V O L U M E 2 8 , N O . 1, J A N U A R Y 1 9 5 6 amount of time for complete color development. -4development time of 10 minutes was chosen as optimum to ensure complete color intensity. The complex between cycloserine and sodium nitritopentacyanoferroate was found to be sensitive toward temperature changes. Although the color develops rapidly a t room temperature, the highest precision and reproducibility were obtained in a temperature-controlled laboratory where the temperature was 25' & 1' C. and the relative humidity \vas 50 f 2y0. Cooling samples below 15OC.before color development resulted in slow color formation, and heating samples above 50OC. completely destroyed the color.
41 acid, methionine, glucurono-lactone, rutin, urea, benzylpenicillin, streptomycin, dihydrostreptomycin, bacitracin, Aureomycin, Terramycin, neomycin, glucose, lactose, and sucrose. These compounds did not give a blue color. -4 positive reaction was given by derivatives of cycloserine which still retain the basic ring structure of cycloserine. The derivatives prepared in the laboratory and tested were: monoacetyl, monobenzoyl, mono-and diisocyanate, and desaminocycloserine. The test appears to be specific for the determination of the csycloserine molecule in a wide variety of samples. SUMMARY
REAGEF-T COIVCENTRATION
The concentration of reagent is not critical, as long as an excess amount is added. 4 2% solution of sodium nitritopentacyanoferroate in 2.000N sodium hydroxide gives a low reagent blank and maximum color. ACID-BASE RELATION TO COLOR
The complex develops in weakly acidic medium. Acetic or phosphoric acids can be used, but stronger acids such as sulfuric, hydrochloric, or nitric destroy the color complex. The optimum amount of acid required was investigated. Several aliquots containing 100 y of cycloserine were treated with 1.0 ml. of color reagent in 2.ON alkali. The amount of 4.0S acetic and the water content were varied. The total volume was held constant a t 5.0 ml. The results are given in Table 11. Three milliliters of a 1.00OS solution of acetic acid was chosen as optimum when 1.0 ml. of color reagent equivalent to 2.000.Y alkali was used. INTERFERENCE
In order to determine the specificity of the nitritopentacyanoferroate method toward cycloserine several compounds !$ere tested. These included degradation compounds, amino acids and other antibiotics. The compounds tested were: u-serine, serine amide, serine hydrosamic acid, hydroxylamine hydrochloride, D-threonine, L-tyrosine, D-valine, L-proline, L-hydroxyproline, D-lysine, u-glutamic acid, glycine, p-alanine, s-meth? 1glucamine, L-hiqtidine, L-cysteine, asparagine, nicotinamide, uric.
A colorimetric procedure for the specific determination of cycloserine, a new antibiotic, has been presented. The proposed test quantitatively determines the antibiotic in amounts as little as 2 to 3 y and has an accuracy within f 2% with a precision ivithin & 1%. The method has been adapted to the determination of crystalline cycloserine and its salts in pharmaceutical preparations, blood plasma, urine, cerebrospinal fluid, other biological fluids, fermentation broths, and all phases of proceseing. Interference has been encountered only in urine. A close correlation has been found between a biological assay and the color apsav. LITERATURE CITED
Cuckler, A. C., Frost, 13. XI., McClelland. L., and Solotororsky, AI., Antibiotics &- Chernofherapy5 , 191 (1955). Epstein, I. G., Sair, K. G . S.,and Boyd, L. J., Antibiotic M e d . 1, 80 (1955).
Harned, R. L., Hidy, P. H., and LaBaw, E. K., A~itibiotic & Chemotherapy 5, 204 (1955). Harris, D. A . , Ruger, AI., and others, Ibid.,5, 183 (1955). Hidy. P. H., Hodge, E. B., and others, J . Am. Chem. SOC.77, 2345 (1955).
Hofman, K. A . H., A m . Chem., Justus Liebigs 312, 1 (1900); Chem. Zentr.. 557-9 1900 I I .
