of absorption profiles are as follows. The computer program can be used for routine drawing of absorption profiles by using directly the absorbance values to produce absorption profiles. The manual drawing of curves as in Figure 1 is completely eliminated, saving time and labor. There is a n enormous saving of time and labor-this is one of the most important advantages; this advantage becomes particularly important when one has to draw a large number of absorption profiles as in the case of references (1-3). The chances of personal errors due to the experimenter’s judgment being a t fault, personal idiosyncrasies, personal fatigue, and other personal factors, normal variations among replicate manual drawings due to human limitations, etc., are all eliminated by the computer-plotting, whereas manual drawings are subject to all these personal errors. Also, in curve-fitting, the manual method which uses one’s best judgment produces
much more error than the method of least squares. Since the absorption profiles are often used for the purpose of comparison, they should be free of all personal errors, or at least the errors should be constant, in order that the comparison is valid. In the manual method, the magnitude and the direction of personal errors change with time, the nature, and quantity of work, making the total error in the manual method variable. Compared to this, the computer program plots the absorption profiles free of all personal errors, thus enabling valid comparisons of different absorption profiles. RECEIVED for review April 12, 1971. Accepted June 25, 1971. The authors are indebted to the National Research Council of Canada for financial support of this research project.
Simplified Wet Ash Procedure for Total Phosphorus Analysis of Organophosphonates in Biological Samples Donald S. Kirkpatrick and S t e p h e n H. B i s h o p Department of BiochcmLstry, Baylor College of Medicine, Houston, Texas 77025
WET ASH METHODS capable of digesting difliculii:,, hydrolyzable phosphorus compounds in the presence of lares amounts of organic material and inorganic cations inc!u;ie refluxin,: in concerltrated perchloric acid or in concenrrakd sulfuric acid with hydroger. peroxide. These methods suffer from difficulty in controlling acid loss due to reflux (without the use of special glassware such as Kjeldahl flasks), due to oxidation of the sample material, and due to neutralization by cation residue. Because al! standard orthophosphate determinations using the phosphomolybdate blue complex are sensitive to acid concentration ( I ) , the amount of acid in the residue after digestion must be adjusted before the subsequent orthophosphate determination. Here we report the composition of a nitric, perchloric, sulfuric acid mixture and time-temperature operating conditions for wet-ashing samples containing 0.040 gram of organic material and 0.6 meq of inorganic cation. The procedure yields a uniform amount of acid in the residue after digestion in test tubes. Orthophosphate (1-50 nmole) can be determined by Bartlett’s ultramicro method ( 2 ) on the digested residue without adjustment of acid concentration. Recovery of total phosphorus from an organophosphate and four phosphonates ranges from 95 to 101 %. Relative standard deviation for replicates averages 1.7 %. EXPERIMENTAL Reagents. 2-Aminoethylphosphonic acid (AEP) was synthesized as described by Kosolapoff (S), and purified by chromatography o n Dowex-50-(H+)-8%. A. F. Isbell, Department of Chemistry, Agricultural and Mechanical College ( 1 ) 0 . Lindberg and L. Emster, “Methods of Biochemical Analysis,’’ D. Glick, Ed., Vol. 111, Interscience Publishers, Inc., New York, N . Y . , 1956, p 1. (2) G. R. Bartlett, J . Biol. Chem., 234, 466 (1959). (3) G. M. Kosolapoff, J . Amer. G e m . SOC.,67, 2112 (1947).
of Texas, Collegt Station, Texas generously supplied N . methyl-AEP and N,N-dimethyl-AEP. 2-Amino-3-phosphonopropionic acid (2A3PPA) was purchased from Calbiochem, Los Angeles, Calif. Phosphoserine was purchased from Sigma Chemical Company, St. Louis, Mo. Orthophosphate standard was prepared from Baker Certified Aminonaphthol sulPrimary Standard KH?POI (99.9 fonic acid (ANS) was purchased from Eastman Organic Chemicals, Rochester, N. Y ., and recrystallized. Concenvacuum distilled) was purchased trated perchloric acid (70 from G. Frederick Smith Chemical Company, Columbus, Ohio. All other chemicals used were analytical reagent grade. Deionized water was used throughout the procedure. Digestion mixture was prepared by stirring 98 ml of concentrated sulfuric acid into 230 ml of wafer. After cooling, 1200 ml of concentrated nitric acid and 120 ml of concentrated perchloric acid were added and the volume was adjusted to 1800 ml with water. This reagent could be stored for at least two months in a used hard glass sulfuric acid reagent bottle without increase in background due to silicate. Ammonium molybdate reagent was 1.18% solution in water. ANS reagent was 0.1 ANS in aqueous NaHSOs (0.548M), N a p S 0 3(0.159M) as described by Bartlett ( 2 ) . Equipment. TUBEHEATER.Test tubes (18 X 150 mm) were heated in a thermostatically controlled aluminum block electric tube heater (Warner-Chilcott Laboratories Instrument Division) loaned by T. E. Nelson of this Department. Temperature was monitored by a thermometer immersed in a tube containing DC-560 silicone oil. SPECTROPHOTOMETER. The Gilford Model 240 Spectrophotometer was used with cuvettes having 1.OO-cm light path and 1.5-ml capacity (Pyrocell No. 1007). Total Phosphorus Determination. DIGESTION PROCEDURE. In each digestion tube is placed a sample containing 1-50 nmole of phosphorus in the presence of less than 0.040 gram of organic material and less than 0.6 meq of cations. Digestion mixture (1.5 ml) is dispensed into a blank tube, a tube containing AEP standard (50 nmole), and each sample tube. The tubes are placed in the heating block for 1.5 hours at
z).
