V O L U M E 21, NO. 8, A U G U S T 1 9 4 9
965
point some of the RSI formed apparently decomposes slowly with the formation of disulfide and free iodine.
2RSI
+RSSR
+
Table 11. Analysis of Commercial Mercaptans for Their Primary and Tertiary Mercaptan Content
(10)
12
This formation of free iodine gives a constantly increasing current a t the microelectrode. For this reason the end point must be determined very rapidly if the amperometric method is used. Such a procedure is not practicable and :he results are uot accurate. Some evidence that all tertiary mercaptans do not react *toichiometrically to form the sulfenyl iodide has been obtained by Laitinen ( 5 ) . A qualitative measure of the tendency to disulfide formation by a tertiary mercaptan can sometimes be obtained a t the completion of the iodometric titration. If Reaction 10 is taking place to any appreciable extent, the microammeter will register the constantly increasing current due to the formation of free iodine. If the rate of increase is smallLe., less than a few tenths of a microampere per minute-the formation of disulfide by tertiary mercaptan is probably slight. By the above test it has been noted that the dimethyl-n-nonylcasbinthiol has a much higher tendency to form disulfide than does the di-n-butyl-n-propylcarbinthiol. Some commercial mercaptans have been analyzed by thP method described in this paper (Table 11). INTERFERENCES
The substances that interfere in the argentometric titration have been discussed ( 2 ) . Substances oxidizable by iodine under the conditions of the titration mill interfere in the iodometric proeedure. I n addition, substances that form insoluble precipitates with lead should be absent or enough excess lead nitrate should be added to precipitate all of the interfering substance. .%]though in many instances the iodometric procedure could be applied to pure mercaptans without the addition of lead, this is undesirable because the iodide formed by the reaction reduces the accuracy of the determination of the equivalence point. When iodide is present the current a t the rotating electrode for any given excess of iodine is greatly reduced (see Table 111). The addition of excess lead as recommended in the procedure precipitate3 the iodide ap lpad iodide, so that iodide desenpitiza-
Mercaptan (Calcd. as CnHzaSH). Mercaptan Titrated Fraction 3B, Sharples
Primary Mercaptan,
%
%
Tertiary Mercaptan,
%
Table 111. Diffusion Currents at Rotating Platinum Electrode with Varying Excesses of Iodine in Some Solutions Solutions to Which Iodine 100 ml. 95% ._ ethanol 0.01 M HClOA 0.001 AM KI
loo ml. 95% .-
ethanol 0.01 .W HClOi 0.005 N I9 sulutlor~ in Excess, SI1. 0.07 0.14
1.7
n , 30
' -
Microamperes
,~---
0.21
--
Is Added 100 ml. 95% ethanol 0.01 M HClOi 0.001 M K I 0.01 iM Pb(NOd1
0.3
1.9
3.2
0.5
3.7
4.7 6.3
0.7
5.3
1.0
7.6
tion is rliininated. In all caseb the eiid point obtained is the same whether or not iodide is present, although less precisely determined when iodide is present. The same electrode should not be used for the iodometric titration as is used for the argentometric titration unless the silver plated onto the platinum has first been removed with nitric acid. LITERATURE CITED
(1) Kimball, J. W.,Kramer, R. L., and Reid E. E., J. Am. C'hern. SOC.,43, 1199 (1921). (2) Kolthoff, I. M.,and Harris, W. E., IND.ESG.CHEM.,ANAL.ED., i8. 161 (1946). (3) Laitinen, H. A,, private communication. (4) Rheinboldt, H., Ber., 72,657 (1939). (5) Tyler, W. P.,and Brown, W. E., B. F. Goodrich Co.. Drivate ,
I
communication.
RECEIVED August 11, 1948. Investigation carrled out under sponsorship of the Office of Rubber Reserve, Reconstruction Finance Corporation, in connectinn with the government synthetir rubber program.
