was added rapidly until the indicator was green. After the needle on the millivolt scale assumed a constant value, additional titrant was added in increments of 0.1 ml. The end point was determined by noting which 0.1-ml. increment gave the largest change in millivolts. At the end point the indicator gradually changed from green to yellow. In the few cases in which i t was tried, back titration with sodium acetate in acetic acid was satisfactory. Figure 1 shows the potentiometric infections obtained for the titration of two compounds. ACKNOWLEDGMENT
F. E. Cislak, Reilly Tar and Chemical Corp., Indianapolis, Ind., gave us
generous samples of many of the Noxides studied. For this help we are very appreciative.
(8) Zbid., pp. 243-53. (9) Wimer, D. C., ANAL.CHEW30, 77-80 (1958). (10) Ibid., p. 2060. ( 1 1 ) Zbid., 34, 873 (1962).
CHESTER W. MUTH ROBERT S. DARLAK H. ENQLISH WILLIAM ALLENT. HAMNER
LITERATURE CITED
(1) Burton, H., Praill, P. F. G., J. Chem. SOC.1950, 1203-6. (2) Ibid., pp. 2034-8. (3) Fritz, J. S., “Acid-By; Titrations in Nonaqueous Solvents, p. 13, a.
Frederick Smith Chemical Co., Columbus, Ohio, 1952. (4) Furukawa, S., Yakugaku Zasshi 79, 492-9 (1959); C . A . 53, 180296 (1959). (5) Jaffe, H. H., Doak, G. O., J. Am. Chem. SOC.77, 4441-4 (1955). (6) Mackensie, H. A. E., Winter, E. R. S., Trans. Faraday SOC.44, 159-70 (1948). (7) Zbid., pp. 171-81.
Dept. of Chemistry West Virginia University Morgantown, W. Va. I n part from Robert S. Darlak’s M.S. Thesis at West Virginia University (1961).
Robert S. Darlak held an N.D.E.A. Fellowship, and William H. English and Allen T. Hamner held N.S.F. Summer Undergraduate Fellowships at West Virginia University during 1960 and 1961, respectively.
Spectrophotometric Ultra microdetermination of Inorganic Phosphorus and Lipide Phosphorus in Serum SIR: Adaptations of the Fisk-SubbaRow microprocedure (6) have been reported (4, IO, 11), but improvement in its sensitivity is desired. Heat has been applied to intensify the color of the mixture (2), and the method has recently been revived for a sensitive phosphorus assay in lipide column chromatography (1). The procedure described in this paper’ involves: (1) increasing the acidity of the sodium trichbroacetate (TCA) filtrate with relatively strong sulfuric acid and a d a p e ing the heating modification of Bartlett ( I ) for a sensitive inorganic phosphorus assay; (2) providing simultaneous lipide extraction after the TCA aliquot is removed, since the plasma phospholipide can be quantitatively precipitated by TCA (13) and extracted by alkalinealcoholic solution (14). I n the method described, the amount of serum used is approximately l/m of that required by the Fisk-SubbaRow procedure. PROCEDURE
Reagents and Materials. Hydrogen peroxide (30%, Baker’s, phosphorus-free) and ammonium molybdate (5% in deionized water) are used. T h e Fisk-SubbaRow reagent is prepared by adding 250 mg. of purified l-amino-2-naphthol-4-sulfonic acid (Eastman Organic Chemicals) with mechanical stirring to 100 ml. of freshly prepared 15% sodium bisulfite (anhydrous), followed by 500 mg. of anhydrous sodium sulfite. The solution is filtered and stored in a dark bottle. It is freshly prepared weekly. All the tubes are soaked in 5Oy0 nitric acid overnight, rinsed with distilled deionized water five times, and dried. 1164
ANALYTICAL CHEMISTRY
Inorganic Phosphorus. From 5 to 10 pl. of serum is added to 100 pl. of water in a 10-ml. centrifuge tube. After mixing, 40 pl. of 40% trichloroacetic acid is added, mixed with a mixer (Vortex Jr.) for 1 minute, and allowed to stand for 5 minutes in an ice water bath. The mixture is centrifuged for 15 minutes a t 2500 r.p.m. in a refrigerated centrifuge and 100 pi. of the supernatant fluid is transferred into another centrifuge tube. Then 300 pl. of 10N sulfuric acid, 250 pl. of water, and 200 p l . of 5’% ammonium molybdate are added. After mixing, 50 pl, of FiskSubbaRow reagent is added a t the bottom of the tube and mixed well. The solution is placed in a boiling water bath for 7 minutes and then cooled in an ice water bath. The color is read a t 830 m r with a Beckman spectrophotometer with a red-sensitive phototube. A microcuvette, 4 X 10 x 3 mm. (Pyrocell Manufacturing Co.), is used. A standard solution containing from 0.1 to 1.0 pg. of phosphorus is made for the preparation of the standard curve. In an alternative procedure for the Coleman Jr. spectrophotometer. 