ANALYTICAL CHEMISTRY
402 minimizes vaporization of titanium and subsequent gettering of evolved gases, and seems to maintain more uniform temperature conditions within the working zone of the extraction crucible. Evidence of the effectiveness of the above-discussed techniques is given by the results of the vacuum-fusion analyses of titanium listed in Table I. Samples 0-1 and 0-2 were double arc-melted iodide titanium with no titanium dioxide addition. The nnalyses of these samples are the base oxygen levels for the arcmelted standards and are a measure of the original oxygen content of the metal plus the oxygen picked up during arc melting. The estimated total oxygen of each standard was the summation of this base value and the weighed addition of oxygen as titanium dioxide. It is general experience that the values obtained for a multiplicate series of vacuum-fusion analyses agree to about 10 relative yo. However, the analyses in the present work, using either
method of analysis, agree somewhat better than this. The experimental results also agree with the estimated oxygen contents within 10 relative 70with an over-all average of about 6 relative c/o. LITERATURE CITED
(1) Derge, G., J. Metals, 1, 31 (October 1949). W., ‘I. Trans. Am. Soc. MetaZs, 41, 870 (1949). (2) iMallett, > and Griffith, C. B., “Vacuum-Fusion Analysis of (3) Mallett, M. W., Molybdenum,” Trans. A m . SOC.Metats, in press. (4) Sloman, H. A,, and Harvey, C. A. (appendix by Kubaschewski, O.),J . Inst. Metals, 80, 391 (1951-52). (5) Walter, D. I., ANAL.CHEM.,22, 297-303 (1950). RECEIVED for review July 21, 1953. Bccepted September 21, 1953. The work described in this paper was part of a program sponsored a t Battelle Memorial Institute by Aeroneutical Research Laboratory, Wright Air Development Center, under Contract No. AF33(616)-103, Expenditure Order No. R-463-5BR-1.
Rapid Microcolorimetric Determination of Dissolved Oxygen W. F. LOOMIS
.
The Loomis Laboratory, Greenwich, Conn.
T
HE Winkler method of determining dissolved oxygen requires samples of water as large as 250 ml. (1, 10-12’). hlthough several micro-Winkler modifications have been described for use with 1-to 10-ml. test samples, they all require the construction of specialized syringe-pipets and take 15 to 30 minutes per analysis (3,4, ?‘,9). The present paper describes a colorimetric method for use with 0.5-ml. samples, which may be completed in about 1 minute. No specialized equipment need be constructed, as a standard tuberculin syringe is adapted for direct use in a Beckman DU spectrophotometer, an instrument so sensitive that changes in concentration as small as 5 2 % saturation Kith air ( f 0 . 2 mg. of oxygen per liter) may be detected over a range that extends from fully anaerobic (0%) to fully aerobic (100%) conditions. The principle of the method lies in measuring the color change from yellow to red that occurs when a solution of reduced indigo carmine is partially oxidized by the oxygen in the test sample, a modification of the color change from yellow to blue that takes place on its complete oxidation as described by Efimoff (2’, 8). APPARATUS
Xitrogen tank, fitted with low pressure reducing valve and gage. A 1-ml, tuberculin syringe, graduated in 0.01 ml., is used. Provision is made for mixing the contents of the syringe on shaking by inserting a small steel ball (or nail head) in the barrel of th; syringe. To avoid exposing the sample to air during the spectrophotometric measurement, the syringe is placed directly (after its needle has been removed) in the borosilicate glass absorption cell (10-mm. light path) of a Beckman DU spectrophotometer. Provision is made for locking the syringe in place by cementing a small block of wood, through which a hole has been made just large enough to hold the neck of the syringe, to the bottom of the borosilicate glass cell; and winding a 5-mm. strip of tape around the middle of the syringe (just below the 0.5-ml. mark) until the barrel of the syringe fits snugly inside the absorption cell. The syringe may be made to lock into position, preferably one with as clear a light path as possible, by making this bushing of tape oval in cross section with vertically placed strips of tape applied to one side. The neck of the syringe and the steel ball are masked from the light path with a 1-em. piece of black tape applied across the front of the lower portion of the absorption cell. Beckman D U Spectrophotometer. As the height of the syringe precludes closing the sample compartment with its usual cover, a substitute is made by stapling 2 inches of black cloth to the edges of a 4 X 5 X 5 inch lightrtight cardboard box which is placed over the syringe during determinations.