Determination of Inorganic Phosphate Modijficat ion of Isobutyl A lco hol Procedure J A M E S B. MARTINLAND D. M. DOTY Purdue C'nirersity Aprirultitral Experiment Station, Lufayette, Ind.
T
HE procedure employing extraction of phosphomolybdic
acid by isobutyl alcohol described by Berenblum and Chain (2) and recently evaluated by Pons and Guthrie ( 4 ) affords a good method for determination of inorganic phosphate in colored solutions. In studies of phosphatases and of phosphorus fractions in alfalfa, the use of this method seemed desirable because colored solutions were encountered. Investigation of the isobutyl alcohol extraction procedure has led to improvements that simplify the color development, shorten the extraction procedure, and eliminate the interference from proteins. In the improved procedure the sample is in contact with an acid solution for only a short time (60 t o 90 seconds). This should enhance the value of the method for the determination of inorganic phosphate in the 1 2
Present address, Procter a n d Gamble Co., Cincinnati, Ohio. Present address. American Meat Institute Foundation, Chicago, I11
preseiice of easily hydrolyzable phosphate compounds. Ferric ions do not interfere in the isobutyl alcohol extraction procedure, REAGENTS
Isobutyl Alcohol-Benzene Solution. Mix equal volumes of isobutyl alcohol and thiophene-free benzene. (Some technical grades of benzene impart a cloudiness t o the solutions after color development.) Molybdate Reagent. Dissoh e 50 grams of ammonium molybdate in 400 ml. of 10 N sulfurir acid and dilute to 1 liter with water. Silicotungstate Reagent. Dissolve 5.7 grains of sodium silicate nonahydrate and 79.4 grams of sodium tungstate dihydrate in about 500 ml. of water. Add 15 ml. of concentrated sulfuric acid, boil for 5 hours, cool, and dilute to 1 liter with water. Stannous Chloride Stock Solution. Dissolve 10 grams of stannous chloride dihydrate in 25 ml. of concentrated hydrochloric acid and store in a small glass-stoppered brown bottle.
ANALYTICAL CHEMISTRY
966
The method of Berenblum and Chain for the determination of inorganic phosphate has been modified. The number of extractions and washings has been decreased from three to one and the shaking tinie reduced to 15 seconds. Color development is effected by direct addition of stannous chloride solution to an aliquot of the extract which has been diluted with ethyl alcohol-sulfuric acid solution. The modified procedure may be applied directly to solutions containing proteins by use of a silicotungstate reagent. m
Stannous Chloride Dilute Solution. Dilute 1 ml. of the stannous chloride stock solution to 200 ml. with approximately 1 X sulfuric acid. Prepare fresh daily. Sulfuric Acid in Ethyl Alcohol. Dissolve 20 ml. of concentrated sulfuric acid in 980 ml. of 99.5y0ethyl alcohol. (Instability of the molybdenum blue color has been attributable at times to some contaminant in 95y0 ethyl alcohol.) Phosphate Standard. Dissolve 0.4950 gram of reagent grade potassium dihydrogen phosphate (dried a t 110" C.) in water and dilute to 1 liter. Dilute 50 ml. of this solution to 200 ml. to obtain a solution containing 28.2 micrograms of phosphorus per ml. ANALYTICAL PROCEDURE
Pipet an aliquot of the solution for inorganic phosphate analysis into a 25 X 200 mm. test tube. (The aliquot should contain 20 to 80 micrograms of phosphorus if a light absorption cell 2 cm. thick is to be used for the final colorimetric reading.) Add water to bring the volume to 15 ml. Add 25 ml. of the isobutyl alcoholbenzene solution. (Use a rubber bulb or other mechanical source of suction for pipetting this solution to avoid inhaling the toxic fumes of benzene.) Add 5 ml. of the silicotungstate reagent followed by 5 ml. of the molybdate reagent. Stopper the tube with a rubber stopper and immediately shake the mixture 15 seconds. Allow the phases to separate. Pipet a 10-ml. sample of the isobutyl alcohol-benzene layer into a 50-ml. volumetric flask and wash the sample from the pipet with alcoholic sulfuric acid solution. (Reproducible drainage of the isobutyl alcohol-benzene solution is difficult to obtain,) Dilute the sam le to about 45 ml. with the alcoholic sulfuric acid, add 1 ml. of dirute stannous chloride solution, dilute to volume with alcoholic sulfuric acid, and mix. Carry a blank through the same procedure. Measure the per cent transmittance of the sample in a photometer adjusted to read 100% transmittance for the blank. A filter system transmitting between 625 and 725 millimicrons should be used in the photometer. The authors have isolated a satisfactory wave length band by the combination of a Corning 243 filter with 2.5 cm. (1 inch) thickness of 2% copper sulfate solution. Prepare a standard curve by the same procedure, substituting aliquots of the dilute phosphate standard for the sample aliquots.
