Microdetermination of Oxalate in Fermentation Media GEOFFREY HALLIWELL Faculty of Technology, Manchester University, Manchester 1, England An improved volumetric method is presented for the determination of small quantities of oxalate, capable of analyzing 3.5 mg. of oxalic acid with a recovery of 100.55%(coefficient of variation 0.24) down to 0.4 mg. with a recovery of 99.71% (coefficient of variation 0.40). Comparisons are made with contemporary techniques. The effect of citrate, original sample voliime (up to 10 ml.), and medium constituents upon the accuracy is also given.
A
METHOD is described for the estimation of small quantities (0.4 to 4.0 mg.) of oxalic acid in the presence of other or-
ganic acids. The method of Pucher, Wakeman, and Vickery (4), on which it was based, entailed precipitation of the calcium salt a t pH 5.0; separation was effected by means of a microcrucible containing special asbestos, prepared as directed by Kirk and Moberg ( 2 ) . Seither this separation, which was unreliable and involved a transference of the precipitate, nor separation by centrifugation was found suitable. Advantageous though the latter process might be in theory (3), it had the disadvantage that floating of the precipitate was troublesome, in spite of the addition of an acid-alcohdl mixture.
Table I.
Standard Method
(1 ml. of oxalate solution in each case. 8 replicates) Theoretical Range of Coefficient ICOOHh Recovery Difference Recoveries of Variation 3.646 3.599 1.370 1.286 0.4061 0,3990
3.666 3.614 1.366 1.281
+0.020 +0.015
0.4040
-0,0012 -0.0001
0.3989
REAGENTS
Sodium oxalate, analytical reagent grade, dried a t 110' C. for 2 hours. Solutions are made up to contain approximately 5.0, 1.0, and 0.6 mg. per ml.
-0.004 -0.005
+0.55 4-0.42 -0.29 -0.39 -0.29 -0.@3
100.3-100.7 100.0-100.7 99.1-100.3 99.3-99.8 99.2-100.3 99.5-100.3
Yacuilm t-
Figure 1. Method of Filtration
0'24
o,37 o.40
The present author employed King filter sticks ( 1 ) in the early part of the work for the separation of the precipitate, but occasional reaction of the fiker paper with the permanganate caused their discontinuance, although they were handier to use. Sinteredglass filter sticks were found most suitable for the separation. Titration of the oxalate with 0.02 N potassium permanganate at 100' C..gsve unsatisfactory end points (variation of +=2.5% for 0.4 mg. and *5.0yofor 7 mg. in 12 ml., 4 ) . Instead, titration was done with 0 10 N or 0.05 A' permanganate a t room temperature (about 2.5' C . )in the presence of manganese sulfate as a catalyst. This was very satisfactory if the volume of liquid to be titrated was kept below about 3 ml. and if horizontal microburets capable of delivering 0.8 to 0.001 ml. or 0.2 to 0.0002 ml. were used.
Potassium permanganate, analytical reagent grade, 0.10 N and 0.05 N . The requisite amount is dissolved in slightly more than the theoretical quantity of distilled water, boiled 30 minutes, then allowed to stand overnight. After being filtered through sintered glass the solution is standardized against sodium oxalate. Permanganate so prepared is stable for a t least 9 months. Citric acid, analytical reagent grade. Solutions are prepared to contain approximately 25.0, 6.0, and 2.0 mg. per ml. Ammonia, 6 N , analytical reagent grade. Acetic acid, 2 N , analytical reagent grade. Sulfuric acid, 3 N , analytical reagent grade. Manganese sulfate tetrahydrate, analytical reagent grade. Bromocresol purple indicator, 0.04'% in distilled water. Calcium acetate, pure, 5 % solution. PROCEDURE
~~
Table 11. Modifications of Filtration and Titration Procedure Filtration Method King filter stick Pucher (4) Pucher (4) King filter stick
(1 nil. of oxalate solution i n each case) Theoretical Repli(COOH)* cates Recovery Titration Method 0.02 N K M n O ~ 10-ml.buret . gradiiated in 0 . 0 1 mi.. 1000 c. (4) Pucher (4) Excess KRlnOi. K I , and titration with Ka&03 in 10-rnl.buret graduated in 0 . 0 1 ml. (5) Powers (5)
Difference
3.519 3.501
8 8
3.593 3.589
t0.075 +?.lo +0.088 + 2 51
3.626 3.588 3.390 3.426
6 6 6 6
3.817 3.773 3.501 3.519
+O 191 +O.l85 +0.111 + O 124
3.521 3,500
5 7
3.577 3.566
+0.056
+ O 066
1184
+5 % +5 1 +3.3 +3.6
Coefficient of Variation
1'7 2,5
+l.tiC
i-1.90
I.'
