This constancy is significant and promising for analytical applications. DISCUSSION
Cheng has reported a molar absorptivity of 33,840 for a 1 : l zirconiumxylenol orange complex in 0.70.U perchloric acid ( 3 ) . Cheng also reports that an excess of xylenol orange causes a decrease in the molar absorptivity, which is opposite to the effect we find for semixylenol orange. The xylenol orange used by Cheng was obtained from Chemapol and was not purified, so it may have been an impure mixture. Babko and Vasilenko (1) reported a molar ahsorptivity of 52,000 for Zr(X0): but did not report the puritv of their reagent. The molar absorptivity of the zirconium-semixylenol orange complex which forms in a large excess of SXO is 59,000 a t 535 mp and is approximately constant
over the raiige of 0.2 to 1.O.V perchloric acid. Therefore, semixyienol orange is 3s sensitive or more sensitive a reagent for zirconium than xylenol orange. The monosubstituted product is easier to purify and seems to be more stable than the disubstituted product. .Icomparison with other zirconium complexes reported in a recent review (13) shows that semixylenol orange is one of the most sensitive known reagents for the determination of zirconium. Because the reagent can be used in large excess and a t high acidity, it should be an advantageous analytical reagent. LITERATURE CITED
(1) Babko, A. K., Vasilenko, V. T., Ukrain. Khim. Zh. 26, 514 (1960). (2) Cheng, K. L., Talanta 2, 61 (1959). (3) Ibid., p. 266. (4) Ibid., 3, 147 (1959). (5) ~, Fletcher. M. H.. ANAL. CREW 32. 1827 (1960).
(6)Fritz, J. S., / h i d . , 26, 1978 (19541 (7) Fritz, J. s., Johrtsoii, M.,fbkd., 27, I@ (1955). (8)Kdthaff, I. M.. '.%cid-Baue Indicators," trsna by C. Kwenblum, pp 108, 119, The Y n e n i i h b Co., Xew York, 19s.
J., C'dl. C'zeck ('hem ('onim. 178'3 (195T). Korbl, J., Prihil, H., C'henkzst-d iknl!/sl (1W). 1, J., Pr&il, R., Chem. Ind. (Londole) 19!57, 233. 12) Korbl, J., Pribil. R.. Emr. E.. Coll. Czecfi. Chem. Comm'. 22; 961 (195i). 13) Milner, G. W. C., Edwards, J. W., Ana&yst 85, 86 _ _ (1960) 14) Wekher, F. J., "The Analytical Uses of Etthylenediaminetetraacetic AeW' D. 241, Van Nostrand. Princeton, 19%: 15) h i e n , A- J., Connick, R. E., J. Am. Chen. SOC.78, 5785 (1956). RECEWEDJanuary 26, 1962. Accepted J m e 25, 1962. Support by the Air Fame W c e of Scientific Research is \ - - - - / .
gratefully aeknowledged. Taken in part from a PLD. thesis of D. C. Olson, Purdue University, 1962.
