of permangnate to give erroneous results. The addition of the hydroxylamine hydrochloride reduces the slight excess of potassium permanganate and ensures that the iron is present in the ferrous form, because bathophenanthroline nil1 not form a complex with iron in the ferric state. Three milliliters of 207, hydroxylamine hydrochloride are sufficient and provide a slight excess. Other reducing agents, such as 207, hydrazine hydrochloride, may be used. Thioglycollic acid is less desirable because of the objectionable odor. Eastman reagent grade hydroxylamine hydrochloride has been found relatively low in iron content. Studies with iron-59 added to urine show that 10 ml. of saturated sodium acetate are necessary for the complete extraction of iron from the aqueous solution into the bathophenanthroline-isoamyl alcohol reagent. However, for complete development of color b y the ferrousbathophenanthroline complex, 14 to 15 ml. of acetate are necessary. This amount brings the p H of the mixture to about 5.5, ne11 within the range of pH 4 to 7 for formation of this complex. Bathophenanthroline is readily soluble in the amyl and hexyl alcohols. I n this procedure, 4 ml. of a 0.0025X solution of bathophenanthroline in isoamyl alcohol are added and the flask is immediately covered with a glass stopper. Smaller amounts of this solution may be used, but 4 ml. provide sufficient sensitivity for microanalysis and more ease in pipetting from the flask. Where the quantity of iron exceeds 7 to 8 y, larger amounts of the bathophenanthroline-isoamyl alcohol reagent will be required. This may be estimated from the intensity of the red color after alcohol extraction. There
Table I. Recoveries of Iron Added to 8 MI. of Iron-Free Urine from Patient with Iron Deficiency
Bdded, 1.0
y
Absorbance 0.102 0.104 0.104
2.0 4.0
0.201 0.202 0.203 0.403 0.404 0.406
Recovery, 70 100.0
101.9 101.9 98.5 99.0 99.5 98.7 99.0 99.5
may be objections to the use of isoamyl alcohol because of the penetrating odor, but this is overcome when mechanical pipetting is carried out in a well ventilated hood. n-Amyl and n-hexyl alcohols are somewhat less odorous but contain larger amounts of iron as a contaminant. Smith et al. suggest a distribution coefficient of approximately 1570 to 1 for the ferrous complex of bathophenanthroline between isoamyl alcohol and water (Y). Iron49 studies indicate that the extraction of iron into a 0.0025M solution of this solvent is complete. Immediate covering of the flask is important, because iron-59 studies show further t h a t evaporation of isoamyl alcohol occurs in a n unstoppered flask and may give erroneously high results. Aspiration of the aqueous leaves the organic solvent in the original flask, from which it may be pipetted, transferred to standard cuvettes, and read on a Beckman DG spectrophotometer a t 533 mp. The color has been shown to be stable for long periods (6). The method described is a simplified one, in that the entire procedure is carried out in one piece of equipment, a n Erlenmeyer flask. It employs a color agent m-ith a molar absorbancy index
of about 22,400 for the ferrous complex and gives further sensitivity by the extraction of iron into a n organic solvent. It is specific for iron a t a p H of 4 to 7. While copper is extracted from the aqueous digest a t a n acid pH, i t does not produce its yellow cuprous-bathophenanthroline color below p H 7 . Cobalt also gives a yellox color with this agent but is not extracted a t an acid pH. Smith el al. have shom-n that other cations do not interfere (7'). The development of pyrophosphates has not been a problem in this procedure, probably because the digestion is carried out at a moderate temperature. Each step in this method was evaluated by studies n ith iron-59 added to urine. Spectrophotometric recovery studies with 1 to 4 y of iron added to iron-free urine from a n iron-deficient patient are detailed in Table I and show recovery values of 98.5 to 101.9%. These compare favorably with recovery studies by radioactive iron analysis. Under carefully controlled conditions, it has been possible to attain a precision to =t2% in the determination of 1 to 4 y of iron. LITERATURE CITED
(1) Case, F. H., J. Org. Chem. 16, 1541-5
(1951). (2) Farrar, G. E., J . Bzol. Chem. 110, 685-94 (1935). (3) Fortune, Mf. B., Mellon, 11. G., IND. ESG.CHEM.,AXAL.ED.1 0 , 6 0 4 (1938). (4) Jackson, S. H., Ibid., 10,302 (1938). (5) Peterson, R. E., h . 4 ~ .CHEM. 25, 1337-9 (1953). (6) Sandell. E. B.. "Colorimetric Deter-
' EXQ. CHEM.,AG'AL. ED.7,'301-5 ('1935). RECEIVED for review February 14, 1988. A4cceptedAugust 13, 1958.
Methods of De Saint Venant and Mohr and Use of Statistical Criteria ANNE G. LOSCALZO and A. A. BENEDETTI-PICHLER City College, New York, N.Y., and Queens College, Flushing, N.Y.
b Parallel application of the methods of de Saint Venant and Mohr was used for comparing the efficiencies of classical and modern statistical treatment of data. It appears that the method of de Saint Venant is at least equal to that of Mohr with respect to accuracy and precision. Adjustment of the pH for Mohr titration by adding an excess of bicarbonate is detrimental to precision. The classical 2018
ANALYTICAL CHEMISTRY
approach and the more laborious modern statistical treatment of the data seem to give essentially equal information. The method of d e Saint Venant deserves consideration for the precise determination of halides.
D
SAINTYEI~ANT ( 2 )points out that he may have been the first to use a change of color for end point detection. I n 1819, he determined chloride in saltE
peter brine by mixing the brine Ivith an equal volume of lime water and titrating with silver nitrate until the color of the mixture changed from bluish white to fawn. Obviously, the end point indication was obtained b y the precipitation of silver oxide a t a p H of 12.3. This titration with the p H adjusted to 11.3 ( 1 ) is compared in the folloving \yith the method of Alohr. It is also s h o m that rapid preparation of
Table I.
.80
Series A
5.000 10,000 25.00 50.00 a, ml.
4 546 8 933 22 168 4-1 21 +o 120
p.60
2+ -2 d W
2ul
y,
5, Sample Volume, 311.
.40
b
0 A0 0 0
0150 0930 076 0.049 0 0027 0 0017
s, ml.
K , ml.
i
La
E
da
.20
La, ml. d b , ml. X
.oo 0.0
M L . CHLORIDE
I.