One niilliliter or less of the solution containing oxalic acid (0.4 to 4.0 mg.) wa8 measured into a microbeaker and made just alkaline to the bromocresol purple (already present) with 6 S ammonia; 2 S acetic acid was then added t,o a rellow color (pH 4.0 to 5.6), followid by 0.5 ml. of 5% calcium acetate in the cold, and was left to stand a minimum of 2 hours, after the beaker had been gently swirled to mix the contents. The supernatant was removed under slight vacuum by means of a 7-mm. I14 sintered-glass filter stick and the preci itate was washed with a saturate$ solution of calcium oxalate prepared a c c o r d i n g to S t a n f o r d a n d Wheatley ( 5 ) . The washings were removed in the same manner and the precipitate was finally sucked dry by appropriate manipulation with the filter stick.
V O L U M E 22, NO. 9, S E P T E M B E R 1 9 5 0
1185
The beaker containing the precipitate and filter stick was now treated with 1 ml. of 3 N sulfuric acid and warmed on the hot plate. The filter stick was used to assist the solution of the calcium oxalate, especially on the sides of the beaker, where there was usually a tendency to upward creep of the precipitate during the washing with saturated calcium oxalate. The resultant solution was then removed under reduced pressure through the filter stick, followed by washing with distilled water. The product was collected in a second microbeaker as illustrated in Figure 1. About 4 mg. of manganese sulfate were now added and shaken to dissolve, and the solution was titrated a t room temperature (about 25’ C.), with 0.1 N o r 0.05 h‘ permanganate using a horizontal microburet. The titration was carried out for two minutes with the usual additional care near the end point. This time was required to obtain the accuracy of determination in Table I.
Table 111. Effect of Titration Temperature (King filter stick, 0.8- or 0.2-ml. microburet, and 0.10 or 0.05 N K M n 0 4 . 1 ml. of oxalate solution in each case) TheoTemp retical RepliCoefficimt C.” (CO0H)r cates Recovery Difference of Variation
Mu. 100
60 ”5
9 6 11
3.525 3.499 3.525 3.499 3.596 3.608
F
4 9
Mu.
.*fu.
%
3.553 3.551 3.529 3.527 3.593 3.618
+0.028 +0.052 +0.004 +0.028 -0.003
+0.79 +1.48 4-0.11 +0.80 -0.08 +0.28
f0.010
0.87
” 23
DlSCUSSlON
The tables show that the standard method of titration a t about room temperature with manganese sulfate as catalyst was by far the most accurate. All other titration techniques gave greater variability and a slightly high result (Table 11). Raising the temperature had, a similar effect on the results of the standard method (Table 111). The volume of solution a t the time of precipitation had a small but definite effect in raising the titer slightly and introducing a little greater variation (Table IV). Although the presence of citrate produced statistically significant differences from the expected titers, these effects were relatively small under the conditions described in Table V. Possibly the additive effect of the larger citrate concentration was due to a slight insolubility of calcium citrate in the small volume of precipitated solution (2 ml.) k e d (but see Vickery and Pucher, 6). I t has never proved serious in the author’s experience in the range indicated. These values of citrate (0 to 26 mg.) and oxalate (3.6 mg.) were chosen to cover the expected values in a research program on the metabolism of Aspergillus niger.
Table IV. Volume 10
0 :5
Table V.
Effect of Volume of Precipitating Solution
(Present standard procedure. 6 replicates) Theoretical (COOHh Recovery Difference 3.476 3.575 3.476 3,575
Coefficient
of Variation
3.490 3.593
+0.014 +0.018
+0.40 +0.50
0.21
3.488 3.586
+0.012 +0.011
+O 34
o,15
+0.31
Effect of Citric Acid on Oxalate Precipitation
(Present standard procedure. 1 ml. of oxalate solution and 1 ml. of citrate solution in each case, save with oxalate alone which contained 1 ml. of oxalate eolution and 1 ml. of distilled water. 7 replicate+. Theoretical (COOH)*, a v . . 3.627 ing.) Citric Acid Recovers Coefficient (Av.) (Av.) Difference of Variation Mg.