Kuehl, F. d.,Jr., Wolf. F. J . , and others, J . A m . Chem. SOC. 77, 2344 (1955).
Stammer. C. H., Filson, .I,X., Holly, F. W..and Folkers, K., Ibid..77. 2346 (1955). ~,
Welch, H.,'Putrnan, L. E., and Rantiall, IV. A , , Aiifibiotic M e d . 1 , i 2 (1955). RECEIVED for review July 18, 1955. Accepted September 18 19%.
Color Reaction of Salicylic Acid and Nitrite with Cupric Ion A . L. UNDERWOOD D e p a r t m e n t of Chemistry, Emory University, Emory University, G a .
The variables which affect the determination of copper h>-the Jorissen reaction (the formation of a red color upon heating a solution containing salicylate, nitrite, and cupric ion) have been investigated. The pH of the solution, the concentration of nitrite and of salicylate, and the duration of the heating period must be controlled if this reaction is to yield reliable results. Under the conditions suggested as a result of this study, the recommended copper concentration range is about 2 to 9 p.p.m. A t the optimal concentration (about 5 p.p.m.) the standard deviation is about 0.04 p.p.m. Although a detailed study of interferences has not been made, the method appears to be fairly selective for copper. Information regarding the identity of the red reaction product is also presented.
T
HE red color fornied upon heating :t solution containing salicylic acid, inorganic nitrite, and cupric ion has been known for many years. It \vas first reported by Jorissen ( 2 ) and later st'udied by Schott ( 4 ) and Sherman and Gross ( 5 ) . More recently, this color reaction has been mentioned by 1Iuller and Burtsell ( 3 ) and has fourid its way into several standard treatises ( 1 , 6, 7') where it, i? suggested as the basis of a colorimetric copper determination and also as a confirmatory test for salicylates. In spite of this attention, the reaction has never been studied criticslly to establish optimal conditions for analytical procedures, nor has the nature of the red reaction product h r e n elucidated. In the present investigation, it has tieen found that the variables affecting the development, of the red color must be controlled much more carefully than previous reports have in-
ANALYTICAL CHEMISTRY
42
dicated in order to obtain rep r o d u c i b l e results. Conditions are suggested under which it is possible to determine copper in concentrations from about 0.2 to 15 p.p.m. The most satisfactory concentration range extends from about 2 to 9 p.p.m., with an optimal concentration of about 5 p.p.m. which can be determined with a standard deviation of about 0.05 p.p.m, The method seems fairly selective in that only relatively large quantities of several other metals imitate the copper reaction. The available data shed considerable light upon the nature of the reaction, but definitive identification of the red substance has proved difficult.
Table I.
Reproducibility at Three Concentrations 5.15 P.P.M. Cu 10.3 P.P.M. Cu
2.06 P.P.M. Cu Absorbance Dev. V8. from mean blank absorbance 0.183 0.006 0.183 0.006 0.177 0.000 0.176 0.001 0.177 0.000 0.177 0.000 0.176 0.001 0.175 0.002 0.175 0.002 0.175 0.002 0.183 0.006 0.177 0.000 0.176 0.001 0.173 0.004 0.177 0.000 0.177 0.000 0.173 0.004 0.175 0.002 0.177 0.000 0.178 0.001
Absorbance Range 0,173-0.183 Median 0.177 Mean 0.177 Av. dev. 0.0019 Std. dev. 0,0029
Cu, p.p.m. 2.02-2.13 2.06 2.06 0.022 0.034