z,
ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
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was hydrolyzed because of exhaustion of oxidizing capacity of the perchloric acid. Exhaustion of the oxidizing capacity of the perchloric acid did not affect the subsequent color reaction since N a 3 . citrate . 2H10 up to 0.1 gram had no effect on phosphorus recovery from orthophosphate standards DIGESTION WITH NITRIC-PERCHLORIC-SULFURIC ACIDMIXTURE. Concentrated sulfuric acid alone at 214 "C (not fuming) did not convert 50 nmole of AEP to orthophosphate. Quantitative conversion proceeded with the addition of 0.1 ml of concentrated perchloric acid to the sulfuric acid. Blanks, orthophosphate standards, and AEP standards (50 nmole) with four levels of Na3.citrate.2H20 (0.0 to 0.1 gram) were digested by 0.10 ml of perchloric acid and three levels of nitric acid (0, 0.40, and 0.80 ml) in the presence of 3.0 meq of sulfuric acid (Table I). Samples were heated with the acid mixtures at 214 "C for 3.0 hours. Orthophosphate determination was performed directly on the residues. I n the presence of 0.020 gram of Na3.citrate.2H20, AEP was hydrolyzed quantitatively by perchloric-sulfuric acid mixture without nitric acid. Addition of 0.40 ml nitric acid to the perchloric-sulfuric acid mixture was necessary for quantitative hydrolysis of AEP in the presence of 0.060 gram Table 11. Effect of Salt on Color Development Reaction of Na3.citrate.2Hz0. Addition of 0.8 ml of concentrated AAQ at 830 nm for nitric acid enabled quantitative conversion in the presence NaCIO4, mmole orthophosphate, 50 nmole of 0.10 gram of Na,. citrate. 2H20. Addition of more than 0.06 0.5 0.972 gram of Nas. citrate, 2 H 2 0 caused a severe drop in absorp1.o 0.962 tivity (orthophosphate) even though the oxidizing capacity 1.5 0.946 of the acid mixture was not exhausted (AEP). 2.0 0.983 EFFECTOF CATIONS ON ORTHOPHOSPHATE DETERMINATION. 2.5 0.954 If more than 1.0 meq of cations remained from ashing salts 3.0 0,935 of organic acids, enough residual sulfuric acid was neutralized a A A = Absorbance of orthophosphate - absorbance of blank to affect subsequent orthophosphate color development. Blanks and orthophosphate standards (50 nmole) containing six levels of NaC104 (0.5 to 3.0 nmole) in 3.0 meq of sulfuric acid were heated at 214 "C for 3.0 hours. Orthophosphate 225 "C. Each tube should now contain in about 0.1 ml of determination was performed directly on tube residues clear, colorless digestion residue, 3.0 meq of concentrated (Table 11). Blanks and orthophosphate standards digested sulfuric acid. in the presence of sodium ion (as sodium perchlorate) equivORTHOPHOSPHATEDETERMINATION. The ultramicro alent to the 3.0 meq of sulfuric acid did not develop the molybmethod of Bartlett is used to determine orthophosphate. denum blue color characteristic of too low an acid concenTo the cooled digestion residue, ammonium molybdate tration. An amount of perchloric acid equivalent to the reagent (0.85 ml) and ANS reagent (0.05 ml) are added to cations present was nonvolatile under these conditions of bring the final volume to 1.00 ml. The tubes are mixed digestion even though the temperature was maintained well by vortex, capped, and heated in a boiling water bath for above the boiling point of perchloric acid in the presence of 10 minutes. Absorbance is measured in the spectrophoconcentrated sulfuric acid. Color development with orthotometer at 830 nm. phosphate was not affected by high levels (0-3.OM) of sodium