Table I. Recovery of Inorganic Phosphate by Isobutyl ..ilcohol-Benzene Mixtures as a Function of Extraction Time Extraction T i me Seconds
5
10 15
P Added Y 56.4 56.4 56.4
P Recovered Y
56.2 56.2 56.3
Table 11. Effects of Sulfuric, Hydrochloric, and Acetic Acids on Stability of Molybdenum Blue Color Developed by Homogeneous Reaction with Stannous Chloride Vol., MI. per Acid Concd. HzSO,
Concd. HCI Glacial acetic
50 hll. 0.1 0.2 0.5 0.2 0.5 2.0 1.0 5.0 25.0
Per Cent Transmittance 15 min. 30 min. 45 min 32.2 32.2 32.2 32.0 32.0 32.2 32.0 32.1 32.7 38.1 39.5 36.4 35.0 36.5 35.8 35.0 35.6 37.0 38.2 39.4 36.1 36.0 36.6 34.8 33.5 33.9 34.2
IYVESTIGATION OF PROCEDURE
Consideration of such factorb as extraction time, number of extractions, method of color development, and interference from proteins led to the modifications embodied in the analytical procedure. A check on the rate a t which isobutyl alcohol would extract high concentrations of phosphomolybdic acid from aqueous solutions revealed that 500 micrograms of phosphorus could be extracted quantitatively in 10 seconds' shaking time. Sormal phosphorus concentrations were recovered quantitatively by as little as 5 seconds' shaking (Table I). In previous methods (2, 4)an acid wash of the isobutyl alcohol layer and color development by shaking with stannous chloride solution are required. Because the stannous chloride solution may be added to the organic solution of phosphomolybdic acid to give a homogeneous, stable, colored solution in the presence of alcoholic sulfuric acid solution (Table 11), i t was possible to eliminate all except the initial extraction by aliquoting from the organlc layer. The color stability was exceptionally good, as per cent transmittance readings after 24 hours were identical with the initial value. Some lots of 95% ethyl alcohol caused the molybdenum blue color to be unstable; however, the use of 99.5% ethyl alcohol has eliminated difficulties of this nature. The mutual solubilities of isobutyl alcohol and water are different. T o overcome the appreciable change in volume of the phases during mixing, an investLgation was made of the extraction of phosphomolybdic acid by mivtures of isobutyl and other alcohols with benzene (Table 111). Dilution of alcohol extractants for phosphomolybdic acid with benzene up to 50% by volume does not actually reduce the amount of phosphorus extracted. Apparently the decrease in phosphorus recovery a t low benzene concentrations results from the higher solubility of water in the organic phase, which causes an error from dilution. At high benzene concentrations the extraction is actually inhibited. The data (Table 111) indicate that n-butyl alcohol or possibly the amyl alcohols could be substituted for isobutyl alcohol as an extractant. KO informatioii has been found in the literature as to equilibrium compositions in the ternary system water-isobutyl alcoholbenzene. Washburn and Strandskov (6) indicate that in the system n ater-n-butyl alcohol-benzene a 1 to 1 mixture of n-butyl alcohol and benzene in equilibrium n-ith water gives a two-phase system having nearly equal mutual solubilities. A mixture of isobutyl alcohol and benzene in the proportion of 1 to 1 by volume has been adopted for use in the proposed procedure for phosphorus determination. Because changes in the composition of the aqueous phase might cause variations in the mutual solubilities, the volume of the organic layer in equilibrium with aqueous solutions of varying pH and salt content has been measured and found to be constant (Table IV). The equilibrium volume of the isobutyl alcohol-benzene phase will vary inversely with the water content of the initial isobutyl alcohol-benzene solution. Protein interference in determination of inorganic phosphorus appears to be of two types: interference due to turbidity resulting