MQ.
,wg.
25.94
3.658 3.641
+0.031 +0.014 +O.C17 +0.027
10.67 5.55
3.24 1.34 0
3.644
3.654 3.633 3.631
+0.006
+0.007
% +O.. 86
+0.39 +o 47 +0.75 +0.17 f O . 19
0.79 n.33 0 39
0.25 0.42
0.23
Table VI.
Recovery of Oxalic Acid in Presence of Large Excess of Citric Acid
(Present standard procedure. 4 ml. of oxalate solution and 1 rnl. of citrate. 74.5 mg. of citric acid per ml. 4 replicates) Theoretical Ranie of Coefficient (C0OH)s Recovery Difference Recoveries of Variation
.\if
g
0 4176 0 4211
Mu.
Mu.
%
%
0.4155 0.4195
-0.0021 -0.0016
-0.50 -0.38
99.2-99.7 99.3-99.9
0.22
Table VI provides data on the recovery, in the presence of a rather greater excess of citrate. This concentration of oxalate (0.4 mg.) was used because it provided the practical minimum for the present improved method of oxalate determination. This weight, however, may be contained in up to 10 ml. of solution with only slight changes in efficiency (Table IV). Titration of less than 0.4 mg. of oxalic acid with permanganate provides difficulties in a poor end point appreciation and the recoveries presented here no longer hold.
Table VII.
Recoveries from Media Containing Citrate
118.3 mg. of citric acid per rnl. of media. Present standard procedure. 1 ml. of oxalate solution and 1 1 1 1 1 . of media in each case. 4 replicates) Recovery Of Coefficient Medium (C0OH)z Added Range of OC (COOH), Added ( C O O H h Difference Recovery X-ariarion Ma. Mu. Mu. Mu. % % 0.1589 1.6811 1.6778 - 0 , 0 0 3 3 - 0 . 2 0 9Y.5-100.3 0.34 0.1963 1.8516 1.8516 0.0 0 . 0 99.5-100.4 0.43 1 , 8 4 3 6 -0.0041 0.1826 1.8477 - 0 . 2 2 99.7-100.6 0.44
Table VI1 indicates that recoveries from fermented media (Aspergillus niger grown on original media containing ammonium nitrate 0.2275, potassium dihydrogen phosphate 0.10%, magnesium sulfate heptahydrate 0.025%, and glucose 10% as carbon source) were in good agreement with theory. To apply the method to very dark colored material, such as molasses or plant tissues, the material is treated as for “preparation of organic acid fraction” (Pucher et al., 4). An aliquot of the neutralized extract is then used for the determinatioq of oxalate as described above. The method has been used for routine analysis in these laboratories over the past year and has proved fully satisfactory, When twenty samples were run together the method required about 5 hours, excluding the time involved in the precipitation and subsequent standing. If a less accurate determination will sufficee.g., coefficient of variation not more than 0.9% (Table 111)then to speed up the titration, the acid solution of calcium oxalate may be heated to boiling on the hot plate with a crystal of manganese sulfate and titrated without further heating, the catalyst compensating for the falling temperature. ACKSOW LEDGMENT
The author wishes to express his sincere thanks to D. J. D. Hockenhull, late of this department, for help and advice. LITERATURE CITED ( 1 ) King, E. J., Analyst, 58, 325 ( 1 9 3 3 ) . ( 2 ) Kirk, P. L., a n d -Moberg. E. G., IND.ENG.CHEM.,ANAL.ED.,5 , 9 5 (1933). ( 3 ) Powers, H. H., a n d L e v a t i n , P., J . Bid. Chen., 154, 207 ( 1 9 4 4 ) . (4) Pucher, G . W., W a k e m a n , A. J., and Vickery, H. B., IND.ENG. C H E M . , h A L . ED.,13, 244 ( 1 9 4 1 ) . (5) Stanford, R. V., and Wheatley, A. K . X I . , Biochen. J . , 19, 710 (1926). (6) Vickery, H. B., a n d Pucher. G . W., C o n n . Agr. Expt. S t a . , Bull. 3 5 2 , 0 4 9 (1933). RECEIVED October 7. 1949.