VARIABLES AFFECTIYG COLOR DEVELOPMENT
Apparatus and Reagents. A411absorbance measurements were performed with a Beckman Model DU spectrophotometer, using 1-em. Corex cells. Measurements of pH were performed with a Beckman Model G pH meter. Reagent grade materials were used throughout the study. The following solutions are employed in the method. STANDAFCD CUPRICNITRATE,about 207 of copper per milliliter, prepared from electrolytic copper foil 1.0 X lO-*M SODIUM SALICYLATE, ACETATEBUFFER,l M , adjusted t o pH 4.2 POTASSIUM NITRITE,50 mg. per ml., freshly prepared Absorption Spectra. Figure 1 shows that the red solutions exhibit an absorption maximum a t about 520 mp. The band is sufficiently broad so that the wave-length setting is not critical. All measurements reported below were performed a t this wave length. (The ultraviolet region of the spectrum was also investigated: Although a prominent absorption maximum was found, it waa almost equally strong in both blanks and copper solutions.) Effect of pH. A series of solutions was prepared, each containing 6 0 of ~ copper, 2 ml. of a lO-8M sodium salicylate solution, 1 ml. of potassium nitrite solution (50 mg. of potassium nitrite per ml.), and acetic acid to a final concentration of 0.1M. The
Absorbance us. blank 0.456 0.459 0.462 0.456 0.452 0.455 0.453 0.452 0.453 0.453 0.457 0.458 0.457 0.463 0.457 0.464 0.454 0.456 0,454
Dev. from mean absorbance 0.000 0.003 0.006 0.000 0.004 0,001 0.003 0.004 0.003 0.003 0,001 0,002 0.001 0.007 0.001 0,008 0,002 0.000 0.002
Absorbance blank 0.872 0,850 0.855 0.855 0.870 0.858 0.868 0,845 0.848 0.859 0.862 0.862 0.838 0.850 0.848 0.865 0.870 0.861 0.857 0.862
from mean absorbance 0.014 0.008 0.003 0.003 0,012 0.000 0.010 0.013 0,010 0,001 0.004 0.004 0,020 0,008 0,010 0.007 0.012 0,003 0.001 0.004
Absorbance 0.452-0.464 0.456 0.456 0.0027 0.0036
C u , p.p.rn. 5.11-5.24 5.15 5.15 0.031 0.041
Absorbance 0.838-0.872 0.858 0.858 0.007
Cu, p.p.m. 10.1-10.5 10.3 10.3 0,085 0.11
V9.
0.009
pH values of the solutions were adjusted with sodium hydroxide or hydrochloric acid as desired, and the solutions were brought to a final volume of 20 ml. A similar series of “blanks,” in which the copper was omitted, was prepared. The solutions were heated for 1 hour in a water bath a t about 80” C., cooled to room temperature, and measured at 520 mp against a distilled water reference. The results of this experiment, clearly showing the very critical necessity for pH control, are plotted in Figure 2. The optimal pH is about 4.1 to 4.2. The unfortunate need for very careful buffering represents one of the serious drawbacks in the use of this method. Effect of Salicylate Concentration. Varying quantities of the 1034 sodium salicylate solution were added to a series of solu-
tions containing 607 of copper, 1 ml. of the potassium nitrite solution, and 2 ml. of the 1M acetate buffer of pH 4.2. The total
0’7L\ 0.6
\ 480 520 560 WAVE LENGTH (MILLIMICRONS)
440
Figure 1. Absorption spectra Upper curve, copper solutions Lower curve, blankn
Dev.
Figure 2.
Effect of pH
Upper curve, copper aolutiona Lower curve, blanks
V O L U M E 28, NO. 1, J A N U A R Y 1 9 5 6
43
0.:
W 0
z
40,;
m K
0
m m
20.2
a
0
m m
0
a
0 _I
BLANKS
I
I
I
100 200 300 MILLIGRAMS OF KNO,
V
I
I I
MILLILITERS Figure 3.
I
OF
I
I
2 SALICYLATE
I
Figure 4.