20 pl. of serum in 100 pl. of water is precipitated with 30 fil. of 50% TCA. The final color mixture is read a t 700 mp, using a microspace adapter (Coleman 6-319). Lipide Phosphorus. After the supernatant fluid is decanted, the precipitate is washed twice with 0.5 ml. of 5y0 TCA. Then 0.2 ml. of 0.2N potassium acetate in absolute ethanol is added to the precipitate and mixed well with the mixer for 1 minute. The tube is allowed to stand 10 minutes a t room temperature, and the contents are mixed and centrifuged. The supernatant fluid is carefully transferred to a borosilicate glass centrifuge tube. using a capillary pipet with constricted end. The precipitate
is re-extracted twice with 0.3 ml. of absolute ethanol and the extract is added to a corresponding centrifuge tube. The collected lipide extracts are evaporated to dryness, 0.5 ml. of 10N sulfuric acid is added, and the extracts are placed in an oven at 230” C. for 10 minutes. After the tube is cooled, 1 drop of H202is added and the tube is placed back in the oven for 5 minutes. Excess peroxide is removed bv adding 1 drop of 5% urea solution and reheating for 5 minutes. When the tubes are cool, 1.3 ml. of water and 0.4 ml. of 5% ammonium molybdate are added to the digest. After mixing, 100 pl. of FiskSubbaRow reagent is added, and the procedure for inorganic phosphorus determination is followed. A standard curve must be prepared. RESULTS AND DISCUSSION
A known control serum (Versatol) was diluted to various concentrations and used to compare the ultramicromethod with the Fisk-SubbaRow method. Each filtrate was individually assayed according to the procedure outlined in Table I. The color produced by the two methods was read both a t 830 rnb with the Beckman DU and a t 700 mp with the Coleman Jr. spectrophotometer. Beer’s law was followed (Figure 1). The spectra of the color produced in these two methods were compared (Figure 2). By these figures, it is clearly demonstrated that the color produced by the present method is about 20 times (at 830 mp) or 5 times (at 700 mp) as intense as that obtained by the Fisk-SubbaRow procedure. The pattern of the spectrum obtained from the inorganic phosphorus on TCA filtrate is very closely correlated with that of lipide phosphorus previously
R\
I .4\ I
I
.21
f \
w 0 1.01
PHOSPHORUS
Figure 2.
IJ 6 .
Spectrum analysis of colored mixtures
Figure 1 . Density of color by two phosphorus assay procedures
reported by Bartlett (1). Thus the present work re-evttluates the heating method, and confirms its increased sensitivity. The color produced is stable for at least 3 hours. However, the density of the color decreases slightly within 2 hours thereafter.
Table 1.
Outline of Two Procedures Reagents and FiskU1:rsi Conditions SubbaRow micro Filtrate, volumes 5 (1)o 1 (1) 10N H B O I , volumes ... 2 . 4 (2) Ammonium molyb- 1 (2) 1 . 6 (4) date, volumes Fisk-SubbaRow 0 . 4 (3) 0 . 4 (5) reagent, volume H20, volumes 3 (4) ; , 3 5 ( 3 ) Final volume of 2.35 mixture, ml. TemDerature of re- 24-25 100 (for aciion, C. 7' min.) 5 (700 Sensitivity of color, 1 (700 ratio mP) mr)
l(830
extraction of lipide with alcohol-ether (10, 11). However, the ether-soluble lipide phosphorus may include phosphorus-containing material other than lipide phosphorus (9). Therefore, the stage of purification a t which the analysis takes place depends largely on the possible contamination of inorganic phosphorus-containing compounds. Since the recovery of lipides (13) or lipide phosphorus (14) after extraction of the coprecipitated protein lipide with TCA has been shown to be complete, the method can provide for the simultaneous extraction of inorganic phosTable 11.
LITERATURE CITED
(1) Bartlett, G. R., 466 (1959).
J. Biol. Chem.
Determination of Phosphorus in Control Serum
Inorganic Phosphorus, Mg. % Versatol Versatol ALabtrol 4271100 Alt. 4294100 LT-21 L Control value Date of Detn.
234,
hide Phosphorus,
Mg. %
Versatol 4271100
...
3.90
2.60
3.35
Av.