Glass bottles are fitted with rubber caps through which No. 22 h podermic needles may be inserted repeatedly without leakage o f air. Sixteen-ounce narrow-mouthed acid bottles with rubber caps (Fisher Scientific Co. 2-922 and 3-225) are very satisfactory; 60-ml. serum bottles (3-220) may also be used. REAGENTS
Sufficient glucose and potassium carbonate are added to a 1.0% solution of indigo carmine (National) to make it 1% with respect to each substance. A serum bottle is half-filled with this solution, the rubber cap wired in place, and the remaining air space flushed with tank nitrogen for 10 minutes through two No. 22 hypodermic needles, after which it is left under 5-pound pressure of nitrogen. The reagent is reduced by incubating it for about an hour in a water bath a t 80” to 90°C. PROCEDURE
A blank absorbance value is obtained by filling the syringe exactly to the 0.50-ml. mark with reagent alone. The experimental absorbance value is determined by filling the remainder of the syringe to the 1.00-ml. mark with a sample of the water to be tested. 2.0
1.6 h
I
1
e
e
0
PO 40 60 80 % SATURATION WITH AIR
100
Figure 1. Calibration Curve Showing Essentially Linear Relationship of Absorbance at 580 MH to Per Cent Saturation with Air
V O L U M E 26, NO. 2, F E B R U A R Y 1 9 5 4 I n practice, the instrument is originally adjusted to zero with distilled water in the syringe. Subsequent drift is prevented by taking occasional check readings against a second borosilicate glass cell containing a dilute solution of indigo carmine, whose absorbance should not change with time. A determination is then made by rinsing the syringe a t least twice with the reagent, filling i t to about the 0.80-ml. mark, shaking it to effect mixing, removing the needle, which is then blown free of excess reagent, wiping the barrel of the syringe with a piece of Kleenex, locking it into the prepared absorption cell, placing the light-tight box in position, and determining the absorbance at 580 mp. The syringe should always be held vertically with the needle uppermost when reagent is drawn into the barrel as well as when its contents are injected into an empty 60ml. serum bottle.
540mu
610rnu
403 580 mp. Being essentially linear, such a curve may be routinely constructed by simply connecting the zero point to the absorbance value obtained with a sample of 100% saturated water, preferably obtained in duplicate. For highest accuracy the 100% saturated test sample of water should be of the same composition as the experimental and should have been equilibratrd with air overnight in a shallow dish a t the same temperature and pressure as the unknown. Saturation values between 0 and 1 0 0 ~ omay be obtained if desired, as in Figure 1, by filling the syringe with varying percentages of reagent (0%) and fully saturated (100%) water. Care must be taken in such measurements to subtract appropriate fractions of the blank absorbances from the experimental. For euample, a 2070 saturation value is obtained by subtracting 90% of the blank absorbance from the experimental value obtained by filling the syringe nith O.!)O ml. of reagent and 0.10 ml of 100% saturated water. I)ISCUSSION
600
500
WAVE
9700
LENGTH, M p
Figure 2. .\bsorption Spectra Chromogens of Indigo Carmine
of
.4. Red partially oxidized chromogen B . Blue fully oxidized chromogen Values in terms of 70absorbance of peak values. .4 was obtained by reducing B with alkaline glucase until the 620 peak had essentially disappeared. When a dilute solution (0.002%)of indigo carmine such as B was completely reduced with dithionite. no measurable absorption waa observed over the range 500 to 700 m p
After the b h k absorbance has been measured, thr nredle is replaced on the syringr and the lunger advanced to eiartly the 0.50-nil. mark. -4stiong light gehind the syringe considerably increases the accuracy of this placement. A sample of the water to be tested is now drawn up into the syringe to exactly the 1.00-ml. mark, and its absorbance is determined without delay as the red color, which develops immediately, fades ~ 1 0 with ~ 1time ~ a t approximately 17, a minute. Duplicate determinations should check each other to within 2 to 5Yc, depending on the ability of the operator to prevent contaminating the reagent with atniospheric oxygen. CALCULATION
Thr net absorbance of a test sample is obtained by suhtmcting one half of the blank absorbance from the experimental. The per cent saturation with air of the unknown is obtained by dividing its net absorbance by the net absorbance of a sample of fully saturated water and multiplying the result by 100. The value of the net absorbance of 100% saturated water depends on the exact concentration of the indigo carmine in the reagent. Rawson's nomogram (6,6,11) may be used, if desired, to convert per cent saturation results to absolute concentrations of dissolved oxygen expressed as either milligrams or milliliters of oxygen per liter.
