per ml. of
C-CHo
from epichloro-
/, ‘\()/
hydrin in a carbon tetrachloride solution containing di-n-butyl ether (8 mg. per ml.) and 1-octene (110 y per ml.) has been detected, using this technique at 2.2 microns. I n this particular case, 10-cm. cells and the 0.0 to 0.1, 0.1 to 0.2 absorbance slide-nire on the Cary Model 14 were used. The detection limit is believed to be about 10 y per nil. of ‘C-CH?
\o/
Table V.
Molar Absorptivity of Epichlorohydrin in Various Solvents Using Cary Model 14 Spectrophotometer
1.6-1Iicron Region 2.2-Micron Region Molar A4bsorptivity Molar Absorptivity ___ Solvent X M ~ ~1.1. , E , liter/mole-cm. Xuax.,p e, liter/mole-cm. 2 207 1.65 1.644 0.214 Carbon tetrachloride 1.92 0.202 2 208 Heptane 1.645 1.35 0.196 ’7 207 Di-n-butvl ether 1 646 Methyl ethyl ketone 1.642 0.175 e Diethylcyanamide 1 642 0.182 Solvent absorbed too much in the 2.2-micron region to observe any bands. Q
in carbon
tetrachloride. I n hydrocarbon samples the limit is about 100 p.p,m. Some a o r k has also been done on the use of near-infrared to determine the purity of different c,po\id(.; To increase the accuracy over the i 1 to 27, which is usually typical of direct determinations in carbon tetrachloride, a high absorbance reference technique was used on the Cary Model 14 equipped 131th a 0 to 0.1. 0.1 to 0.2 absorbance slide-wire. Preliminary data indicate that determination of the purity of epoxides to *0.5% or better i; powble. ANALYSIS OF MIXTURES OF UNSATURATES AND EPOXIDES
It has been pointed out that both the 1.6- and 2.2-micron regions could be used to analyze mixtures of terminal epoxides and terminnl unsaturates.
Several synthetic mixtures of epichlorohydrin and 1-octene were analyzed using the Cary RIodel 14 spectrophotometer. Calibration curves for the analysis of binary mixtures, using the 2.2-micron region give good Beer’s law plots. Keither compound contributes to the absorption a t the maximum for the other in this region. The mixtures were also run in the 1.6micron region, but the two bands, epichlorohydrin a t 1.644 microns and 1octene a t 1.635 microns, overlap appreciably. Also, the molar absorptivities are lo^, indicating that a high concentration of sample is needed for analyqis. From previous work, it is evident that other types of compounds containing a terminal methylene group n-ould give better results than 1-octene when mixed Kith terminal epoxides, because most of them absorb a t shorter wave length; which are further from the epoxy band
at 1.65 microns. For instance, vinyl ethers have a band a t 1.615 microns, acrylic-type compounds a t 1.620 niicrons, and allyl acetate has a band a t 1.626 microns. Thus. it should be relatively simple to analyze mixtures of these olefins with epoxides in both the 1.6- and 2.2-micron regions. The determination of both functional groups in reaction mixtures and polyfunctional compounds by this technique should have many advantages over existing methods. LITERATURE CITED
(1) Durbetaki, A. J., A r a ~ CHEXI. . 29, 1666 (1957). (2) Goddu, R. F., Ibid., 29, 1790 (1957). (3) Henbest, H. B., Neakins, G. D.,
Xichols, B., Taylor, J. K., J . Chem.
SOC.1957, 1459.
RECEIVEDfor review January 10, 1958
A4cceptedJuly 16, 1958.
Spectrophotometric Determination of Iron,in Urine, Using 4,7Dipheny I- 1,lO- phenunt hroIine MARYIN
J. SEVEN’ and RALPH E. PETERSON
National Institute of Arthritis and Metabolic Diseases, Bethesda,
b A simplified procedure for the determination of iron in urine employs as a color agent the compound, 4,7diphenyl-1,lO-phenanthroline, which has a molar absorbancy index of 22,400 for the ferrous complex. The entire procedure is carried out in one flask. Extraction of the colored complex into isoamyl alcohol after wet ashing further increases the sensitivity and avoids interference from other ions commonly found in biological flJids. Spectrophotometric and radioactive iron recovery studies indicate that complete recovery of iron i s achieved when the proper proportions of reagents are used. For 1 to 4 y of iron, a precision to &2y0can be achieved.
