds containing a nitrogen-nitrogen bond. Results reported here indicate that though not all heterocyclics containing a nitrogen-nitrogen bond react with TPF, the reagent does form a colored complex with pyrazole. The reaction is sufficiently sensitive and specific to serve for the analysis of the compound in bacterial media. EXPERIMENTAL
Apparatus. A Beckman DK-2 spectrophotometer with 1 .O-em. cells was used to obtain absorption spectra.
Routine analyses were done with a Beckman DU spectrophotometer. Reagents. Pyrazole (hldrich Chemical Co. Milwaukee, Wis.) w a s recrystallized from benzene. d standard solution containing 1.0 pmole per ml. was prepared daily by dilution of a 0.1M aqueous solution. The stock solution was stored in the cold and remained stable for six months. Aqueous solutions of 0.2Oj, trisodium pentacyanoaminoferrate (Fisher Scientific Co., Pittsburgh 19, Pa.) and 10% N a N 0 2 were prepared immediately before use. Preparation of Standard Curve. The standard pyrazole solution was added in amounts from 0.1 to 1.5 ml. to test tubes containing 1.0 ml. of 0.2% TPF. Water was added t o bring the total volume to 3.0 ml. A tube containing no pyrazole acted as the reagent blank. With mixing, 0.1 ml. of KaNO2 was added followed immediately with 0.1 ml. of glacial acetic acid. The absorbance was read at 458 mp. d plot of absorbance us. concentration yielded a straight line intersecting the origin. This served as a standard curve for the determination of pyrazole in the range 0 to 1.5 pmole per sample tube. Determination in Microbiological Media. The sample was centrifuged
to precipitate bacterial cells. If the supernatant was cloudy, it was filtered through a G7 filter (Gelman Inytrum a t Co., Ann Arbor, Mich.). A portion of clear supernatant containing 0.1 to 1.0 pmole of pyrazole wah added to 1.0 ml. of 0.2yGT P F and treated as described above. The concentration of pyrazole w&< found b). comparison with the standard curve.
I
I
W
z 4
cc v)
m
4
::[ 0
I
I
I
I
350
400
450
500
WAVELENGTH
Figure 1 .
Absorption spectra
A. 0.1% TPF in water 6. Product of reaction of 1 pmole pyrazole with pentacyonoaminoferrate, N a N 0 2 , and acetic acid
RESULTS
The absorption spectra for the yellow complex of pyrazole and TPF exhibited a different spectrum than TPF itself (Figure 1). The reagent blank was almost colorless. The yellow color formed immediately after mixing the reagents, and was stable for several hours. A molar absorptivity of 2300 was calculated. Heating the reaction mixture 10 minutes a t 60" C. did not increase the absorbance. Without the addition of nitrite, the color formed only slowly. Ammonium persulfate, potassium ferricyanide and potassium periodate were tested as oxidants. When these were used pyrazole formed a yellow color with TPF, but the blank was colored. The procedure described above also produced acid stable colors with indazole, 3 - amino - 4 - cyano pyrazole, 3methyl pyrazole, 4 - bromo - 3 - methyl pyrazole, 4 - bromo pyrazole, 3, 5 - dimethyl pyrazole and 4-bromo-3,5-dimethyl pyrazole (Figure 2). N-Hydroxy-methyl pyrazole and l-hydroxymethyl-3,5-dimethyl pyrazole produced colors indistinguishable from those with pyrazole and 3,5-dimethyl pyrazole; the hydroxy-methyl group was probably split off in the procedure. Colors formed with 4-amin0-3~5-dimethylpyrazole, 3,4-diamino-5-hydroxy pyrazole and 4 - amino pyra~ole(3~4-d)pyrimidine faded after addition of acid. No color was produced by 1-phenyl-3-methyl pyrazole or b(pyrazoly1-N)-alanine. When 0.1-ml. samples of solutions containing 0.05% pyrazole and 0.5% of pyrazolyl-alanine or various nutrient media (Difco) were tested, it was found that pyrazolyl-alanine did not interfere with the assay, and that pyrazole could be determined in the presence of ten times its weight of nutrient media (Table I). The nutrients when tested alone produced only a slight absorption. Analysis of the nutrients showed that pyrazole was absent. The slight absorption was caused by methionine which reacted with TE'F under the re-
action conditions to produce a pink color absorbing maximally a t 515 mp. Most microbial studies were carried out in a medium (16) containing 1 gram of pyrazolyl-alanine, 0.1 gram of Difco yeast extract, 1.5 grams of K2HPO4, 0.5 gram of K H 2 P 0 4and 0.1 gram of MgS04 per liter of water. When solutions containing 0.2 to 5 pmole of pyrazole per milliliter of medium were prepared and determined by the described procedure, recoveries varied from 91 to 102y0. The lower limit of sensitivity was 0.04 pmole per determination. Table I1 lists amino acids and nitrogen-containing heterocyclics tested at a level of 0.1 mg. None produced an appreciable absorption. Methionine and histidine a t higher concentrations interfered with the assay. Since the aza-analogs of nucleic acids did not give a reaction, it is apparent that the presence of the nitrogen-nitrogen bond is not a sufficient requirement for the reaction with TPF.
