ANALYTICAL CHEMISTRY
1968 Table I. Quantitative Analysis of Spruce Pulp Hydrolyzed with 7270 Sulfuric Acid Volume of Hydrolyzate Determined Calcd. Sugar Applied t o Weight Concn. in Chromatogram, of Sugar, Hydrolyzate, Sugar PI. 7 Mg. /Ml. Glucosea 5 12.2 24.4 24.9 24 9 28.3 1.30 38.2 1.40 Xylose b 39.0 0.97 55.0 0 92 Sum of av. of sugar in hydrolyzate, 27.0 mg. /nil. Hydrolyzate diluted 10 times; calculated sugar values corrected
Mannose b
10 20 30 40 60
or dilution. b
Hydrolyzate undiluted.
variations in the shade of this color for different sugars, however, indicate that the reaction mechanism may be somewhat more complex than this. The absorption maxima of the brown-colored compounds formed with aldohexoses and 6-deoxyaldohexoses were a t 400 nip and 385 mp, respectively. The colors obtained with these sugars were relatively stable, but for best results, the absorbance should be measured shortly afler color development. The compound produced by reaction of aldopentoses or hexuronic acids with the p-anisidine hydrochloride reagent was rather unstable, but the addition of stannous chloride to the eluting solution enhanced the color stability. Nevertheless, for a pentose determination, it is advisable to measure the absorbance of the solution within a period of 30 minutes after the development of the color on the chromatogram.
The determination of standard sugars together with the unknowns on the same paper chromatogram has alleviated the use of exactly standardized conditions, so that small variations in the procedure given will not affect the final results. Some typical examples of curves obtained with standard sugars are shown in Figure 1. The errors are within &4%. Table I shows data obtained for the quantitative analysis of spruce pulp hydrolyzed with 7’2% sulfuric acid, using the procedure developed by Saeman and coworkers ( 3 ) . In order to determine glucose, which was present in a high concentration in the pulp, the hydrolyzate was diluted 10 times before it was applied to the chromatogram. Mannose and xylose determinations were made using undiluted hydrolyzate. A determination of the total sugar present in the hydhlyzate was carried out by cautious evaporation of a known volume a t 50” C. under reduced pressure to a constant weight sirup. This gave a value of 27.3 nig. per nil. On this basis the figure given for the total sugar concentration in Table I represents a 99% recovery. ACKNOWLEDGMENT
The author wishes to thank Maija Lakstigala for her interest and criticism. LITERATURE CITED
(1) Hough, L., in “Methods of Biochemical Analysis.” T’ol. I, D. Glick, ed., p. 205-42, Interscience, New York, 1954. (2) Hough, L., Jones, J. K. K.,Wadman, W. H., J. Chem. SOC.1950, 1702. (3) Saeman, J. F., Moore, W.E., hlitchell, R. L., Alillett, AI. 1.. Tappi 37, 3 3 6 4 3 (1954). RECEIVED for review .4pril7, 1956. Accepted August 16,1956.
Colorimetric Estimation of Dialkyl Phosphites in Presence of Trialkyl Phosphites, Phosphates, and Phosphonates SAMUEL SASS and JAMES CASSIDY Chemical Research Division, Chemical W a r f a r e Laboratories, A r m y Chemical Center, Md.
A method for the detection and quantitative estimation of dialkyl hydrogen phosphites in the presence of other phosphatics is based on the reaction of these phosphites with trinitrobenzene to form a red color measurable on a colorimeter or spectrophotometer at 465 m p . The method is sensitive to 0.2 to 0.5 y of dialkyl hydrogen phosphite per ml. of solution.
