Co Io rimetric
Estimat io n of Phe noxy methy Ipenici IIin (Penicillin V) and Phenoxyacetic Acid in Samples from Penicillin Fermentations J. BIRNER Commonwealth Serum laboratories, Melbourne, Australia
,The routine determination o f the phenoxyacetic acid content of fermentation liquors, during production of penicillin V, furnishes a means of regulating the quantity of precursor needed for an optimal yield of the product. A method i s given whereby phenoxyacetic acid and penicillin V may b e estimated quantitatively. Both compounds, nitrated b y 10% nitric acid in concentrated sulfuric acid, form a phenol derivative which i s yellow in ammonia solution. The colored derivative is estimated colorimetrically. Phenoxyacetic acid in a saturated aqueous solution o f ammonium sulfate at pH 2 is extracted quantitatively with benzene. Any penicillin V in the fermentation broth i s converted quantitatively to phenoxymethylpenicilloic acid by treatment for 15 minutes with 2N sodium hydroxide. In this form it remains in solution during the benzene extraction. As little as 5 y of phenoxyacetic acid can be estimated. By substituting methyl isobutyl ketone for benzene, both phenoxyacetic acid and penicillin V can b e extracted quantitatively, and b y using the two extraction procedures the quantity of penicillin V can b e found b y difference.
p
acid gives by nitration a phcnolic derivative ( 2 ) which on neutralization ivith alkali. produces a yelloiv compound. The nitration of phenouyacetic acid has been used as a procedure for estim a t’ion of that compoiiiid in urine ( 3 ) . Stoughton i 7 ) , in describirig his method for estimating phenols, claimed that by heating the reactants, nitrosophenols are formed as a consequence of nitratioii n ith dilute nitric acid in the presence of sulfuric acid. According to Lykken ( 4 ) the nitrosophenols resulting from the nitration of phenol form. in the presence of ammonia, the highly colored quinonoid radical. It has now heen observed that nitrstion of dry phenoxyacetic acid with lo(,”, potassiuni nitrate in concentrated sulfuric acid and subsequent neutralizaHESO\TACI.TIC
tion with ammonia produces a similarly colored compound. The yellow derivative is governed by Beer’s law and hence is amenable to colorimetric estimation, within the range of from 5 to 50 p.p.ni. in a volume of 7 ml. Phenoxymethylpenicillin, when similarly treated, yields also a derivative which, with ammonia, produces the yellow substance. The intensity of the color prdduced by nitration of penicillin TT corresponds stoichiometrically to the phenoxymethyl group in the molecule. The lowest limit for its estimation is about 12 Y. SEPARATION OF PHENOXYACETIC ACID A N D PENICILLIN V
Separation of phenoxyacetic acid and penicillin V prrsents difficulties, because of similarity in their behavior. A search for solvents suitable for selective extraction was unsuccessful. It was knon-n t h a t a method involving differential extraction has been used in the estimation of benzylpenicillin and benzylpenicilloic acid ( 5 ) and it seemed likely that a siniilar procedure might be used to separate phenoxyacetic acid from phenoxymethylpenicilloic acid using methyl isobutyl ketone as the solvent, but the separation was found to be incomplete. However, as shonn in Table I, it is possible to extract phenoxyacetic acid quantitatively by lienzene a t p H 2 in the presence of phenoxymethylpenicilloic acid formed from penicillin T’, n-hen the solution is saturated with animoniuni sulfate. The phenoxymethylpenicilloic acid is insoluble in the extractant \Then the organic solvent phase and aqueous phase are in the ratio 10 to 4 but, under the same conditions, both compounds are extracted quantitatively when methyl isobutyl ketone is substituted for benzene. Based on these observations a method has been developed whereby the tn-o compounds can be determined colorimetrically. The fermentation liquor is treated