-
Std. dev. Relative std. dev., % Dev. from control serum value
3.76 3.84 3.94 4.04 4.00 4.04 4.35 4.20 4.02 *0.29 6.2
2.85 2.7.9 2.80 2.72 2.64 2.68 2.72 2.84 2.76 50.08 2.9
3.34 3.44 3.40 3.40 3.30 3.25 3.30 3.40 3.35 *0.07 2.1
5.92 5.30 6.16 5.92 5.72 5.82 5.72 5.52 5.76 f0.27 4.7
*0.23
10.19
*0.07
...
2/2/1962
20 (830
mfi) Number in parentheses indicates order of addition of reagent. mP)
2/5/1962
0
2/7/1962 2/9/1962
Previous workers (1, 6, 8) expressed the belief that, during heating, some inorganic orthophosphate may be converted into inorganic phosphate in the presence of phosphate esters. However, Buell (3) and Benedict and Theis (2) found no acid-soluble organic phosphorus in the serum filtrate even after its hydrolysis with strong acid or heat. The views of these investigators (6,3, 7) are supported by the recovery study on the known control serum. The present method is not recommended when whole blood or serum from badly hemolyzed blood is employed, because the heating and strong acid may hydrolyze some of the organic phosphorus compounds contained in the corpuscles. Some micromethods for the determination of lipide phosphorus involve
of that used by the original Fisk-SubbaRow procedure for lipide and inorganic phosphorus, can be a n important contributing factor in the study of the ultramicro quantities of serum obtainable from infants and small experimental animals. It also provides for a practical estimation of phospholipides in highly diluted body fluids, such a s cerebrospinal fluid, where the aveiage value of lipide phosphorus is only a few hundredths of that found in other human sera (18).
phorus (supernatant fluid) and lipide phosphorus (precipitate). The method has advantages over the alcohol-ether extraction method, in that the lipide extracted is relatively free from ethersoluble nonlipide phosphorus. Table I1 shows the reproducibility of the inorganic and lipide phosphorus determination on three control sera during one week. Inorganic and lipide phosphorus or phospholipide levels were also determined for some patients (Table 111). The present method, which reduces the amount of serum to to l / p o o
Table 111.
Determination of PhosDhorus in Human Serum
Inorganic Phosphorus, Serum Mg. % 2.94 3.12 3.60 3.78 4.26 3.42 4.50
Lipide Phos-
Phosphclipide, Mg. yo Mg. % '
phorus,
5.60 10.68 17.36 7.20 10.00 8.72 7.36
VOL 34, NO. 9, AUGUST 1962
140 267 433 180 250 228 184
1165
Benedict, S. R., Theis, R. C., Ibzd., (2) Bene( 61, 63 (1924). (3) h e llll, M. V., Zbid., 97, lvi (1923). (4) Clayton, M. M., .4dams, P. A, Mahoney, G. B., Randall, S. W., Schwarta, E. T., Clin. Clzn. Chem. 6 , 426 (1959). (5j Fisl;, C. H., SubbaRow, Y., J . Riol. Chem. 66, 375 (1925). ( 6 ) Griswold, B. L., Humoller, F. L., McIntyre, A. R., ANAL.CHEM:23, 192 (1951). (7) Hawk, P. B., Oser, T3. L., Summerson,
W. H., “Practical Physiological Chem-
istry,” 13th ed., McGraw-Hill, New York, 1954. (8) Horecker, B. L., Ma, T. S.,Hass, E.,
J. Bid. Chem. 136. 775 (1940). (9) LeBreton, I. W.,’Hall,‘R. J:, Bzochem. J . 67, 400 (1957). (10) McDonald, I. W.! Hall, R. J., Ibzd., 67, 400 (1957). (11) .!atelson, S.,“Microtechniques of Clinical Ckmistry for the Routine
Laboratorv. Charles C Thomas. Springfield,’Ill., 1958. (12) Shin, Y. S., Lee, J. C., Clin. Chem., In press.
Tourtellotte, W. W.,’I-srider, A . J., Slrrentny, B. rl., DeJong, R. S . , J . L a b . Clin. Med. 52, 481 (1958). (14) Zilversmit, D. B., Davis, A. K., (la)
Ibid., 35, 155 (1950).
Y U N GS.SHIS’ Department of Biochemistry St. .4nthony Hospital Terre Haute, Ind. Present address, Biochemistry Laboratory, St. Mary’s Mercy Hospital, Gary, Ind.
