of the cyanoethylation procedure and the reliability of the measurement used (the measurement of pH) are also very advantageous. Observational uncertainties are inherent in other methods in which precipitation, separation of oils, gas evolution, etc., are utilized. The major limitations of the cyanoethylation method are its failure to distinguish primary amines from secondary amines, to detect some secondary amines and thereby distinguish them from ammonia or tertiary amines, and to detect primary or secondary amines, when in mixtures with a too high
proportion of ammonia or tertiary amine. LITERATURE CITED
(1) American Cyanamid Co., Xew York, N. Y., “Chemistry of Acrylonitrile,” 1951. ( 2 ) Bruson, H. A., Org. Reactions 5 , 79 (1949).
, - - - - I
( 3 ) Burckhalter, J. H., Jones, E. M., Holcomb, W. F., Sweet, L. A.; J. Am. Chem. SOC.65, 2014 (1943). (4) Duke, F.R., IND. ENG.CHEbf., ANAL. ED. 17,196 (1945). (5) Feigl, F., “Qualitative Analysis by
Spot Tests,” Elsevier, New York, 1947.
( 6 ) Hinsherg, O., Kessler, J., Ber. 38, 906 (1905). (7) Soloway, S.,Lipschitz, A., J . Org. Chem. 23,613 (1058). (8) St,evenson, G. R’., \\-illismson, D., J . Am. Chem. SOC.80, 5943 (1958;.
:9) Riedeman, 0. F., Montgomery, W. H., Zbid., 67, 1994 (1945). RECEIVEDfor review August 31, 1959. Accept,ed October 6, 1959. From portions of a thesis submitted hy Sherwood H. Biers to the University of California, Los Angeles, in partial fulfillment of t,he requirements for the degree of master of science. Investigation supported by research grant B-1106 from the Sational Institute of Neurological Diseases and Blindne~sof the h’ational Iristjtutes of Heal.th, U.S.Puhlic Health Errvice.
Microdetermination of Phosphate in the Range of 1 to 10 Micrograms ARTHUR A. HIRATA’ and DAVID APPLEMAN College o f Agriculture, University of California, tos Angeles, Calif.
b A reliable method was needed for determining phosphorus in biological materials containing less than 10 y of phosphorus per available sample. The method of Bernhart and Wreath lacked the sensitivity required for determining such small amocnts. A detailed study of factors affecting the phosphomolybdate chromogen formation led to changes in concentrations of reagents and measuring at a different wave length. The modified method had greatly increased sensitivity; it is possible to determine phosphorus in biological and other materials in the range of 1 to 10 y with high accuracy and precision.
color development. Therefore, a detailed study was mad? of the various factors that influence the formation of the phosphomolybdate complex. The absorbance of the complex was measured at different wave lengths. As a result, changes were introduced in the concentra.tions of the reagents used to form the phosphoinolybdate complex, and in the wave length at which the absorbance of the complex is measured. These modifications increase the sensitivity of the Bernhart and Wreath method many fold, extending its usefulness into the micro region. It is now possible to determine phosphorus in the range of 1 to 10 y with a high degree of accuracy and precision.
and Wreath (2) developed a method for the determination of phosphate, taking advantage of the fact that acetone enhances the absorbance of the phosphomolybdate complex. The method has simplicity of operational procedure, stability of the color developed, excellent reproducibility, and stability of the reagent over several months. These points have been confirmed in this laboratory. The method, however, lacks the sensitivity necessary to determine with a high degree of :muracy less than 10 y of phosphorus. The original report (2) gives little information on the conditions that affect
REAGENTS
ERSHART
1 Present address, Division of Chemistry and Chemical Engineering, California
Institute of Technology, Pasadena, Calif.
MOLYBDATE REAGEST. Dissolve 1.545 grams of ammonium molybdate, (NH4)6M07024 4H20, in 400 ml. of distilled water with the aid of 0.8 mi. of 70% HCIO, and dilute to 1 liter with water. ACETONE,double distilled, stored in deep freeze. PERCHLORIC ACID, 7OY0 (w./v.) , 11.7N1reagent grade. AMMONIUM HYDROXIDE, concentrated, reagent grade (approximately 14N). PHOSPHATE STAKDARD. Dissolve 219.7 mg. of reagent grade potassium dihydrogen phosphate (dried a t 110” C.) in water and dilute to 1 liter (50 y of phosphorus per ml.). Dilute this stock solution with water to obtain standard phosphate solutions 1.0 ‘to 10 y of phosphorus per ml.