Effect of nitrite concentration Upper curve, copper solutions Lower curve, bianks
Effect of salicylate concentration
volume was 20 ml. in each case. A similar series of blanks was prepared, and the solutions were heated for 1 hour a t 80" C. The results of this study are shown in Figure 3. hlaximal color development requires about a twentyfold molar excess of salicylate with respect to copper. Effect of Nitrite Concentration. Varying quantities of the potassium nitrite solution were added to a series of solutions containing 607 of copper, 2 ml. of the 10-*M sodium salicylate solution, and 2 ml. of the buffer. The total volume was 20 ml. in each case. A similar series of blanks was prepared, and the solutions were heated for 1 hour a t 80" C. Figure 4 shows that the nitrite concentration has a marked effect on the color development, and that this effect is a complex one, with maximal color when about 50 mg. of potassium nitrite is present, Effect of Duration of Heating. A series of solutions was prepared containing 607 of copper, 2 ml. of the buffer, 2 ml. of the sodium salicylate solution, and 1 ml. of the potassium nitrite solution. The volume was 20 ml. A series of blanks was also prepared, and the solutions were placed together in a water bath at 80" C. At certain time intervals, solutions were removed from the bath and quickly cooled in ice n-ater. The absorbances of the solutions are plotted in Figure 5, where it is seen that 1 hour is sufficient t o ensure maximal color development The practical difficulties in reproducing the heating and cooling make it necessary to run standards and unknowns through a determination together. Stability of Colored Solutions. The red solutions, allowed to stand a t room temperature, maintained constant absorbanre values through an observation period of 2 hours. AlALYTlCAL RESULTS
A Beer's law plot obtained under optimal conditions as elaborated above was linear from 0 to about 8 p.p.m., after which a slight negative deviation appeared. A Ringbom plot of these same data showed that the minimal photometric error occurred when the copper concentration was about 5 p.p.m. Concentrations of about 2 and 9 p.p.m. embraced the range over which the method exhibited very nearly its maximal reproducibility. Three groups of about 20 solutions each were prepared, containing 2.06, 5.15, and 10.3 p.p.m. of copper. The results obtained with these solutions are presented in Table I, where the range, median, mean, average deviation, and standard deviation are shown for each group. It seems clear from these results that the variables influencing the reaction are under reasonable
V
I IO
Figure 5.
I
I
I
20 30 40 TIME IN MINUTES
I
50
Effemt of duration of heating
Upper curve, copper solutions Lower curve, blanks
control when conditions are regulated according to the above studies. Interfering Metals. The changes in the absorbance values of solutions containing 5.15 p.p.m. of copper caused by the presence of 500 p.p.m. of several other metals were measured. Assuming the interference t o be proportional t o the concentration of interfering metal, the concentration of each metal was calculated which would produce an absorbance change of 0.0036 ( l ~ , Table I). These values also represent the concentrations of the metals which would produce the same absorbance value as 0.04 p.p.m. of copper. The tabulation (Table 11) shows that the method is much more sensitive for copper than for the other metals tested. I n addition t o those metals in the table, iron(III), bismuth, and aluminum were tested. These three metals, a8
44
ANALYTICAL CHEMISTRY
expected, interfere by precipitating during the heating period at p H 4.2. Concentrations of bismuth and of aluminum which do not lead to precipitation do not interfere. Iron(III), on the other hand, leads to green rather than red solutions even when present to the extent of less than 1 p.p.m, NATURE OF RED REACTION PRODUCT
I t is necessary to heat solutions containing nitrite, salicylate. and cupric ion in order to obtain the red color within a reasonable period of time, It was observed, hov-ever, that the red color forms immediately a t room temperature upon the addition of copper to a solution containing nitrite and salicylate n hich has been heated in the absence of copper. This eliminates from consideration such possibilities as a catalytic effect of cupric ion on some organic reaction between salicylate and nitrite. By breaking down the reaction into two stages, one involving nitrite and szlicylate a t elevated temperature and the other the reaction of copper with a product of the first reaction, insight has been gained into the mechanism of the color formation.