2016
ANALYTICAL CHEMISTRY
Md.
iron determinations are beset TT ith problems. Some, inherent in trace metalanalyses,include the rendering large quantities of apparatus iron-free, the removal of contaminating iron from reagents, and the lack of sensitirity of color agents. Others are peculiar to iron determinations in biological materials, such as the adequate digestion of organic matter and the complete recovery of iron without interference by pyrophosphates Irhich may develop during digestion procedures (2-4, 6). The method detailed here overcomes these problems by a highly specific and simplified procedure employing 4,7-diphenyl-l,lO-phenanthroline (bathophenanthroline) as 3 color agent. Because the procedure is carRIXARY
ried out in one flask, large quantities of equipment are eliminated. Small amounts of urine are required. Reagents are readily made iron-free by redistillation or by extraction x i t h a reducing agent and the phenanthroline reagent in isoamyl alcohol. Spectrophotometric and iron-59 recovery studies indicate complete recovey of iron when the proper proportions of reagents are used. Interest has de\ eloped recently in the use of 4,7-diphenyl-1, 10-phenanthroline for iron determinations. This compound was introduced by Case (1) and shown by Smith, McCurdy, and Diehl 1 Present address, Hahnemann Medical College, Philadelphia, Pn.
( 7 ) to have great value in overcoming some of the problems encountered in the determination of iron in n ater. More recently, it was found to provide a sensitive and highly specific means for the determination of iron in small amounts of serum (6). Because i t has a molar absorbancy index of 22,400 for the ferrous bathophenanthroline ion, this reagent is a more sensitive color agent for iron than thiocyanate and other ferroin reagents (6). Smith el al. have demonstrated that extraction of the ferrous complex from a n acid solution into an organic solvent such as isoami 1. n-amyl, or hexyl alcohol, increases the sensitir ity and rules out interference b y the coninion anions and cations found in biological fluids (7'). APPARATUS AND REAGENTS
Fume ducts (refluxing still heads) t o fit 250-nil. Erlenmeyer flasks ( 8 ) . Concentrated nitric acid, redistilled in an all-glass apparatus and stored in borosilicate bottles. Sodiuni acetate, saturated aqueous solution, reagent grade (bIerck &- Co., Inr.), made iron-free by adding a few milliliters of 20% hydroxylamine hydrochloride (Eastman Chemical Products, Inc.) and shaking n-ith 5 to 10 nil. of the bathophenanthroline reagent in isoamyl alcohol, stored in a polyethylene container. Bathophenanthroline, 0.0025M in a good reagent grade or redistilled isoamyl alcohol (G. Frederick Smith Chemical Co.). Standard iron solutions. Dissolve iron wire (99.87,) in a few milliliters of redistilled concentrated nitric acid and redistilled or reagent grade concentrated hydrochloric acid. Dilute with ironfree water to give a concentration of 1 mg. per ml. From this solution dilute standards may be prepared to contain 1 to 2 y per ml. These should be preserved with a few milliliters of reagent grade (Merck & Co., Inc.) concentrated sulfuric acid. Water used in the reagents, determinations, and for TI asliing purposes must be redistilled in an all-glass apparatus or passed through a satisfactory ion exchange column, then stored in borosilicate g1a.s or polyethylene containers. PROCEDURE
All glassn are must he scrupulously cleaned with conrentrated nitric or hydrochloric acid and rinsed three or four times 11-ith iron-free n ater. Pipet 5 t o 10 nil. of urine into a 250ml. borosilicate Erlenmeyer flask, add 0.75 ml. of concentrated reagent grade sulfuric acid (Merck 6- Co., Inc.) and 5 ml. of redistilled concentrated nitric acid. Cover flask with refluxing still head and digest on hot plate (Lindberg) a t an intermediate temperature (approximately 250" C.) until the thick brown fumes almost disappear and a nearcolorless solution remains. Cool partially and n-ash down still
head and inside of flask with iron-free water. Add 5 ml. of 30% hydrogen peroxide (Merck &- Co., Inc., Superoxol) through the still head and heat at an intermediate temperature until ebullience disappears. Cool partially and add 2 ml. of 30% hydrogen peroxide. Heat for 1 hour a t an intermediate temperature (not over 250' C.), periodically lifting still heads to permit accumulated fluid to run to the bottom of the flask. If heat is unerenly distributed on the hot plate, rotate positions of the flasks a t least once during this heating period. Cool partially, wash down still head, and remove it. M7ash down inside of neck and sides of flask until a t least 20 ml. of iron-free water have been added. Add 0.2% potassium