VOLUME
ii, N O . 8,
AUGUST
967
1949
Table 111. Extraction of Phosphomolybdic Acid from Aqueous Solutions by Organic Alcohols and Esters Alone and in Mixtures with Benzene Organic Solvent
Vol. Proportion, Solvent : Benzene
COMPARISON OF ,METHODS 7‘ /r 35.8 33.0 32.0 34.2 40.5 91.0 46.0 32.4 c
lsobutyl alcohol
,,-Butyl alcohol
10: 1 3:l 1:l 1:2 1:4 1:9 l:o 1:l 1:4 1:9 l:o 1:l
If the molybdenum blue color shows evidence of instability, the ethyl alcohol stock should be considered as a likely source of the difficulty and other supplies of alcohol should be tested.
P Added Y
56.4 56.4 56.4 56.4 56.4
56.4 56.4 56.4 56.4 56.4 28.2 56.4 28.2 56.4 28.2
P Founda Y 50.8 55.0 56.4 53.2 44.8 4.6 38.6 55.9
Deriation
%
-
10.0 -2.5 0.0 -5.7
-13.0 -92.0 -32.0 -0.9 -10.0 -64.0 -4.6
36.0 50.7 66 7 20.2 lsoamyl alcohol 58.3 26.9 33.5 54.1 -4.1 27.0 -4.3 58.1 Benzyl alcohol l:o 7.3 -87.0 86.3 1:4 Amyl acetate l:o 59.6 25.7 -9.0 1:4 100.0 56.4 0.0 -100.0 Ethyl acetate l:o 33.0 56.4 55.0 -2.5 1:4 100.0 56.4 0.0 -100.0 % transmittance ‘I Aliquots taken from organic layer a f t e r extraction. referred t o standard curve prepared using 1: 1 isobutyl alcohol-benzene extraction.
from coagulation of protein, and IOSS of phosphorus as phosphomolybdic acid precipitating with the protein during coagulation. These sources of error from proteins have been eliminated by the modifications incorporated in this method. When a sample is taken from the isobutyl alcohol-benzene layer rather than the entire layer withdrawn for analysis, the coagulated protein that ~ 0 1 lects a t the interface is not a source of turbidity in the solution after color development. To eliminate the interference from prot,ein precipitation of phosphomolybdic acid it was necessary t o find a satisfactory protein precipitant that could be added to the solution prior to the formation of the phosphomolybdic acid. Silicotungstic acid, recommended by Mitchell, Shaw, and Frary ( 3 ) as a good precipitant for gelatin, was satisfactory. Good recoveries of quantities of inorganic phosphate added to protein-containing solutions were obtained by the addition of the reagent prior to the addition of the molybdate reagent (Table V). The silicotungstic acid did not interfere in the colorimetric determination, for it contributed no color to a phosphate blank solution. The effect of variations in the use of the silicotungstate reagent is worth noting. The boiling treatment of the si1icotungstat)eproduces a reagent which has no tendency to form a complex with the phosphate of the solution. Optimum reproducibility is obtained whrn additions of silicotungstate and molybdate reagents and the estraction with isobutyl alcohol-benzene are performed in successive operations with minimuni intervening time intervals. Good recovery of the phosphate may be obtained in the presence o f proteins even when the order of addition of silicotungstate and molybdate reagents is reversed, provided the extraction time is inrreased to approximately 2 minutes. Silicotungstate appears to replace phosphomolyhdatc i n a precipitated protein phosphomolybdate complcs.