ANALYTICAL PROCEDURE
PROCEDURE A. Pipet a maximum of 3.3 ml. of inorganic phosphate solution (ivater for blank) into a 10-ml. volumetric flask. Add 0.3 to 0.6 ml. of 707, perchloric acid. Add water to bring the total volume to approximately 4 ml. and mix, Add 2.0 ml. of molybdate reagent and mix thorouglily. ildd 4.0 ml. of ice-cold acetone, inserting tlie tip of the pipet beneath the surface of the solution. Add water to the 10-nil. mark. Stopper and mix thoroughly. A piece of Saran Wrap (The Dow Chemical Co., hlidland, Xich.) may he used as a stopper for the flask. LIeasure the absorbance a t 320 mu in a Ueckinan I)U spectrophotometer against the blank. The absorbance of the blank against r a t e r should read 0.570 with 1-cm. light path. The ahsorhance of the phosphomolybdate-acetone complex against the blank is 0.324 per 5 y of phosphorus, and remains stable for 8 hours when the phosphorus content in 10 ml. is less than 4 y . With more than 4 y of phoqphorus per 10 ml. the absorbance increases 3531, in 8 hours. PROCEDTJRE 13. Three instead of 4.0 ml. of acetone may be used. I n this case, however, the perchloric acid should. not exceed 0.4 ml. per 10-ml. total volume. The absorbance of the blank should read 0.410. The absorbance of the phosphomolybdate-acetone complex remains the same as in Procedure A. PHOSPHORUS DETERMINATION IN ORGANIC COMPOUNDS
Only organic phosphorus compounds that were hydrolyzed by treating with 707, perchloric acid for 40 minutes a t VOL. 31, NO. 12, DECEMBER 1959
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Figure 2. Effect of perchloric acid concentration on absorbance of phosphomolyhdate-acetone complex a t 320 mp read against blank 350
W a v e length, m p
5 y of P, 10 ml. toto1 volume, 4 ml. of acetone, 2 ml. of molybdate, 1 .cm. light path
Figure 1. Absorption spectra of phosphomolybdateacetone complex and its reagent blank Blank read against water. Complex read against blank. 5 y of in 10-ml. total volume, 4 ml. of acetone, 2 ml. of molybdate reagent, 1 -cm. light path
P
203' C. were investigated. Ribose and deoxyribose nucleic acids should be hydrolyzed by this treatment ( I ) . It is convenient to digest the sample in a 15 X 125 mm. silica tube. Procedure. Add 0.33 to 0.66 ml. of 70y0 perchloric acid t o 2 t o 4 ml. of tissue extract containing 1 t o 10 y of phosphorus. (If the tissue has been previously extracted with perchloric acid, the amount of perchloric acid present in the extract must be subtracted from the amount of 70% perchloric acid added.) Add a boiling chip and heat at 203" C. (the boiling point of 70% perchloric) for 40 minutes. This generally gives a clear solution. Transfer the digest into a 10-ml. volumetric flask. Rinse the silica tuhe several times. The final volume a t this stage should be 4 nil. [Allen ( I ) reported that ahout 10% of the perchloric acid is lost upon heating. This has been confirmed; thus \\hen 0.33 t o 0.66 ml. of the 707, perchloric acid is heated about 0.03 to 0.06 ml. is lost.] Add 2 ml. of molybdate reagent and mix thoroughly. iidd 4.0 ml. of acetone and fill to the 10-ml. mark with water. Mix thoroughly and read a t 320 nig against a blank. FACTORS AFFECTING THE REACTION
Selection of Wave Length of Measurement a n d Molybdate Concentration. Klien 3 to 4 ml. of acetone per 10-ml. total volume was used, t h e phosphomolybdate-acetone complex gave a maximum absorption a t 320 mp (Figure 1) and this wave length was selected for the measurement of the reaction. The absorbancp of the blank 2098
ANALYTICAL CHEMISTRY