ANY phosphorus compounds used for insecticides and
related purposes are made in several synthetic steps which require alkyl phosphites or yield them as intermediates. No micromethod was available for distinguishing between dialkyl hydrogen phosphites and the monoalkyl and trialkyl phosphites, among others. I n this particular case a method was required for the estimation of dialkyl hydrogen phosphites in the presence of larger quantities of compounds with varying similarity in chemical properties, Evidence that the dialkyl hydrogen phosphite exists as an equilibrium between the hydrogen phosphite and phosphonate came from unpublished infrared and nuclear magnetic resonance studies made in these laboratories, and from its hydrolytic characteristics (Reactions 1, 2, 3), studied here and also reported by Fox (S), among others.
CHIO
90 %
CHIO
\P
CHIO/
10%
0
P-H+HOH
M
/
/
CHiO
CHIO
\7
HCL
-+
P
HO’
0
+ CHsOH
(2)
H ‘
However, its oxidative characteristics in the presence of iodine or bromine show an apparent 100% trivalent phosphorus character or 100% as the apparent hydrogen phosphite form. The tautomeric character of the dialkyl hydrogen phosphite appeared to be reminiscent of the enol keto equilibrium of carbon ketones, thus suggesting the use of aldehyde and ketone techniques. The use of dinitro- and polynitrobenzenes to form colored compounds with ketones ( I , 9,4 ) was of interest in these latora-
1969
V O L U M E 2 8 , NO. 1 2 , D E C E M B E R 1 9 5 6 tories because it helped to supply a technique for the determination of small quantities of chloroacetophenone. Bost and Nicholson ( 2 ) suggested the use of ketones t o distinguish between mono- and polynitrobenxenes and toluenes. Baerstein (1) used the reaction to determine bcnzene and toluene in solvent mixtures by introducing a differential oxidation technique, followed by treatment with butanone. The mononitro con~pounds did not form color. The suggested mechanism ( 1 ) was a condensation between the enol form of the ketone and the aci form of nz-dinitrobenzene with the elimination of a molecule of water. Exploratory and developmental work done in these laboratories had shown that small quantities of ketones, including chloroacetophenone, could be estimated if conditions were modified to maintain a measurable stability of color. When expanded to the dialkyl hydrogen phosphites, it was found that 771-dinitrobenzene, while forming a color n i t h these compounds, did not allow for a sensitivity in the desiicd area (at least 1 y per ml. of solution) n i t h satisfactory accin acy. 2,4,6-Trinitrobenxoic acid, which u as available, produced a greater sensitivity. 1,3,5Trinitrobenzenc produced an even greater sensitivity with the dialkyl hydrogen phosphite. Whitmore (6)stated that 1,3,5trinitrobenzene reacts n ith sodium alcoholateq eventually to give replacement of one S O , by nlkoxyl. “The intermediate product is a highly colored compound probably of quinoid structnrc” (Reaction 4).
NO*
1
1
DISCUSSIOY
Trinitrobenxene was made to react with the dialkyl hydrogcn phosphites in the presence of alcoholic sodium hydroxide. The postulated mechanism for the formation of colored product, extended from the work of Baerstein ( I ) and Whitmore ( 5 ) with ketones, is shown in Reaction 5.
NO*
+ S a O E t or SttOH
k,,,(,
Reagents. Absolute ethyl alcohol. Sodium hydroxide, 0.005-V in absolute ethyl alcohol. Glacial acetic acid, 0.058’ solution in absolute ethyl alcohol. 1,3,5-Trinitrobenzene (TNB), melting point 121-122’ C., Eastman Kodak Co., 0.05% solution in absolute ethyl alcohol. Dialkyl hydrogen phosphite. Apparatus. Absorbance measurements were made on a Cary recording spectrophotometer. Measurements of concentration us. instrument units (absorbance) were made on a Klett-Summerson colorimeter using Klett filter No. 44 (410 to 480 mp), and on a Beckman DO spectrophotometer a t a wave length of 465 mp. Procedure. Prepare a solution of the dialkyl hydrogen phosphite in absolute ethyl alcohol, such that a 5-ml. aliquot mill represent from 5 to 50 y of compound. Pipet 5 ml. of the specimen solution into a Klett tube, add 1 ml. of trinitrobenxene solution, and mix. Then add 1 ml. of 0,005X ethanolic sodium hydroxide, mix, and allow to stand for 1 minute. Add 3 ml. of 0.05&Vethanolic acetic acid, mix, and allow to stand for 6 minutes. Measure the developed color on a colorimeter using Klett filter S o . 44 or on a spectrophotometer a t 465 mp. Run a suitable reagent blank. For specimens containing materials of relatively high acidity, adjust to pH 10.5 ~ i t hethanolic sodium hydroxide after the addition of trinitrobenxene. Proceed with ethanolic acetic acid, etc. The sensitivity by this procedure is approximately 0.5 y per ml. of solution. The sensitivity can be doubled by restrictr ing the total volume of reagents and specimen to 5 ml. Preparation of Standards. Prepare standards containing 5, 10, 15, 20, 25, 30, and 40 y of dialkyl hydrogen phosphite per 5 ml. of alcohol. The curve, which is reproducible, follous Beer’s law through 40 y.