The chief difficulty in measuring the 0.1 to 5.0 y of oxygen dissolved in the small test sample of water lies in the danger of eontaminating the sample with oxygen from the air. This is avoided by carrying out the reaction, including its spectrophotometric measurementj in an airtight syringe and by using a hermetically sealed reagent bottle kept under positive pressure of nitrogen. As some air is unavoidably iritroduced into the reagent bottle on repeated usage, the dye is made up in a continuously acting reducing solution that returns the rragent on standing to the fully reduced state. The reducing system must not be so strong, however, as to cause the red color to fade rapidly during a determination. An adequately stable red color, one that fades only about 1% per minute, is obtained by balancing the amounts of dye and glucose in the reagent so that only a gentle reducing system remains after the initial reduction of the concentrated indigo carmine has taken place. Adjusting the strength of the reducing system by lowering the pH (11.5 with potassium carbonate) was found to introduce a delay in the tlevelopmen t of the red color. The UPP of methylene blue as redox indicator was found to be unsatisfactory, as reported by Efimoff ( 2 , 8 ) , as the leuco dyt: is uiistable under the alkaline conditions necessary for its rapid r+ osidat ion. Indigo carniinrl \vjis satisfactory in this respect, but unlike methylene blur, formcd a red fiemiquinone on passing from the reduced form to the blue of the fully oxidizeil dye. Tho absorption peaks of this t\vo-p?iase color shift are illustrated iri Figure 2 . The only method of obtaining a linear color changc, therefore, seemed to lie in isolating the yellow-to-red shift that :iccompanies its partial oxidation to the semiquinone. This was accomplished by increasing the (wnwntration of the dye to 1 yo, where even the oxygen dirxolvrd in a 100% saturated sample of water could oxidize only i %of the reduced reagent, and by enipirically choosing a wave length slightly above the red pe:~k, such that an essentially 1ine:ir curve r r d t e d as in Figure 1. I t was observed that wave lengths above 5SO mp resulted in curves that were concave u p m r d s ; below 580 mp, concave downwards. The use of as conrentratcd a dye solution as 1 % was made possible by the observation that reduced indigo carmine is considerably more soluble than the oxidized form.
CALIBRATIOX
As the molar concentration of 10070 saturated water (10 mg. prr liter) is 0.33 X 10-3AlIwith respect to oxygen, a 1% solution of indigo carmine (21.5 X 10-3M) is over 60 times as concentrated. Since one molecule of oxygen can oxidize four molecules of dye to the semiquinone, even 100% saturated water oxidize? only one out of fifteen molecules (7%) of indigo carmine from yellow to red. As a consequence, the blank absorbance with an oxidized sample is essentially the same (93%) as that for a reduced one.
Figure 1 illustrates the almost linear relationship that exists between per cent saturation with air and net absorbance at
iilthough the reaction described here is as specific for dissolved oxygen as the Winkler method, compounds capable of oxidizing
404
ANALYTICAL CHEMISTRY
or reducing indigo carmine, such as nitrates, chlorates, nitrites, iron salts, and sulfites, will interfere with the method unless previously removed (1). Calibrating the reaction by constructing a linear curve from the net absorbances at 0 and 100% saturation as described here provides a simple and rapid method adequate for most purposes; the slight change in blank absorbance between reduced and oxidized samples introduces an error of less than 1%. The practical difficulties involved in preparing a graduated series of water samples of known oxygen content, protected from the air and in sufficient quantity for determination by the Winkler method, would seem to make this method less advisable for routine use. LITERATURE CITED
(1) Am. Pub. Health Assoc., New York, “Standard Nethods for the Examination of Water and Sewage,” 9th ed., 1946.
(2) Efimoff, W. W., Biochem. Z., 155, 371 (1925). (3) Fox, H. XI., and Wingfield, C. A,, J . Ezptl. Bid., 15, 437 (1938). (4) Kirk, P. L., “Quantitative Ultramicroanalysis,” ii’ew York, John Wiley & Sons, 1950. (5) Ramon, D. S., Limnological Soc. Am., University of Michigan, Ann Arbor, LIich., Spec. Pub., 15, 1944. (6) Ricker, W. E., Ecology, 15, 348 (1934). (7) Roughton, F. J. W., and Scholander, P. F., J . Biol. Chem., 148, 541 (1943). (8) Snell, F. D., and Snell, C. T., “Colorimetric Methods of Analysis,” p. 137, S e w York, D. Van Nostrand Co., 1945. (9) Thompson, T. G., and Miller, R. C., Ind. Eng. Chem., 20, 774
(1928). (10) Treadwell, F. P., and Hall, TV. T., “Analytical Chemistry,” 9th ed., Vol. 11, p. 700, Xew York, John TViley & Sons, 1951. ( 1 1) Welch, P. S., “Limnological Methods,” p. 366, Philadelphia, Blakiston Co., 1948. (12) Winkler, L. W., Ber., 21, 2843 (1888). RECEIVED for revicw June l:, 1953. Accepted October 10, 1953.