with sodium hydroxide, the excess alkali is neutralized with sulfuric acid, and the solution is saturated n i t h animonium sulfate a t p H 2. By extraction with benzene the phenoxyacetic acid present is removed almost quantitatively while the phenoxymethylpenicilloic acid, which is formed quantitatively from the penicillin V present in the liquor, remains in solution. B y extraction-still a t a p H of 2--with methyl isobutyl ketone both penicillin V (or the phenoxyinethylpenicilloic acid formed from it) and the phenoxyacetic acid are completely removed. By nitration and treatment with ammonia yellow derivatives are formed. A measure of the intensity of their color provides a measure of the concentration of the phenoxyacetic acid. Nevertheless, the color is to some extent influenced by the concentration of ammonia used in neutralizing the acid added after nitration. I n routine analyses. it is advisable to run a series of standard estimations simultaneously with the sample estimations and to use ammonia solution of the same strength throughout all the tests. The method has been used successfully in these laboratories for more than a year and i t is valued for both its accuracy and speed. I t s results for penicillin V agree to nithin =t5% of those furnished by biological methods and i t is possible to carry out determinations on several samples in less than 2 hours. EXPERIMENTAL
Standard Reference Curve Determination. REAGENTS REQUIRED. lo%;,
potassium nitrate in concentrated sulfuric acid. Phenoxvacetic acid, analytical reagent grade. Prepare a stock solution containing o.oo570 phenoxyacetic acid in benzene-i.e., 50 y per ml. or 50 p.p.ni. Penicillin V. Prepare a stock solution containing 0.0115Y0 penicillin T‘ in chloroform. Ammonia, specific gravity 0.880, diluted 1 to 2. From the stock solution of phenoxyacetic acid in benzene, further dilutions in benzene, containing 40, 30, VOL. 31, NO. 2, FEBRUARY 1959
271
Table I.
Extraction of Free Phenoxyacetic Acid Alone and Mixed with Penicillin V and Extractions of Penicillin V from Solutions Saturated with Ammonium Sulfate
(Solvent-water volume ratio
=
2.5) Sum of Free POAA
Before hydrolysis
Solvent Used for Extraction Benzene Methyl isobutyl ketone
After hydrolysis
Benzene
Methyl isobutyl ketone
20, 10, and 5 y per ml. are made. One-milliliter quantities of the stock solution and of each dilution are pipetted in duplicate into suitable (preferably conical) test tubes. One drop of 0.LV sodium hydroxide is added to each tube and the benzene is evaporated to dryness on a water bath at a temperature of from 60" to 70" C. By blowing a gentle current of filtered air into each tube the solvent is removed very rapidly without the solid contents of the tubes being lost by spattering (6). One-half milliliter potassium nitrate (lo%, in concentrated sulfuric acid) is now added to each tube, TThich is then immersed in boiling water for 15 minutes. ilfter cooling, 1.5 ml. of distilled mater is run into each tube from a buret. The solutions are mixed by swirling and the tubes are cooled in water. Ammonia (1to 2), 5 ml., is added graduallyand the contents of the tubes are again well mixed. The intensity of the color, which develops rapidly, is measured in a colorimeter using a blue filter of a n absorption range of 400 to 465 mp. (A KlettSummerson photoelectric colorimeter was used in the work a t these laboratories, but the estimation may be performed as conveniently spectrophotometrically at 415 mp.) I n precisely the same way a stock solution of penicillin V in chloroform is used to furnish a standard reference curve for that substance. I n preparing the standard curve due allowance is made for a n y color which may be derived from the reagents by running blank tests on the solvents to which no phenoxyacetic acid or penicillin V have been added. By plotting concentration of phenoxyacetic acid (or penicillin V) against the net color intensity-as measured in colorimeter units-the standard curves are established. As the ratio of the molecular weight of penicillin V and phenoxyacetic acid is 350 to 152.14 or 2.3, it follows that 272
ANALYTICAL CHEMISTRY
Extract Pen. V only
P0.kA4 Added, ?/hll.