Deielopment and Validation of Digestion Procedure. perchlorate. ON ORTHOPHOSPHATE EFFECT OF ACID CONCENTRATION TIMEAND TEMPERATURE REQUIREMENTS FOR DIGESTION. DETERMINATION. Bartlett's ultramicro orthophosphate deAEP standards (50 nmole) containing 0.060 gram of Na3. termination was performed on blanks and orthophosphate citrate.2H20 were digested by heating with 1.5 ml of digestion mixture at either 213 or 225 "C for varying times. After standards (50 nmole) over the perchloric acid concentration range 1.16 to 4.64N and the sulfuric acid concentration cooling, orthophosphate determination was performed range 1.0 to 5.ON. The usable range of acid concentration directly on the residues in the tubes. The digestion time to was found to be 2.0-3.ON for both sulfuric and perchloric quantitatively digest a sample containing 0.060 gram of acids. Naa.citrate.2H20was 1.5-3.0 hours at 213 "C and 1.0-2.0 DIGESTION WITH SULFURIC ACID ALONE. Digestion with hours at 225 "C. EFFECT OF N a 3 . C 1 ~ ~ ~ ~ € . 2ONH 2 SENSITIVITY 0 AND sulfuric acid must be performed at fuming temperature for LINEARITY. Total phosphorus determination was performed about 1 hour to quantitatively convert organophosphonates on a series of six AEP standards (0-60 nmole) in the presence to orthophosphate. In this laboratory it has not been possible of four levels of Na3.citrate.2H20(0 to 0.060 gram). AS without the use of Kjeldahl flask to obtain a reproducible Na,.citrate.2H20 increased from zero to 0.060 gram, amount of acid residue after digestion of AEP standards absorptivity decreased 5.2% from 1.92 X l o 4 to 1.82 X IO4. with fuming sulfuric acid. DIGESTION WITH PERCHLORIC ACIDALONE. Blanks, orthoThe standard curves from which the absorptivities were phosphate standard (50 nmole), and AEP standards (50 derived were linear from zero to 60 nmole phosphorus nmole) were digested in the presence of foul levels of Na, . (to A 1.2). RECOVERY OF TOTALPHOSPHORUS.Total phosphorus citrate 2 H 2 0 (0.0 to 0.1 gram) by refluxing 20-25 minutes determination was performed on a blank and 50-nmole in 15-ml Kjeldahl flasks with 0.250 ml of concentrated perstandards of orthophosphate, AEP, N-methyl-AEP, N,Nchloric acid (2.9 meq). Orthophosphate determination was dimethyl-AEP, 2A3PPA, and phosphoserine. All standards performed directly on residues in flasks. Samples containing were prepared from reagents dried overnight over P z O ~in no Na, citrate . 2 H 2 0 gave complete recovery of total vacuo. Six replicate determinations of total phosphorus phosphorus from AEP. With AEP samples containing 0.020 were performed on orthophosphate and the organophosgram of Na, . citrate 2H20, only 36% of total phosphorus Table I. Effect of Na3.Citrate.2Hz0on Digestion of AEP by Mixed HN03-HC1O4-HZSOa Ada at 830 nm OrthoNaa . citrate HNO, phosphate, AEP, 2H20, g (70% ml 50 nmole 50 nmole 0.000 0.00 1.019 1.040 0.020 0.00 1.017 1.029 0.060 0.00 0,928 0.319 0.100 0.00 0.839 0.000 0.000 0.40 0.960 1.022 0,020 0.40 0.978 1.054 0.060 0.40 0.954 0.930 0 . 100 0.40 0.807 0.775 O.Oo0 0.80 0.999 1,101 0.020 0.80 0.960 1.087 0.060 0.80 0.931 0.902 0 . 100 0.80 0.815 0.838 a A A = Absorbance of standard - absorbance of blank.
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ANALYTICAL CHEMISTRY, VOL. 43, NO. 12, OCTOBER 1971
Table 111. Total Phosphorus Recovery Substance, 50 nmole
Recovery, 2
Relative standard deviation,
101 97 97 95 97
1.7 1.4 2.4 I .4 1.5
AEP N-Methyl-AEP N,N-Dimethyl-AEP 2A 3PPA Phosphoserine a Average of six determinations.