Analyses for inorganic phosphate obtained by the modified istr butyl alcohol method have been compared with analyses on the same samples by the Berenblum and Chain procedure ( 2 ) , and the A.O.A.C. sulfite reduction methods (1)(Table VI). The A.O.S.C. method gave consistently higher values for illorganic phosphate in the tissue extracts. The color of the extrarts introduced errors that .could not be eliminated. During color development a turbidity often formed which was probably related to the protein content of the sample. This turbidity could be largely removed by centrifuging. Differences between results by the Rerenblum and Chain niethod and the modified method were small and erratic. Apparently the effect, of proteins in the extracts was not a factor in this study. The modified method showed t h c least deviation between difftlrent extracts. PRECISION OF METHOD
Sixteen analyses of five extracts of one alfalfa sample gavc an average of 0.127% phosphorus with a standard deviation of 0.0016%. Tabie V. Recovery of Inorganic Phosphate in Presence of Proteins by Use of Silicotungstic Acid Reagent 1’ added Y
0.0 .i6.4 84.6
Phosphate Found in Presence of 50 mg. 10 mg. 10 mg. gelatin blood albumin hemoglohia. Y Y Y Y 0.5 7.8 54.4,53,3 .j1.0,54,4 55,;;:5,.757.Aig5.0 8 3 . 8 , 8 2 .;1 83.3,79.8 78.8,79.4 82.8,82.8 10 mg. gelatin
Table VI. Sample0
Inorganic Phosphorus in ilfalfa A.O.A.C.b, -Mg./g
BerenblumChain -Mg./g.
c
Modified Ifg./g
c
‘I All h samples were obtained b) boiling ground alfalfa after pimapholipide extraction for 5 minutes with 0.1 .V sodium acetate buffered to pH 4.8,filtering, washing, a n d di1iit;ng t o volume; 13 by GO-:uinute room temperature extraction with l2.5Y0 trichloroacetic acid, filtering, washing, a n d diluting t o volume. b Solutions t h a t showed turbidity were centrifuged before wading in photometer. C -411 values averages of a t least triplicatr rolorimetrir deterinination\ OII each extract.
ACKNOWLEDGR.1ENT
The authors wish to express their appreciation to Mary Dell Springer for her assistance with some of the analyses performed during t,he course of this investigation. LITERATURE CITED
Table 1V. Volume of Isobutyl Alcohol-Benzene Phase in Equilibrium with Aqueous Phases
25 25 25 20
HzO
.\:HzS04 1.0 .\ H&OI H20 5 AIolgbdate reagent 04
10 H?O 5 Molybdate reagent 10 Silicotungstate reagent
Volume of Isobutyl Alcohol-Benzene After Initial equilibrium M1. Ml. 25.00 24.00 25.00 23.90 25.05 24.05 25.05
24.00
25.00
23.95
Offic. Agr. Chemists, “Official and Tentative Methods of Analysis,” 6th ed., p. 137, 1945. (2) Berenblum, I., and Chain, E., Biochem. J . , 32, 295 (1938). (3) Mitchell, D., Shaw, E. H., Jr., and Frary, G. G., Proc. S . Dakota (1) Assoc.
Acad. Sci., 24, 108 (1944). (4) Pons, It7. A., Jr., and Guthrie, J. D., IND. ESG. CHEY.,h r a ~ . . ED.,18, 184 (1946). (5) Washburn, E. R., and Strandskor, C. V.,J . Phys. Chem., 48, 24f (1944). RECEIVED July 7, 1948. Presented before the Division of Agricultural a n 8 Food Chemistry a t t h e 111th Meeting of t h e AMERICAN CHEMICAL SOCIETY, Atlantic City, K. J. Journal paper 298 of t h e Purdue University .Igricultural Experiment Station. Herman Frasch Foundation for .Igriculturab Research paper 232.
