chromatographed successively. Identification of the pher, ylacetic acid was made by direct comparison with the standard. The acid was shown to have a retention time of approximately 5 minutes. Quantitation of the phenylacetic acid necessitated the, interpolation of base lines beneath the response curves of sample and standwd and the subsequent measurement of peak height. Proportionality between peak height of standard and sample plus the inclusion of a recovery factor to compensate for incomplete extraction over the single pass led to an estimate of phenylacetic acid concentration in the fermentation media.
Table 1. Recovery of Phenylacetic Acid Added to Fermentation Samples
Phenylacetic acid, mg./ml. R Added Found 0.40" O.4Ob 0.4OC 0.50a 0 . 80a 0.80* 0.8OC 1 .ooa 1.20b 1 .50b 2 . ooa
0.37 0.36 0.38 0.49 0.74 0.73 0.77 0.95 1.15 1.46 1.90
~
92 93 90 95 98 93 91 96 95 96 97 95
RESULTS A N D DISCUSSION
Broth taken before precursor addition. Broth taken at the approximate midpoint of fermentation. Harvest broth.
Under the conditions specified, it was found that a lines,r response was generated ranging from 0 to 1.8 pg. of phenylacetic acid per pl. It was shown further that linearity tan be extended to a t least 3.5 pg. thrclugh attenuation. This range is more than adequate for covering the levels of phenylacetic acid encountered in the usual benzylpenicillin fermentations. Two points need t c be clarified concerning the single pass extraction of phenylacetic acid. First, the use of XaC1 as a salting-out agent is preferred over the more commonly used KanSOd. The use of T\'asSOc led to emulsions which were very difficult to break, even with centrifugation. The use of NaC1 has eliminated this problem, making centrifugation necessary only for oc-
casional samples but desirable as a timesaver for all. Second, it was determined that 95 =t 5Yo of the phenylacetic acid added to broth a t all stages of the fermentation was recoverable with a single pass extraction. The reproducibility of the recoveries from day to day (&50J,) warranted the use of the recovery factor mentioned in the statements regarding phenylacetic acid quantitation. Based on data collected over several months, the precision of the method is estimated to be =t2Y0. The accuracy is judged to be zt5% as determined from recovery experiments run in broth, some values of which are listed in Table I.
a
Two of the most likely encountered compounds related to phenylacetic acid are the hydroxyphenylacetic acids (2,~ 4) and~ N-methylphenylacetamide. ~ ~ ~ r e i t h e r interferes since their relative retention times are approximately 0.44 and 1.31, respectively. For periods up to 48 hours, fermentation broths can be safely stored under refrigeration with or without cells. Beyond that time, a rise in phenylacetic acid level is noted, showing an increase of roughly 2y0per day thereafter. I t is noteworthy that high specificity is inherent in this technique making the method unique for the determination of phenylacetic acid in fermentation media. ACKNOWLEDGMENT
The author gratefully acknowledges the technical assistance of R. J. Mark. LITERATURE CITED
( I ) Ishima, hf., Tanno, T., J . Anfibiotics J a p a n 44, 169 (1951). ( 2 ) King, N. K., Hnmbly, A. S . , Roy. Australian Chem. Inst. J . & Proc. 17,
403 (1950). 131 . , Metcalfe. I,. D., .Tuture 188. 142 (1960) (4) Sishikida, T., J . Antibiotics J a p a n 4,299 (1951). ( 5 ) Pan, S. C., Perlman, D., ANAL. CHEIM. 26, 1.132 (1954). (6) Saias, C ildretti, C., ,4nn. Pharm. Franc. 12,'!i3 (1954).
A. 0 . NIEDERMAYER
Squibb Institute for Medical Research New Brunswick, 3 . J.