I.o
8
W V
z
.6
a
m
a
0 v)
m
a
.e
2
0 400
Absorbance at 4.58 mp Plus Alone 50 pg. ( 0 5 mg.) pyrazole Nutrient broth 0 02 0 50 Yeast extract 0 01 0 51 Casamino acids 0 03 0 53 Proteose-peptone 0 01 0 52 Bacto-casitone 0 01 0 51 Pyrazolyl-alanine 0 00 0 51 Pyrazole alone 0 51
Table II.
eo0
roo
WAVELENGTH
Figure 2. Absorption spectra of products of reaction of 1 pmole pyrazole derivative with TPF, N a N 0 2 , and acetic acid A. 6. C. D.
Table I. Effect of Various Nutrient Additives on Development of PyrazoleTPF Colored Complex
500
E. F. G.
3-Methyl pyrazole indozole 4-Bromo pyrazole 4-Bromo-3-methyl pyrazoie 3-Amino-4-cyano pyrazole 4-Bromo-3,5-dimethyl pyrazole 3,5-Dimethyl pyrazole
TPF has been used for the detection or determination of thioketones (7, I6), primary aromatic amines (9), hydrazides (14), hydrazones (IO), isonicotinic acid (8), cyanamide (2), isobutyl alcohol (If), and guanidines (4). It also forms colored complexes with several unknown compounds in extracts of some legume seeds ( I ) . Most of these complexes are colorless in acid. However,
Compounds Giving Negative Reaction with TPF Analysis for Pyrazole
Amino acids Alanine Arginine Glutamic acid Glycine Histidine H ydroxyprolinf: Isoleucine Leucine Methionind Ornithine Phenylalanine Pyroline Tryptophan Tyrosine Valine Nucleic acid derivatives Adenine Adenosine Cytidine Guanine Guanosine Hypoxanthine Inosine Thymidine ITridine Xanthine Xanthosine
Heterocyclic compounds containing adjacent nitrogens Phthalic hydrazide Maleic hydrazide 1,2,4-Triazole 8-Azaguanine 8-Azahypoxanthine 8-Azauridine 8-Azaadenine 8-Aza-2-thioxanthine 8-Aza-2,6-diaminopurine 8-Azaxanthine 6-Azauracil 6-Azauridine 6-Azathymine .Heterocyclic compounds Alanine anhydride Aspergillic acid Glycine anhydride Piperazine Pyrazine 2,3-dicarboxylic acid 3-OH, 2,5-dimethyl pyrazine Pyrrole 2-Quinolinol Quinoxaline Quinoline
VOL. 3 7 , NO. 2, FEBRUARY 1965
247
considering the ease with which TPF forms colored complexes, it is perhaps fortuitous that we found no interfering substances in our work. Dunnill and Fowden (3) extracted pyrazole from root tissue by benzene extraction and Takeshita, Nishizuka and Hayaishi (16) used ion exchange resins to separate pyrazole from protein and amino acids. Pyraaole may be precipitated as a sparingly soluble silver salt (12). If necessary, one or more of these methods might be used to separate pyrazole from interfering compounds. According to Feigl (7) and Herington (9) the reactions of TPF depend on the replacement of NH1 by a molecule of the reacting compound to form a colored complex. No color was formed when sodium nitroprusside replaced TPF in the procedure. A solution of penta-
cyanoaquaferrate, Fe(CH)sH1O-3, obtained by irradiating alkaline nitroprusside with ultraviolet light ( 7 ) , formed a yellow complex with pyrazole. Presumably in this case pyrazole displaced the water molecule. No attempt was made to isolate or characterize the products of pyrazole and the prussic salts. LITERATURE CITED
( 1 ) Bell, E. A., Biochem. J . 70,617 (1958). ( 2 ) Buyake, 0. A., Downing, V.,ANAL. CHEM.32, 1798 (1960). ( 3 ) Dunnill, P. M., Fowden, L., J. Exptl. Botany 14, 237 (1963). ( 4 ) Fearon, W. R., Analyst 71, 562 (1946). ( 5 ) Fearon, W. R., Bell, E. A,, Biochem. J . 59. 221 11955). ( 6 ) Feigl, F.; Anger, T., Frehden, O., Mikrochem. 15, 184 (1934). ( 7 ) Feigl, F., “Qualitative Analysis by
Spot Tests,” pp. 321, 349, 381, 385, Elsevier, Xew York, 1946. ( 8 ) Herington, E. F. G., Analyst 78, 174 (j-.-_, 195R\.
( 9 ) Herington, E. F. G., J . Chem. SOC. 1956, 2747; 1958, 4863, 4771.
(10) Ishidate.