0It
SOzSa
I
R E 4 G E N T S , 4PPARATUS, 4YL) PROCEDURE
A
Spectral Characteristics. The spectral characteiistics of the color produced by the reaction of trinitrobenzene with dialkyl hydrogen phosphite were determined on a Beckman DC‘ spectrophotometer and later on a Cary recording spectrophotometer in the range 300 to 600 mp. Figure 1 shons the absorption curve when dimethyl hydrogen phosphite is treated under the procedural conditions. Figure 2 shows the calibration curves obtained for a number of different alkyl hydrogen phosphites rising the Beckman DU at a wave length of 465 mp, the sensitivity in micrograms per instrument unit being proportional to the molecular weight of compound. Effect of Water. The red colored product can be forincd in an aqueous medium, but under less stable conditions (approximately one half the stability of the nonaqueous medium).
-f
0.20
P-OH
R0/ OzN -/\ N o t RO H
+
or
w
V
HO
0,
z 4
NOzNa
+ €I& or ROH
EOJO 0
(5)
cn
m 4
/
One difference is that the ketone product, while showing up as color in the basic solution, loses the color in the acid. The color formed 11ith the dialkyl hydrogen phosphites remains after acidification. I n fact, the proposed method is based on an acidification after previous reaction and color formation when basic. This treatment was required, because the trinitrobenzene itself is strongly colored in basic solution.
0.0t
I
00
400
WAVELENGTH,
1
I
500 600 MILLIMICRONS
Figure 1. Spectral absorbance curve for 10 y of dimethyl hydrogen phosphite and trinitrobenzene
1970
ANALYTICAL CHEMISTRY
Effect of pH, Time, and Temperature. Specimens of dimethyl hydrogen phosphite were dissolved in water, ethyl alcohol, and methyl Cellosolve. Trinitrobenzene, in the proper proportions, was added to each and the resulting mixtures were treated with sodium hydroxide in the range of pH 7 to 12. A color formed rapidly through most of this pH range (pH 8 to 12) a t ambient temperature (25’ C.), but maximum results were obtained at pH 10.5, if the specimens were kept alkaline for no longer than 1.5 minutes. Good sensitivity resulted, but the reagent trinitrobenzene blank was relatively high. Tests made on the acidification of the colored material between pH 7 and 2 showed maximum stability for the developed color a t pH 6.5, with less than 1.570 color decrease after 40 minutes.
Table I.
Specificity of Method for Diallryl Hydrogen Phosphites
Sample Trimethyl phosphite (1000 Y) Triisopropyl phosphite (1000 y ) Bis (hydroxymethylphosphonic acid) anhydride (150 y ) Methylphosphonic acid (120 y ) Triethyl phosphate (1000
y)
Dimethyl niethylphosphonate (200 y) Phosphorous acid (100
y)
Monomethyl hydrogen phosphonate (1000 y )
Dimethyl Hydrogen Phosphite, y Added Recovered 0