Spectrophotometric Microdetermination of Nitrate with Chromotropic Acid Reagent SIR: The analytical method described here stemmed from a desire to determine micro amounts of nitric acid in aqueous mixtures containing micro amounts of formic acid, formaldehyde, and methanol. The method is a modification of the cne employed by West and Lyles for the estimation of nitrate (3). I n their method, a yellow color was developed when nitrate was treated with chromotropic acid in the presence of concentrated sulfuric acid, and the method reliecl on the presence of chloride ions for full color development. Absorption was measured a t 357 mp, and the method yielded results which obeyed Beer's law in the concentration range 0 to 1 p.p.m.; a t higher concentrations (1 to 5 p.p.m.), or in the absence of chloride, the results did not obey Beer's law. The method of Wetit and Lyles was not directly applicable to the aqueous mixtures containing formaldehyde because the formaldehyde develops a n
~
intense violet-pink color in the presence of chromotropic acid in sulfuric acid ( 2 ) . A method was sought, therefore, which overcame this interference. To this end, the formaldehyde was oxidized to formic acid by passing it through a silver oxide column ( I ) . .is the emergent solution contains silver ions, with which chloride would be incompatible, an alternative method of enhancing the color development was sought. I n the proposed method full color development is rapidly achieved in the absence of added chloride ions by a controlled heating and cooling procedure. The method does not require the addition of chloride ions for full color development and therefore is not troubled by interfering substances such as silver. Color development obeys Beer's law over a much wider concentration range (0 to about 6.5 p.p.m. nitrate), and the absorption is measured in the visible region of the spectrum.
The color intensity is unaffected by small variations in the prescribed temperature, but escessive heating causes a reduction of color. The color intensity is also sensitive to the acid concentration a t concentrations lower than those prescribed in this method, that is, below 60 to 70yoacid. EXPERIMENTAL
Reagents and Apparatus. A solution of chromotropic acid was prepared by dissolving 0.1 gram of the dry powder (May and Baker, Ltd., London-Laboratory Chemicals) in 25 ml. of concentrated sulfuric acid and filtering if any insoluble material was present. Although this solution gradually changed color on standing, as far as color development was concerned it was perfectly stable for one week. As no difficulties were experienced with the above reagent, the chromotropic acid was not purified before use. VOL 36, NO. 4, APRIL 1964
939
,
A Perkin-Elmer Spectracord Model 4000 recording spectrophotometer was used to determine the variation in absorbance of the colored solution with wavelength. The absorption spectrum indicated that the difference in absorbance between the blank and the colored solution was appreciable at wavelengths of about 400 mp, and that there was a plateau in this difference in this region. For these reasons the wavelength of 400 mp was chosen. Procedure. The sample to be analyzed should contain less than about 6.5 pap.m.nitrate. The test solution, 3 ml., is transferred t o a 10-ml. volumetric flask, and the solution cooled by swirling the flask in a n ice bath while 5 ml. of cold concentrated sulfuric acid are added slowly. Chromotropic acid reagent, 1 ml., is added to the solution and mixed, and the flask placed in a water bath at 60' C. for 15 minutes and swirled occasionally. The flask is cooled in a n ice bath.
Table 1. Standard
Absorbance of Prepared Solutions of Nitric Acid
Absorbance, 4-cm. cell,
Nitric acid concn., pg./ml.
400 mp
0.38
0.047 0.084 0.095 0.155 0.194 0.282 0.368 0.560 0.695
0.76
1.01 1.52
2.03
3.04 4.05 6.08 8.10
A portion of the colored solution is transferred to a 4-cm. glass optical cell and allowed to stand for about 5 minutes to permit any bubbles to rise. The absorbance of the solution is then determined against a reagent blank with a spectrophotometer at 400 mp. The nitrate equivalent for this figure may be determined with a calibration curve drawn from the data presented in Table I. LITERATURE CITED
(1) Bailey, H. C., Knox, J. H., J . Chem. SOC. 1951, p. 2741.
Concentrated sulfuric acid is added while the flask is being agitated to bring the level just below the mark of the flask. When the solution is at room temperature, the volume is made up to the mark with concentrated sulfuric acid, the stopper adjusted, and the flask shaken.
(2) Bricker, C. E., Johnson, H. R., IND. ENG.CHEM.,AKAL.ED. 17, 402 (1945). (3) West. P. W.. Lvles. G. I,.. Anal. Chim. Acta. 23, 227 (1g60).'