Pen. V only POXA and pen. V POAA only POA.4 and pen. V Pen. V only POAA only P04A and pen. V
500 100 500 1000 500
Penicillin V Added, y/ML 500 1000 500 1000 500
and POAA from
Penicillin V Added, y/Ml. 217 434 217 434 717
500
500
500
717
a colorimetric value for penicillin V will be 2.3 times that of its equivalent for phenoxyacetic acid. Extraction and Estimation of Phenoxyacetic Acid. PROCEDURE. Five ml. of fermentation liquor are pipetted into a 10-ml. volumetric flask and 1 ml. of 2N sodium hydroxide is added. The contents are mixed a t a temperature of about 22" C. and after 15 minutes are neutralized with 1 ml. of 2 N sulfuric acid. The volume is then adjusted to 10 ml. with distilled water. One milliliter of this solution is pipetted into a suitable centrifuge tube, 1 ml. of 1N sulfuric acid is added, and the solution is saturated with 2 grams of dry powdered ammonium sulfate. This solution, a t approximately p H 2, is mixed by swirling; 5 ml. of benzene are added from a pipet and, with the thumb closing its mouth, the tube is shaken vigorously for 45 seconds and then centrifuged for 5 minutes. After separation in this manner a n appropriate aliquot is removed for drying and subsequent color development as described for the preparation of the standard curve. The volume of the benzene (or methyl isobutyl ketone) extract taken for evaporation and color development should be such that its phenoxyacetic acid content does not exceed 50 y. The developed color is measured in the colorimeter, but as the fermentation medium contributes slightly to its intensity, a blank value for it must be determined (by a procedure similar to that described above) and subtracted. I n a n experiment in which a strain of penicillium was gromm in a skimmed milk medium (1) Lvithout precursor, samples were taken a t 24-hour intervals and color estimations were made in this manner. The fermentation medium, without precursor, if treated like the sample, yields a color which is dependent on the type and concentration of the medium used. If the same type and con-
Hours 0
--
'79
46
io
Recovery, % 68
2
58 46
425 740
2 2
103
100
2
100
5.85
98
960 480
2
100 98 96
2
96
Si1 100 80 740
2 2
100
Table 11.
Time,
pH 2
254
490
1000 100 100
POh.4 Found, y/Ml. 147.5
2
5.85 2
0 80
103
Fermentation in Medium without Precursor
Colorimeter Reading Benzene Methl-1 isobutyl extract ketone extract 15 19 18 14 10 12 15 10 10 15 12 18 13 19
94 120 144 12 168 18 Corresponding colorimeter readings. 25 for 5 y POAA, 50 for 10 y POA4-l.
centration of the medium is being used for fermentation, the value once estimated is constant and as proved does not change during the fermentation. Therefore !Then the same medium is being used in all fermentors, the blank value need be determined on only one sample of the bulk medium without precursor and this value subtracted from the data for samples with precursor. The data collected in Table I1 show that the values do not change during fermentation in the medium and they are equivalent to between 30 and 35-7 of phenoxyacetic acid per ml. Calculation. I'S
= volume of aliquot of or-
R1
ganic solvent extract of sample = volume of aliquot of organic solvent extract of medium blank = colorimeter reading for V s = colorimeter reading for
Ca
=
Cb
=
Vnz R1
Vm POAA equivalent of R1
from standard curve POAA equivalent of Rz from standard curve
CS
=
Cm
=
Cs
- Cm
=
POAA equivalent in original sample, y/mL = Ca x lO/Vs P O L 4 equivalent in medium, y/ml. = Cb X 1O/ V m real concentration of POAA, 7,’ml.
Extraction and Estimation of Penicillin V. PROCEDURE. One milliliter of t h e treated sample, TI hich was used in t h e estimation described above, is pipetted into a suitable centrifuge tube and further treated with 1 ml. of I S sulfuric acid and 2 grams of dry, powdered ammonium sulfate to furnish a saturated solution of the salt at about p H 2 . Five milliliters of redistilled methyl isobutyl ketone are added, and the tube is shaken vigorously for 45 seconds and then centrifuged for 5 minutes. ilfter separation in this manner a suitable aliquot is removed for drying and subsequent color development. The value taken from the standard curve represents the sum of the values for free POAA and P O A h
derived from penicillin T’ and after making due allowance for the values obtained in the blank for a similarly treated aliquot of methyl isobutyl ketone alone, is calculated in micrograms per milliliter (or parts per million) in the manner described above. If B y per ml. represent the value so obtained and A micrograms per ml. the value obtained for free phenoxyacetic acid the amount of penicillin V in micrograms per nil. may be calculated from the formula 2.3 ( B - A ) or read directly from the standard curve for penicillin V. I n the presence of phenoxymethylpenicilloic acid-formed under certain conditions from penicillin V in fermentation broth-the acid will be extracted by methyl isobutyl ketone and hence estimated as penicillin. ACKNOWLEDGMENT
The author gratefully acknowledges
the advice and encouragement received from E. C. Mason throughout the course of this work and wishes to thank J. R. Garnet for help in the preparation of this paper. LITERATURE CITED
(1) dnderson, G., Davis, L. J., Davey, V, F., Australian Patent 215,879. ( 2 ) Beilstein, E. K., “Handbuch der organischen Chemie,” Yol. 6, 4th ed., p. 161, Springer, Berlin, 1923.