New Benzidine Procedure for Determination of Manganese in Biological Samples K. SIVARAMA
SASTRY, NATESAN RAMAN, and P. 5. SARMA
Department of Biochemistry, Indian Institute of Science, Bangalore 7 2, India
,A simple and accurate method for the determination of 0.25 to 1.0 pg. of manganese (in samples containing 1 to 4 pg, of manganese) has been developed by modifying the conditions for the reaction between permanganate and benzidine. Manganese is oxidized with potassium periodate in orthophosphoric acid and selectively estimated in the presence of excess oxidant with benzidine in formic acid. The procedure is applicable for estimation of manganese in biological samples, with recoveries in the range 97.5 to 1O6.1%,
M
IcRomTHoDs for estimation of manganese have been based on the ability of permanganate to oxidize suitable organic compounds such as N,S-diethylaniline (S), 4,4-tetramethyldiarninotriphenylmethane (4) (by catalytic oxidation), and benzidine (5) to intensely colored products. The Fore and Morton procedure (3) is very sensitive but cumbersome, and the method of Gates and Ellis (4) has been criticized as being temperamental in color development (2). One of the more convenient methods applicable to biological materials is that of Wese and Johnson ( 5 ) , based on the reaction between benzidine and permanganate, 1302
ANALYTICAL CHEMISTRY
in the absence of any other oxidizing agent. As Wiese and Johnson pointed out, the obligatory removal of sodium bismuthate in their method makes the permanganate very unstable, necessitating filtration into benzidine when the excess oxidant is to be eliminated. While the procedure is applicable for samples containing 1 to IO pg. of manganese, with samples having 1 to 5 pg. of the metal, the results are very variable. The present modification has been evolved to achieve higher precision and reproducibility in this range of manganese concentration, and the manganese is assayed in such samples by operating with aliquots containing between 0.25 and 1.0 pg. The present procedure is completely different from Wiese and Johnson's method, except for the use of benzidine. Potassium periodate is used for oxidizing manganese and the permanganate formed is selectively determined with benzidine. This has been possible, because in 83% formic acid, permanganate reacts completely with benzidine in 60 seconds, unlike periodate which requires a much longer period. APPARATUS
Manganese was oxidized a t 95" f 0.5" C. in a water bath. Colorimetry was performed in a Klett-
Summerson photoelectric colorimeter with a 42 filter, adapted for use with 3.0 ml. of solution. REAGENTS
STANDARD MASGANESE SOLUTION. Analytical reagent grade MnSO, (British Drug Houses), heated until it is anhydrous, is used t o prepare a standard solutioii in metal-free water containing 1 mg. of manganese per ml. and suitably diluted prior to use. PERIODATE SOLUTION. A 2 mg. per rnl. solution of KIOI (BDH, analytical reagent grade) is aged by keeping at 70" to 80" C . overnight and making up to original volume. The aging is essential to obtain reproducibility. The solution can thereafter be used up to 2 weeks. BENZIDINE REAGENT.Recrystallized benzidine is dissolved in 100% formic acid (Merck 98 to 100% acid purified by recrystallization a t 0" C. before use), to make a 0.1% w./v. solution. Analytical grade acids and metal-free water are used throughout. EXPERIMENTAL
Sample Preparation. Biological tissues were subjected to wet digestion and 1 ml. of with 10 ml. of " 0 3 HClOd (60%, density 1.54, Merck) per gram of sample, in 50-ml. borosilicate glass conical flasks on a sand bath. After complete evaporation,
the procedure was repeated with 5.0 ml. of a mixture of H N 0 3 and HC1 (1 to 1 by volume). Heating was stopped just before coniplete cvaporation, and the digests were made up t o suitable volume with ~ a t e ror , when excessive amounts of solid residues were present, with 5% HXO,. Aliquots containing 1 to 4 pg. of manganese were then pipetted into 5-ml. borosilicate glass conical flasks and taken to dryness a t 100" C. [This procedure is essential to remove nitric and perchloric acids, which otherwise would interfere with the assay. \Yet digestion also prevents loss of mangancse, common to dry-ashing procedures ( I ) .] Oxidation of Manganese. For standards, 1.0 to 4.0 pg. of manganese was taken in 5-1111. borosilicate glass conical flasks in a volume of 1.0 ml. Flasks containing dry residues of acid digests were charged with 1.0 nil. of water. When the acidh used contained small amounts of manganese, an acid digestion. blank was included. All subsequent operations were carefully timed, as indicated below. To the blank, 0.5 nil. of periodate was added; a stop watch was started simultaneously. The contenta were rapidly mixed and 0.5 ml. of orthophosphoric acid (89y0,Nerck) ivas run in. The flask was then rapped tightly with aluminum foil. At the cnd of 90 seconds the flask was inserted in the water bath (95" f 0.5" C.). T o the next flask, periodate was added exactly 30 seconds after insertion of the previous flask in the water bath. Other operations were exactly similar. Each flask was sequentially rwnovrd nftw 30
Table 1. Calibration Data for Estimation of Manganese
Manganese,a Pg.
Colorimeter Units* (Absorbance X 500)
0.25 0.50 0.75
41 zk 1 80 2 120 f 4 164 f 7
1.00 Manganese in each colorimeter tube. * Mean and standard deviation from. 10 replicate determinations.
Table
II.
RESULTS AND DISCUSSION
2oor
m
'5011 I25
I4
/
75
I
2
4
I
I
I
6
8
1
0
TIME, MIN.