M . . Sakaauchi. T., J . Pharm. Sdc. Javan 7& 308 11950): . ,, C.A. 45, 971e (1951). ‘ ( 1 1 ) Kutzelnigg, A,, Z. Anal. Chem. 77, 349 (1929). (12) Loudon, J,.,D.>“Chemistry of Carbon Compounds, E. H. Rodd, ed., Vol. IV, p. 251, Elsevier, Amsterdam, 1957. (13),Pinkerton, M. K., Lauer, J. M., Diamond, P., Tamas, A. A., Am. Znd. Hyg. Assoc. J . 24, 239 (1963). (14) Scardi, V.,Clin. Chem. Acta 2, 134 (1957). (15) Schwechten, H. W., Ber. 65, 1734 (1932). (16) Takeshita, ?*I., Nishizuka, Y., Hayaishi, O., J . Biol. Chem. 238, 660 (1963). RECEIVED for review September 21, 1964. Accepted December 14, 1964.
Spectrophotometric Determination of Nitrate Ion Using Rhenium and a-Furildioxime R. A. BLOOMFIELD, J. C. GUYON, and
R.
KENT MURMANN
Departments o f Chemistry and Agricultural Chemistry, University o f Missouri, Columbia, Mo.
b Nitrate ion may be determined in the range 3 to 35 p.p.m. by its interference with the complex formed between rhenium and a-furildioxime in the presence of SnC12. A standard deviation of 0.19 was calculated. The method is highly sensitive and selective, and i s easily adapted to high speed multi-analysis. Most common ions do not interfere. In most cases the NOz- interference is small enough to b e neglected. The method has been used for analysis of water and forage extracts without complications.
D
of small amounts of nitrate ion in water, soils, forage samples, and biological systems is of considerable importance. Nitrate ion has been spectrophotometrically determined by nitration of a suitable phenolic compound to form the corresponding nitro compound, oxidation of a suitable reagent by nitrate ion, and reduction of nitrate ion to nitrite ion or ammonia followed by a determination of these substances by conventional means. The formation of the yellow &nitro2,4-sylenol by nitration of 2,4-xylenol has bwn Qtudied in some detail (4, I S , 21, 23). Hartley and .hai ( I O ) have thoroughly invedgated the nitration of 2,6-xylenol as a technique for the deter248
ETERMINATION
ANALYTICAL CHEMISTRY
mination of nitrate ion. Several workers (5, 18, 19) have studied the nitration of the 2,4-disulfonic acid derivative of phenol as a method for nitrate ion. Other reagents, such as 2,4-benzenedisulfonic acid (1) 1,5-naphtho1 sulfonic acid (15) and 3,4-xylenol (9) have also been nitrated. These methods all suffer from halide ion interferences, require close temperature control, and, with the exception of the 2,6xylenol reagent, require in excess of 30 minutes per determination. I n addition, they often produce nonlinear working curves. I3rucine is oxidized by nitrate ion to produce a yellow color which is proportional to the concentration of nitrate ion present. This reaction has been studied by several workers, the latest being Jenkins and Medsker (11). In addition to being empirical and dependent upon the source of reagents, the method is seriously influenced by time and temperature. Reduction of nitrate to nitrite (7, 16, 22) and ammonia ( 2 , 1 7 )and subsequent determination of the reduction products has received limited application because of difficulty in controlling the reductive process and the time required for analysis. This paper presents a novel approach to the determination of nitrate ion based on its selective interference in determination of rhenium by a-furildioxime (14).
EXPERIMENTAL
Apparatus. Spectral measurements were made on either the Beckman DU or the Bausch and Lomb Spectronic 20 Spectrophotometers. Reagents. All reagents used were of t h e highest quality obtainable. The a-furildioxime was purchased from Eastman Kodak Co. and used without further purification. A solution of this ligand was prepared by dissolving 0.3500 gram in 100 ml. of reagent grade methanol. The stability of the ligand solution was greatly increased by using methanol instead of acetone as recommended by Meloche, Martin, and Webb (14). The stannous chloride solution, 8.5y0 was prepared by dissolving 10.0 grams of SnCl2.2H20 in 10 ml. of concentrated HCl and diluting to 100 ml. with water. The K R e 0 4 was obtained from the University of Tennessee. Analysis by means of tetraphenyl arsonium chloride indicated a purity ~of 99.9%. Recommended General Procedure. Prepare a calibration curve by adding up to 15 wg. of nitrate ion to 10-ml. volumetric flasks and bringing to a total volume of 2 ml. with water. T o each flask add 0.200 ml. of aqueous potassium perrhenate solution, 0.45 ml. of concentrated hydrochloric acid, 3.0 ml. of methanol, and 1.0 ml. of 8.5yc tin(I1) chloride. Mix and allow the solution to stand for exactly 10 minutes and add 1.0 ml. of the CYfurildioxime solution. Dilute to 10.00 ml. with wat,er. After exactly 10