Volumetric Determination of Aluminum in Steels, High Temperature Alloys, and Nonferrous Alloys SIR: Investigation of the acidimetric determination of aluminum using potassium fluoride as in Viebock and Brecher’s method (S), and subsequent modifications (1, 6, 4, 6) revealed that the method was effective for the determination of aluminum in ores. Adaptation for its use in metal samples seemed practical. Following the method outlined here, aluminum can be determined in high temperature alloy, bronze, and steel in less thaa one hour. Aluminum is initially separated from its base metal and alloying constituents by mercury cathode electrolysis. Since Ti, V, and Zr are not separated by electrolysis, their effect on the recovery of aluminum was investigated (Table I). W, Tal and Nb are hydrolyzed on fuming with perchloric acid, and are filtered off. NH3+ interferes because of the buffering action of the solution. The concentration of KF is not critical as long as a twofold excess is maintained. p H 8.2 was found to give maximum deflection of the p H meter as well as faster reaction rate of K F with the aluminum. Apparatus. p H meter, standardized with p H 7.0 buffer. Mercury cathode cell.
Reagents. Standard HCl solution, 0.2N t o 0.3N. Ascorbic acid. Standard sodium or lithium hydroxide, 0.2N to 0.3N, standardized for equivalent consumption of the 0.2 to 0.3N HCl. Sodium gluconate, 30% water solution, C.P. grade obtained from Pfanstiehl Laboratories, Waukegan, Ill. Thymol, phenyl mercuric acetate, or mercury (0.1 gram per liter) may be added as B preservative. Potassium fluoride solution, 30% water solution stored in polyethylene and adjusted to p H 8.2 before each use. Sodium or lithium hydroxide, approximately 2N water solution. Phenolphthalein, 1% methanol solution. GENERAL PROCEDURE
Acid-Soluble Aluminum. Dissolve a l,.O-gram sample in 20 ml. of aqua regia. When solution is complete, add 12 ml. of HClOl and bring to fumes. (If chromium is present, it may be advantageous to volatilize it with HCl to cut down electrolysis time.) After fuming, wash the flask with 25 ml. of HzO and heat t o dissolve salts. Filter off any insoluble and hydrolyzed material. (Reserve the residue.) Transfer to a mercury cathode cell with water, making the final volume approximately 100 ml. Electrolyze a t 15 amperes for 20 t o 30 minutes. Check
for completeness of electrolysis with spot tests for iron and nickel. Upon completion of electrolysis, transfer the solution from the cell into a 1liter beaker and make up to approximately 500 ml. with water. Add 40 to 50 ml. of 30% sodium gluconate and approximately 0.1 gram of ascorbic acid to complex and reduce traces of Fe and Mn that are not removed by electrolysis. Add 10 drops of phenolphthalein indicator and make a rough neutralization to a red color with approximately 2.ON LiOH or NaOH. Then with the p H meter in place, adjust the p H back to exactly 8.2 with ”21. Add 35 ml. of 30y0 KF, previously adjusted to p H 8.2, and stir for 1 to 2 minutes. Titrate with standard HC1 until p H is 8.2. Record consumption. Test the final end point with an additional 10 ml. of 30y0 KF. If a deflection occurs, not enough K F was added initially, and the sample should be started again. Total Aluminum. Proceed as for acid-soluble aluminum and ignite the reserved residue in a platinum crucible. Fuse the oxides with sodium carbonate and dissolve in 50 ml. of 10% HC104. Evaporate the solution to strong fumes of perchloric acid. Dilute with water, heat to dissolve salts, and filter. Combine filtrates and proceed as described above for acid-soluble aluminum. DISCUSSION
The basic reaction involved is: Al(0H)s 6KF + K3.41Fs 3KOH
+
Table 1.
Effect of Elements Not Separated by Electrolysis
Amt. Element Titanium
Used, Mg.
Aluminum, Mg. Taken Found
Bias, Mg.
50 25
67.5 67.5
67.0 67.2
-0.5 -0.3
Vanadium
50 25
67.5 67.5
67.5 67.5
0 0
Zirconium
50 30 20 10
67.5 67.5 67.5
71 . O 69.6 68.9 68.2
+3.5 +2.1 +1.4 $0.7
1166
ANALYTICAL CHEMISTRY
67.6
% Recovered 99.26 99.56 100 100 105.19 103.11 102.07 101.04
+
The reaction is not stoichiometric when a direct titration is employed; 2.9 instead of 3.0 moles of hydroxide are released per mole of aluminum. The reaction is stoichiometric, however, if the titration is carried past p H 8.2 with the standard acid, then backtitrated with the standard base and its equivalent amount of acid is deducted from the net titration. However, since the mole ratio of 1 to 2.9 is achieved constantly when titrating directly to p H 8.2, it is not necessary to introduce the overtitration step simply to obtain