permanganate dropn-ise (usually 1 t o 3 drops) until a faint pink color persists. Add 3 ml. of 20y0hydroxylamine hydrochloride; mix b y rotation, then add 15 ml. of saturated sodium acetate and mix by rotation. Add 4 ml. of bathophenanthroline in isoamyl alcohol and immediately cover flask with glass stopper. Shake vigorously by inversion until no further red color development is apparent, usually 60 to 90 seconds. When alcohol and aqueous phases have completely separated, aspirate the aqueous and discard. Cover the flask immediately n-ith the glass stopper. Draw off enough isoamyl alcoholbathophenanthroline solution to fill a cuvette and read a t a wave length of 533 niM in a Beckman D U spectrophotometer against a reagent blank set a t 100% transmittance. Carry the reagent blank and standards containing 2 to 4 y of iron through t h e above procedure with the unknown samples. Carry out all determinations in triplicate. CRITICAL EVALUATION
Acid Digestion. T h e urine mas digested in Erlenmeyer flasks on a hot plate rather t h a n in Kjeldahl flasks because spattering from t h e neck occurs n-ith t h e latter type, even on careful heating. Covering t h e flasks n i t h refluxing still heads provides three important functions; adequate cover, a n easy means of addition of reagents, and an adequate fume vent ivhich prevents the loss of fluid droplets. -4n ordinary glass funnel can be used, but droplets may spatter through the small opening, and the narrow portion tends to become partially filled with condensation fluid or reagents, n hich destroys adequate vent action. Funnels also require reagents to drop suddenly into the center of the digestion mixture rather than slonly down the side of the flask. Hot plates of the Lindberg type supply adequate temperature ranges and easy adjustments, but have the disadvantage that the distribution of heat to the plate surface is unequal. This may be overcome by periodically rotating the
flasks or covering the surface of the hot plate n i t h a heat-transmitting material. The volume of the urine sample should be selected according to the expected iron content, usually 5 to 10 ml. fur samples rvithin a norniiil range. Sulfuric acid is added to provide the residue necessary for a wet digestion. The 0.75 nil. provides the minimum necessary to cover the Lottoni of the flask durin2 the heating process and minimizes the ainount of sodium acetate required to raise the p H later in the procedure. Kitric acid is added in as sniall a n amount as is consistent ITith complete digestion, usuall>- one half the urine volume, because even redistilled acid contains s m e iron. Glass beads or boiling chips are not necessary if the digestion is carried out a t an intermediate temperature. If considerable charring occurs during the digestion, it may be necessary to add further increments of nitric acid and continue the digestion until the solution is clear and colorless or s h o w only a light yelloiv tinge. A t this point, the refluxing still head and inside of the flask are washed down with n a t c r to return the spatterings of the digest to the bottom. Hydrogen peroxide is the oxidizing agent used to complete the digestion because i t can be completely removed from the final digest b y heat and oxidation with permanganate. The peroxide is added in tiyo stages, but further increments may be added, if necessary, to produce a clear and colorless final digest. Partial cooling before each increment prevents bumping. Color Development. T h e heating period of 1 hour is designed t o remove excess peroxide. Heating for shorter periods a t a higher temperature is effective b u t results in some loss of sulfuric acid, greater creeping of t h e acid u p t h e sides of t h e flask, and a resultant adherent precipitate of inorganic salts on t h e bottom of t h e flask. Enough water is added in wishing d o n the refluxing still head and sides of the flask to provide a t least 20 ml. of solution, because the next rragents to be added may be destroyed b y concentrated acid solutions. Potassium permanganate is added dropwise to ensure complete ovidation of any residual traces of peroxide. This represents the only variable quantity in the method, but because the iron content of this reagent is very low and only small amounts are required, the variation is not significant. If heating has been inadequate a t 250" C., large amounts of permanganate are required, and this reduced permanganate imparts a yellow color to the solution which is extracted by the isoamyl alcohol but does not interfere with readings a t 533 mp. However, enough iron may be added with large amounts VOL. 30, NO. 12, DECEMBER 1958
2017
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