Polarographic Determination of o-Phenylenediamine in the Presence of Other Isomeric Phenylenediamines SIR: A recent investigation of the isomeric phenylenediamines by anodic voltammetry showed that it was not feasible to analyze for an individual isomer in the presence of one or both of the other isomers by this technique (2, 6). The diimine product of the electro-oxidation of the more easily oxidized isomer of the mixture reacts chemically with the other isomer(s) t o give a species which is also readily oxidized at the potential already applied. Thus, the peak current obtained is considerably larger than expected, which leads t o considerable error. This paper reports a successful electrometric method for the determination of trace amounts of the ophenylenediamine isomer in the presence of up to a 10-fold excess of either or both the m- and p-isomers. This method is based on the catalytic polarographic prewave [thought t o involve the reduction of a Ni complex which is regenerated in a cyclic manner ( I ) ] observed when Ni(I1) is reduced at a dropping mercury electrode in the presence of small quantities of o-phenylenediamine.
reference electrode, and its electrical contact with the sample solution in the polarograph cell was made through a n agar-agar KC1 bridge. The polarograms were obtained with a Leeds and Northrup Type E Electrochemograph with no damping. Reagents. The isomeric phenylenediamines were purified by recrystallization from concentrated hydrochloric acid ( 2 ) . Because of their susceptibility to air oxidation, stock solutions were prepared as needed with air-free distilled water. All other solutions were prepared with distilled water and reagent grade chemicals. Procedure. The phenylenediamine sample solutions were diluted with 10
940
ANALYTICAL CHEMISTRY
I
I
1
I
RESULTS AND DISCUSSION
E, volts vs. S.C.E.
EXPERIMENTAL
Apparatus. T h e dropping mercury electrode (D.M.E.) used in these experiments had a drop time of 3.10 seconds a t a height of 62.8 cm. of mercury in 0.1M KC1 with no applied potential. Under these conditions, the outflow of mercury was 2.25 mg. per second. A saturated calomel electrode (S.C.E.) was used as t h e
I
a n equal volume of a n air-free 6 X lO-3M X(Ac)2, 12 X lO+M Ca(Ac)z, and 2.OM acetate buffer (pH = 5.5) solution and the polarograms of the resulting solutions were measured. All current values are reported as the peak current obtained during drop-life (corrected for residual current). All solutions were deaerated with nitrogen gas purified according t o the standard practice (6). The Ca+2 ion was added as a maximum suppressor for the Ni(H20)6+2background wave (3) and did not effect the limiting current of the prewave but did increase the definition of the prewave by decreasing the rate of rise of the foot of the background wave. As the variation of the limiting current of the prewave was not quite linear with [o-phenylenediamine], a calibration curve was constructed using known standards.
Figure 1. Effect of the isomeric phenylenediamines on the foot of the Ni(Hz0)6+2 reduction wave X IO-aM, [ C O + ~ ]= 6 X [acetate buffer] = 1.OM (pH = 5.5) -----Residual current: [Ni(H20)B+2 wave] A. [o-Phenylenediamine] = 7.3 X B. [in-Phenylenediamine] = 3.2 X C. [p-Phenylenediamine] = 3.2 X [Ni+2] = 3
lO%i, 10-%4 10-2M
IO-W
Figure 1 shows the polarograms obtained for solutions of 3.00 X 10-3M Ni(Ac)2, 6.00 X 10-3M Ca(Ac)z, and 1 . O M acetate buffer (pH = 5.5) which were 7.3 X l O - 5 M in o-phenylenediamine, 3.2 X 10-2M in m-phenylenediamine, and 3.2 x 10-2M in p-phenylenediamine. The solution containing the o-isomer exhibits a well defined prewave with limiting plateau. The m-isomer (in much larger concentration) has a different effect which apparently only shifts the position of the foot of the Ni(H20)6+2 background wave (dashed line), and the presence of the p-isomer has almost no effect at all on the position of the foot of the wave. Previously it