increases sharply a t the shorter wave lengths (Figure 1). T o decrease the value of the blank absorbance, the molybdate concentration was reduced to the final concentration of 3.09 mg. per 10-ml. total volume (2 ml. of reagent per 10-ml. total). Increasing the concentration of the reagent did not increase the absorbance of the complex (Table I). Acetone Concentration, Acetone concentration was the most critical factor in this reaction. T o facilitate accurate delivery and t o counteract t h e heat of solution, acetone was chilled in a n ice bath before use. An increase in acetone concentration increases the absorbance of the blank. Thus, for a final volume of 10 ml., measurrd a t 320 mp against water, the absorbance values when 3, 4, and 5 ml. of acetone are used are 0.410, 0.570, and 0.825, respectively. There was no difference in absorbance of the phosphomolybdate-acetone complex n hen 3 to 4 ml. of acetone in a final volume of 10 ml. n as used. When 5 ml. of acetone mas used, the absorption niavimum of the complex shifted to 315 mp. ,4t this nave length the absorbance of the blank was very high (1.40 against water). It is best to niake all measurements using an acetone concentration of 30 to 40% by volume. Neasurement was made at 320 nip because a t this wave length the highest sensitivity is attained. Even though th; blank at this wave length is high, when the samples are read against the
blank, the precision is not seriously affected. Perchloric Acid Concentration. When 4 ml. of acetone was used, t h e absorbance of the blank decreased with increase in perchloric acid content, 0 to 0.35lN, and remained a t a plateau value thereafter, 0.351 to 1.7N. At the same concentration of acetone, the absorbance of the phosphomolybdate-acetone complex showed a plateau betaeen the acid concentrations of 0.351 and 0.702.V (Figure 2). Therefore, the acid concentration should be confined within these values. mhen 3 instead of 4 ml. of acetone per 10-ml. total volume was used, the acid concentration had similar but more pronounced effects on the reaction. There was no difference in the blank absorbance betn een the acid concentrations of 0.117,Y and 0.55LYJ but the absorbance of the phosphomolybdateacetone coniplex shon ed a 4iorter plateau: 0.117 t o 0.35l.Y. Temperature Variation. JYlien the reaction n a s performed a t room ternperature (20' t o 25" C.) and then incubated for 30 minutes a t 37" C., the absorbance of t h e phospliomolybdate-acetone complex decreased 9%. Incubation a t a lower temperature (5" C.) had a similar effect. T h e depressing effects on the aLsorbance by either higher or loner temperaturcs nere slonly reversible. It is evident t h a t the reaction should i I c carried out at room temperatuie. Ammonium Hydroxide. T h e perchloric acid concentration should not evceed 0.705, because higher concentrations depress the absorbance of the pliosplioniolydate-acetone complex.
However, wherelarger quantities of t h e acid must be added, t h e acid concentration can be brought down t o 0.70N by addition of ammonium hydroxide. T h e maximum a m o u n t of ammonium hydroxide that can he added m-it'hout affecting the absorbance of the complex \vas found to be 0.3 nil. of approximately 11N pcr 10-m1. total volume; addition of lnrger quantities increased the nbsorb:mcc. Tissue Extract. Possible interferences by tissue constituents were t8wtcd \\-it11 avocado fruit est!.net. Acetone powder of the avocado (avocado fruit tissue acetone-extracted anti pondered) was extracted with 1N p~w!iloricacid a t 70' C. for 20 minutes a n d centrifuged. T h r e e milliliters of the superlintant n'ns hcnted for 40 niiniitcs :It 203" C. with 0.5 nil. of 70% p~wliioric:ickl; phoephorus cortent \Y;S fount1 t o be 6.22 y per 3 d.of siipcmxitnnt. Aliquots of 3.5 y of phosphorus (as RHJ'OI) were atcltld to 3-nlI. :tlicjiwts of the supernntant anti !ihosplioriis contcnt, was dctermincd irig for 30 minutcs at 203" C. The rccovei'J- of thc n[ltlml phosphorus w:is 94.2 to 96.74r,, DISCUSSION
The llrcwnt method is much more
Table
1. Effect of Molybdate Concentration on Absorbance
Molybdate Reagent, 111. 1 1 2 3 4
0 5 0 0 0