phorus compounds. Table I11 shows total phosphorus recovered from the organophosphorus compounds relative to total phosphorus recovered from orthophosphate standard along with relative standard deviations of the replicates. DISCUSSION
The residual acid from the digestion procedure must be controlled for subsequent orthophosphate determination by Bartlett’s ultramicro method. The digestion mixture is designed to provide 0.8 ml of concentrated nitric acid, 0.1 ml of concentrated perchloric acid, and 3.0 meq of sulfuric acid to each tube. Nitric acid provides the reserve of oxidizing capacity to oxidize 0.040 gram of organic material without exhausting the oxidizing capacity of the perchloric acid. Perchloric acid provides the oxidizing potential necessary to break the C-P bond of organophosphonates and neutral-
izes residual cations. Larger amounts (than 0.6 meq) of cation can be handled by increasing the amount of perchloric acid and the digestion time. By digesting at a temperature significantly above the boiling point of nitric and perchloric acids but well below that of sulfuric acid, excess nitric and perchloric acids are volatilized from the tubes during the digestion period. The remaining sulfuric acid reproducibly provides 3.0 meq of free acid residue over wide ranges of operating conditions and sample tontents, thereby eliminating adjustment of acidity prior to analysis. The digestion procedure presented here has been applied in this laboratory to measure total phosphorus in amino acid analysis chromatograms containing between 2 and 1000 nmole/ml each of various organophosphates and aminophosphonic acids. Effluent collected from the amino acid analy7er contained between 0.0196 and 0.0343 gram of Na,. citrate 2 H s 0 per 1.O ml fraction ( 4 ) . Although our primary concern is with phosphorus analysis, the digestion procedure may find wider application in the preparation of biological and environmental samples for many analytical procedures.
RECEIVED for review April 30, 1971. Accepted June 8, 1971. Supported by Grant 4294 from the Robert A. Welch Foundation. (4) D. H. Spackman, “Methods of Enzymology,” C. H. W. Hirs, Ed., Vol. XI, Academic Press, Inc., New York, N. Y . , 1967, p 3.
Analysis of Insect Chemosterilants Action of Phosphate Buffers on Aziridine J. George Pomonis, Ray F. Severson,’ Patricia A. Hermes, Richard G. Zaylskie, and Andrew C. Terranova Metabolism and Radiation Research Laborator),, Agricultural Research Serrice, U.S . Department of Agriculture, Fargo, N . D . 58102 WHENCHEMICALS, especially alkylating agents, are to be used to produce sterile insects, it is essential to measure the period of persistence of these substances and their decomposition products. Several methods of analysis for chemosterilants such as aziridine-containing compounds (1-5) and alkylating agents such as alkyl-halides and sulfonates (6, 7) have been reported and have been widely used in degradation and metabolic studies. These kinetic studies were performed in conjunction with those used in the development of an auto-
mated analytical procedure for insect chemosterilants (8, 9). Aziridine, a product of hydrolysis of tris(1-aziridiny1)phosphine oxide (tepa) (9,was selected for extensive study. We report here the kinetic effect of phosphate buffers on aziridine and show that a modification of the standard colorimetric method of assay (1) for aziridine is suitable for the collection of kinetic data.
* Predoctoral assistant, Entomology Research Division, Agricul-
Apparatus. All measurements of absorbance were made with a Cary Model 14 spectrometer at ambient temperature. All measurements of pH were made at a temperature of 27 =t 0.1 “C on a Radiometer Model 22 pH meter equipped with a Model PHA 630 Pa scale expander and a combined glass and calomel electrode assembly (Radiometer GK 2021C). A 10gallon constant temperature bath was maintained at 27 + 0.05 “C with a Bronwill Thermo-regulator-stirrer for the hydrolyses. Materials. Aziridine from commercial sources was purified by distillation from NaOH and stored in dark bottles
tural Research Service, USDA. North Dakota State University. Fargo, N. D. 58102. (1) D. H. Rosenblatt, P. Hlinka, and J. Epstein, ANAL. CHEM., 27, 1291 (1955). (2) R. M. V. James and H. Jackson, Bioclient. Pharmacal., 14, 1947 (1965). (3) J. Epstein, R. W. Rosenthal, and R . J. Ess, ANAL. CHEM., 27, 1435 (1955). (4) A. W. Craig, H. Jackson, and R. M. V. James, Brit. J. Plrarrnnco(., 21, 590 (1963). (5) M. Beroza and A. B. Borkovec, J . Med. Clwm., 7,44 (1964). (6) E. Sawicki, D. F. Bender, T. R. Hauser, R . M. Wilson, Jr., and J. E. Meeker, ANAL.CHEM., 35, 1479 (1963). (7) T. A. Connors, L. A. Elson, and C. L. Leese, Biochem. Pharmacol., 13,963 (I 964).
EXPERIMENTAL
(8) A. C. Terranova, J. G. Pomonis, R. F. Severson, and P. A. Hermes, Proc. Techiiicoii N . Y . Symp. Automrrtion in Anal. Cliem., I, 501 (1967). (9) A. C. Terranova, J . A g r . Fond Clien?.,17,1047 (1969).
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