(3) Levey, K., Leurs, H. B., J . Bid. C h n . 168, 213-21 (1947). (4) Lykken, L., Treseder, R. S.,Zohn, \-., ISD. ESG. CHEM,~ A L ED. . 18, 103 (1946).
( 5 ) Pan, S. c., .kS.\L. CHEX. 26, 1438 (19,541 \ - - -
- 3 .
(6) Pan, S. C., Perlman, D., Zbid., 26,
1432 (1954). (7) Stoughton, R., J . Biol. C h e m 115, 293 (1936). RECEIVED for review June 5, 1958. h c cepted September 8, 1958. Kork performed a t the Commonwealth Serum Laboratories, Melbourne, Australia. Published with the approval of the director.
Chelometric Analysis of Manganese-Magnesium and Manga nese-Magnesium-Zinc Mixtures Fluoride Ion as a Demasking Agent WILLIAM G. SCRIBNER Research and Engineering Division, Monsanfo Chemical Co., Dayton, Ohio
b The use of fluoride ion to demask magnesium selectively from a mixture of manganese and magnesium (ethylenedinitri1o)tetraacetate (EDTA) chelonates permits rapid analysis of manganese-magnesium mixtures b y consecutive titration of a single sample solution. Total manganese and magnesium (and zinc) are first determined by direct EDTA titration a t a pH of 10 employing Eriochrome Black T as indicator. Addition of a soluble fluoride causes formation of magnesium fluoride with the liberation of EDTA equivalent to magnesium, which is titrated with standard manganese. Zinc, if also present, can b e determined by demasking the zinc-EDTA with cyanide and titrating the liberated EDTA with standard manganese. Initial manganese in either case is calculated by difference. The method has been successfully applied to ferrites -e.g., manganous oxide-magnesium oxide-ferric oxide and manganous oxide-magnesium oxide-zinc oxide-
ferric oxide. Extension to other metal combinations is suggested.
I
interest in ferrites [mixed oxides with magnetic iron(II1) oxide] has necessitated the development of analytical methods for their estimation. Determination of iron in ferrites by cerate or permanganate oxidimetry presents no difficulty. However, analysis of certain mixed oxides by the classical methods may involve tedious, time-consuming separations, and precipitations. I n the author’s laboratory, titration with (ethylenedinitri1o)tetraacetate (EDTA) was investigated for the routine analysis of divalent metal ions in manganese-magnesium ferrite and manganese-magnesium-zinc ferrite, Because iron in high concentration is difficult to mask, and it interferes with E D T A titrations in alkaline solution, a prior separation is necessitated. The cupferron extraction method of Fritz. KCREASIKG
Richard, and Bystroff (4) effects rapid, efficient separations of iron from divalent metal ions. K N O W N EDTA M E T H O D S F O R M A N G A N E S E M A G N E S I U M MIXTURES
For the E D T A analysis of a manganese-magnesium mixture, the pH effect alone cannot be utilized to achieve selectivity when the end point is determined with the aid of a metallochromic indicator (3). Analysis by potentiometric E D T A titration of manganese a t a pH of 5 followed by titration of magnesium at a pH of 9 is possible (8). However, if zinc is also present, it cotitrates with the manganese. Although for the E D T A titration of magnesium or calcium, triethanolamine can be employed as a masking agent for small amounts of manganese (e), in ferrites the ratio of manganese to niagnesium prohibits its use. Separation of manganese as the dioxide or sulfide is possible. Flasclika VOL. 31,
NO. 2, FEBRUARY 1959
273