Absorbance Complex Blank nith 5 y of against P agninst water bhnk 0 0 0 0 1
409 470
5iO
750 800
0 072 0 280 0 .121 0 ,321 0 320
sensitive than the original of nernhart and Wreath ( 2 ) . Parallel dctcrminntions made by the two niethocls using a Beckman DLT spectropliotoriic~ti~r gxve specific absorptivities of 4.3s Y, 104 sq. em. per gram of phosphorus nt 430 mu and 6.48 X los sq. em. per g r u n of phosphorus at 320 m p : approsiinstely a 15fold incrcnse in sensitivity. I n the concentration range of 1 to 10 y of phosphorus thc reaction folloirs Bccr-I,anibert's la^. ,2 typical determination on sis replicate?, c i c h having .i y of phosphorus in a total volume of 10 nil., had n rmxn ahsorhaiicc of 0.324 -t- 0.001. This gives a precision within 0.3C;;. The accuracy of this method may be judgcd from the results of a typical
Table II. Phosphorus Determination by Bernhart and Wreath and Modified Methods
(Results in micrograms of phosphorus) Bernhart and Wreath 1lodifcd Added F a .Idded Found 25
21.9 50.9 99.2
50 100
2.51 4.93 10.0%
2.5 5.0 10 0
determination (Tublo 11). 130th tcstz w m made using th(a same staii(1:ird solution of KI12P04. This method has iic~,nsucwssfully used for t1etormin:~tion of ! ~iiosphorus in p h n t mitochonc1ri:i ant1 in niicroorganisnis such ns Tetr;ihymcna and Chlorclla. LITERATURE CITED
(I) Allen, E. J. L., nlbchern. J .
34,
(1940). ( 2 ) Bernhart, D. S.,\\-rc:ith, .?s.IL. CIIEAI, 27, 4-10 (I!J%),
:I. It.,
858
RECEIVETI for review Ftrbru:try 23, 1939. t\ccegted .higust 2 i : 1959. Invcstigstion supported i n part 113' a grant from the Cancer Rcsenrcli Funds of the Univcrsity of Cdiforiii:i.
Spot Tests for Phenol Vapors and for Aromatic Compounds Containing Oxygen FRITZ FEIGL and ERWIN JUNGREIS laboratorio do Produccio Mineral, Ministeria da Agricultura, Rio de Janeiro, Brazil Translated by RALPH E. OESPER, University o f Cincinnati, Cincinnati, Ohio
b The Gibbs indophenol reaction for the detection of phenols by means of 2,6-dichloroquinone-4-chloroimine can be used in spot test analyses for the detection of volatile phenols or phenol vapors carried over by steam. This test involves the action of phenol vapors with filter paper impregnated with the reagent, and subsequent exposure to ammonia vapors. With the aid of this procedure it was shown that the pyrolysis of compounds of phenolic nature and also aromatic compounds containing oxygen in open or cyclic side chains splits off phenols. This finding is the basis of a useful preliminary test for such compounds. Examples are given to illustrate the usefulness of the pyrolytic production of phenols cnd their detection in the testing of materials.
c1
T
HE procedures used previously in spot test analyses for the detection of dissolved and undissolved phenols cannot be employed with vapors of phenols. However, in the preliminary and actual tests of materials, it is often of practical importance to distinguish between volatile and nonvolatile phenols in the vapor phase and in some cases t o detect phenols split off during the pyrolysis of solid materials. This laboratory found that the color reaction reported by Gihbs ( 5 ) can be employed for the detection of phenol vapors, in which phenols condense with 2,6-dichloroquinone-4-chloroiniine to yield colorcd indophenols. I n the case of the simplest phenol, phenol (carbolic acid), the condensation may be represented:
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'cl 1-arious workers have applied this indophenol reaction for the colorimetric determination of phenols in aqueous solution with niaintenancc of slight basicity. Boyland and his associates (2) have pointed out the utility of this color reaction for the detection and distinguishing of phenols in aqueous solution. They also shorred that phenols react only in the free para-position. VOL. 31,
NO. 12, DECEMBER 1959
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