Determination of Phenanthrene in Coal-Tar Products LEENDERT BLOM
and
WILHELMUS J. VRANKEN
Staatsmijnen in Limburg, Central Laboratory, Geleen, The Netherlands
E
XISTIKG methods for the gravimetric determination of phenanthrene via phenanthrenequinone give unreliable results, chiefly because of the shortcomings of the procedures recommended for oxidation of phenanthrene to the quinone with iodic acid. The determination of phenanthrene in coal-tar products by chemical methods has not been studied extensively. Williams (9) suggested a method based on the ovidation of phenanthrene. The phenanthrenequinone produced is precipitated as toluphenanthrazine in an acetic acid medium with 3.4-tolylenediamineJ and the precipitate is filtered, dried, and weighed. X correction of about 15%, found by experiments, is added, being the weight of the toluphenanthrazine in solution. According to Williams, the oxidation is quantitative. KhmelevskiI and Postovskii (6) reported an improvement of Williams’ method. The oxidation procedure is essentially the same; however, the resulting quinone is dissolved in a saturated sodium bisulfite solution, forming an addition compound. After filtration the quinone is precipitated with ammonia and potassium permanganate, filtered, dried, and weighed. S o solubility correction need be made. These authors also state that the oxidation of phenanthrene to form phenanthrenequinone is quantitative. KhmelevskiI and Levin (4)suggested a second improvementj precipitating the quinone from the bisulfite solution with an aqueous solution of o-phenylenediamine dihydrochloride. The difference from Williams’ method is the bisulfite step, which renders the solubility correction superfluous. I t is remarkable that these authors introduce a correction factor (1.136) due to the incomplete oxidation to form phenanthrenequinone, which they ascribe to iodination. Essentially the same method was reported by KhmelevskiI and Postovskii (5), who also used the correction factor mentioned above. Pavolini ( 7 ) used chromium trioxide in glacial acetic acid as an oxidant, and estimated the resulting phenanthrenequinone gravimetrically as the cobalt complex of the monooxime. DEVELOPMENT O F METHODS
Melting points were taken on the proposed standard apparatus developed by W. M. Smit for the Stichting Centraal Instituut voor Physisch-Chemische Constanten in the Netherlands. Materials. The phenanthrenequinone had a melting point of 209.2’ C. An estimation with 2,4-dinitrophenylhydrazine showed 99.1% phenanthrenequinone.
A coal-tar product chiefly composed of anthracene, carbazole, and phenanthrene was dissolved in benzene and successively extracted with 72 and 90% sulfuric acid to remove carbazole. The solution was concentrated, most of the anthracene crystallizing out. After filtration, crude phenanthrene was obtained by evaporation. This was treated three times with maleic anhydride (to remove anthracene), followed by a threefold recrystallization from ethyl alcohol and drying in vacuo over calcium chloride. The phenanthrene obtained in this way had a melting point of 99.0 “C. The ultraviolet spectrum of the sample is shown in Figure 1 and is in excellent agreement with the spectrum shown in the compilation of ultraviolet spectral data of the American Petroleum Institute ( 1 ) except in the case of curve C, where the authors used a concentration that was ten times larger to obtain nearly the same curve. They believe this discrepancy is due to a printer’s error. Determination of Phenanthrenequinone. A small number of preliminary experiments showed the method of KhmelevskiI and Levin (4)to be the most promising, with regard to speed and accuracy. This method is based on the reaction of phenanthrenequinone, dissolved in a saturated solution of sodium bisulfite, with o-phenylenediamine dihydrochloride to form phenanthrophenazine (I), which is very insoluble in the reaction medium.
I The original method of Khmelevskiy and Levin fied as described below:
( 4 ) was modi-
The acetic acid solution of the uinone was cooled before the sodium bisulfite solution was ad?ed. The results were made higher and more reproducible in this way. The o-phenylenediamine dihydrochloride was added as an aqueous solution instead of in the solid form. The precipitated “azine” was dried a t a temperature of 120’ C. instead of 105’ C. I n this way much time is saved. Twenty-four hours of heating a t 120’ C. did not alter the weight of a 400-mg.