Figure 1 . Rates of reaction of permanganate and periodate with benzidine-formic acid A. B.
Permanganate, 2 pg. Mn Periodate, 0.5 mg.
minutes in the water bath and cooled, and 0.5-ml. aliquots were pipetted into Klett colorimeter tubes. (Each colorimeter tube contained at this stage 0.25 t o 1.0 pg. of manganese as permanganate.) Color Development. Permanganate was assayed in each tube exactly 30 minutes after completion of oxidation, in the following manner : To the blank tube (29 minutes after removal from the water bath), 2.5 ml. of the benzidine reagent was added by blowing in from a pipet. The contents were vigorously shaken for 30 seconds. This tube was used to set the instrument to read zero (100% transmittance), 60 seconds after benzidine addition. All other tubes were treated sequentially likewise and color values recorded in each case, 60 seconds after benzidine addition. After the standard curve had been defined in routine assays, the inclusion of two standards with each set of samples was found adequate.
Applicability of Procedure to Estimation of Manganese in Biological Samples
No. of
Sample Aspergillus niger
mycelium
Tecoma stuns leaves Lathyrus sativus seeds
Samples Assayed" 4 5 4 4 4
Manganese Content, rg./G. Mean Range 28.82c 6.638 14.50. 4.086 2.41c
26,50-30.66 5.80-8.02 13.80-15.20 3.80-5.10 2.13-2.71
Mean Recovery of Manganese Added-with Each Sample,* 102.1 f 0.26d 97.5 Z t 3 . 5 106.1 f 1 . 8 101.8 f 1 . 5 100.0 f 1.41
Rat liver Rat spleen All assays performed irn duplicate. * Each sample assayed €or recoveries at aliquot levels in range 0.90 to 3.56 pg. of manganese and with 1.0 pg. of standard manganese added. c pg./g. dry weight. d Standard deviation of mean. e pg./g. fresh weight. 0
The present procedure has been evolved to overcome the chief drawbacks of the original method (5)namely, the errors caused by the obligatory removal of the oxidant, resulting in extreme instability of permanganate. In the present modification, oxidation of manganese has been generally based on conditions suggested by Willard and Greathouse (6), concentrations of reagents and the time factor being stepped up to ensure completion of oxidation at the low levels of manganese handled. When the conditions described herein are carefully followed, the assay is very reproducible and ensures maximal stability of permanganate formed. Ten to 12 samples can be conveniently analyzed in one run. The present procedure has been possible because of the observed differences in the rates with lvhich benzidine reacts with permanganate and periodate, in 83y0 formic acid. Fresh aqueous permanganate equivalent to 2 pg. of manganese was used for deriving curve A and 0.5 mg. of KIOd for curve B (Figure 1). Permanganate reacts completely in GO seconds, whereas KIOl begins to react much later. The blank therefore is small and small amounts of manganese can be assayed. The actual color yield shown in Figure 1, with pure permanganate only, is lower than the expected values when the present method is followed (Table I). This shows the extent of destruction that would occur in the absence of stabilizing agents. The intensity of the yellow color formed in this method obeys Beer's law and has a single peak at 430 mp, the absorption curve being symmetrical in the region 380 to 480 mp. The calibration data for standard manganese are given in Table I. The procedure has been applied for the estimation of manganese in some representative biological samples. Recoveries have been tested by including manganese a t the stage of digestion. The values shown in Table I1 indicate that the method is satisfactory and useful for the purpose. LITERATURE CITED
(1) Bentley, 0. G., Snell, E. E., Phillips, P. H., J . Biol. Chem. 170, 343 (1947). (2) ~, Coteias. G. C.. Phvsiol. Revs. 38. 503 (1958). ,r (3) Fore, H., Morton, R. A,, Biochem. J . 51, 594 (1952). (4) Gates, E. M., Ellis, G. H., J . Biol. Chem. 168, 537 (1947). (5) Wiese, A. C., Johnson, B. C., Ibid., 127, 203 (1939). (6) Willard, H. H., Greathouse, L. H., J. Am. Chem. SOC.39, 2366 (1917). I
"
RECEIVEXI for review February 12, 1962. Accepted May 28, 1962. VOL 34, NO. 10, SEPTEMBER 1962
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