7
Cu SOLN
3
I
I
4
5
I
PH Figure 6. Effect of pH on isolated chelation step at room temperature
First, it a a s possible to show that once the reaction at elevated temperature is over, nitrite is no longer needed for color development. This was done simply by heating a solution containing nitrite and salicylate, then adding a large quantity of urea t 3 the hot solution, followed by cooling it and adding the copper. The addition of urea, which is a well known method for destroying nitrites, did not obstruct the development of the red color. Thus it is certain that the red substance is not a nitrito complex of any s w t ; the possibility of reduction of cupric copper by nitrite is also eliminated. Secondly, studying the affect of p H on the two separated phases of the reaction proved informative. A series of solutions containing nitrite and salicylate was buffered at various pH values and heated. After cooling to room temperature, all the solutions were adjusted to p H 6.0, and copper nas added to each. The resulting curve of absorbance us. p H was essentially the same as that for the over-all p H effect shown in Figure 2. A curve of this shape suggests the operation of opposing factors, which in the
present case might be the effect of pH upon the ionization of salicylate acid and a t the same time upon the stability of some necessary nitrous intermediate. On the other hand, a different curve is obtained for the effect of pH upon the reaction with copper a t room temperature. 9 nitrite-salicylate solution, buffered a t pH 4.2, was heated, then cooled, and aliquots were added to a series of copper solutions of various p H values. The final pH values, plotted against absorbance, are shown in Figure 6. The S-shaped curve thus obtained is typical of pH-dependent chelation reactions in general.
Table 11. 3Ietal Ion
Interfering 3Ietals Concn. Causing Error of l o in Determining 5 . 1 5 P.P.31. of c u , P.P.11. 78 46
39 13 10 2
’
It appears reasonable, then, to postulate that the red color in the Jorissen reaction is formed as follows: Nitrite and salicylate react a t elevated kmperature to form a chelating agent which can then react with copper to form a red product even at room temperature. The identity of this Chelating agent has not been estahlislied. Authentic samples of 5-nitrososalicylic acid, 3-nitrosalicylic acid, and %nitrosalicylic acid do not give a red color with cupric ion. All of the observations which have been made during an attempt to find its nature are consistent, with the interpretation that the free chelating agent is unstable, but that chelation with copper greatly stabilizes the molecule. The red solutions are stable for at’least 2 hours. On the other hand, copper must be added almost immediately to the cooled nitritesalicylate reaction mixture if the maximal quantity of red product is to be obtained. (This instability renders nonfeasible any improvement of the analyt’ical method by preparing a solution of the chelating agent and later developing the color with copper at room temperature in calibrated glassware.) The red material can be extracted int’o certain organic solvents, ahhough it is sufficiently soluble in water that quantitative extraction cannot be made part of the analytical method. I t is soluble in chloroform or ether, but does not extract into carbon tetrachloride or petroleum ether. When a red solution in chloroform is shaken with dilute acid (pH 1 to 2), the red color gives n a y to yellow in the organic pha,se, and copper appears in the aqueous layer. If no more t’han a few minutes intervene, the red color can be partially regenerated by shaking the yellow chloroform solution Tith aqueous copper at p H 6. However, the length of time required to process a large scale preparation (or even to measure the absorption spectrum with a manual instrument) is sufficient to destroy the chelating agent to the point where no red color with copper can be formed. LITERATURE CITED
(1) dssociation of Official hgricultural Chemists, Washington, D. C . , “Official RIethods of Analysis of the Association of Official Agricultural Chemists,” 7th ed., p. 469, 1950. ( 2 ) Jorissen, BUZZ.m a d . roy. SOC. BeZg. [3] 3, 259 (1882). (3) Muller, R. H., and Burtsell, -1.T., Mikrochemie Ter. Mikrochim. Acta 28, 209 (1940). (4) Schott, F., 2. Sahr.-Genziss?n. 22, 727 (1911). (5) Sherman, H. S., and Gross, A . , J . Ind. Eng. Chern. 3, 492 (1911). (6) Snell. F. D., and Snell, C. T., “Colorimetric RIethodsof Analysis,” 3rd ed., vol. 2, p. 136, Van S o s t r a n d , S e w Y y k , 1949. (7) Welcher, F. J., “Organic Analytical Reagents, rol. 2, p. 123, Van Xostrand, S e w York. 1947. RECEIVED for review July 21, 1955. Accepted October 3, 1925,
