ACKNOWLEDGMENT We thank Mary Kinsley of Brookhaven for her assistance and informative conversations. We also acknowledge A. J. Alkezweeney and J. Hales of Battelle Pacific Northwest Laboratories for operating our sampler during their Milwaukee plume experiments, and P. 0. Lioy of the Interstate Sanitation Commission for supplying the High Point, N.J., samples. We are further grateful to Roger Tanner and Mary Phillips of BNL for the turbidimetric sulfate analyses.
LITERATURE CITED “Health Consequences of Sulfur Oxides: A Report from CHESS 1970-1971”, US. Environmental Protection Agency, Publication No. EPA-650/1-74-001, Research Trlangle Park, N.C. (May 1974). M. 0. Amdur, T. R. Lewis, M. P. Fitzhand, and K. 1. Campbell, U S . Environmental Protectbn Agency Publiilkn No.AP-111, Research Triingle Park, N.C. (July 1972). R. L. Tanner and L. Newman, J. Air foilut. Control Asscc., 26, 737 (1976). J. Forrest and L. Newman, J . Air Pollut. Control Assoc., 23, 761 (1973). T. G. Dzubay and R. K. Stevens, Environ. Sci. Techno/., 7 , 663 (1975). “Comparison of Wet Chemical and Instrumental Methods for Measuring Airborne Sulfate”, US. Environmental Protection Agency, Publication No. EPA-600/2-76-059, Research Triangle Park, N.C. (March 1976), p 57. A. P. Altshulier, J . Air Pollut. Control Assoc., 26, 318 (1976). L. Newman, J. Forrest, and B. Manowitz, Atmos. Environ., 9, 959, 969 (1975). (9) W. E. Wilson, R. J. Charlson, R. 8. Husar, K. T. Whitby, and D. Blumenthal, 69th Ann. Meeting of the Air Pollution Control Assoc., Portland, Ore. (June 1976).
(10) Division of Biomedical and Environmental Research, U.S. Energy Research and Development Administration, Washington, D.C. (1975). (11) H. Small, T. S. Stevens, and W. C. buman, Anal. Chem.,47, 1801 (1975). (12) J. Mulik, R. Puckett, D. Williams, and E. Sawicki, Anal. Lett ., 9, 653 (1976). (13) D. A. Lundgren and T. C. Gunderson, J. Am. Ind. Hyg. Assoc., 36, 866, (1975). (14) I. St. Lorant, Z . Physiol. Chem., 185, 252 (1929). (15) C. L. Luke, Ind. Eng. Chem., Anal. Ed., 15, 602 (1943). (16) C. L. Luke, Ind. Eng. Chem., Anal. Ed., 17, 298 (1945). (17) C. L. Luke, Anal. Chem., 21, 1369 (1949). (18) L. P. Pepkowitz and E. L. Shirley, Anal. Chem., 23, 1709 (1951). (19) C. M. Johnson and H. Nishita, Anal. Chem., 24, 736 (1952). (20) L. Gustafsson, Taianta, 4, 236 (1960). (21) J. B. Davis and F. Lindstrom, Anal. Chem., 44, 524 (1972). (22) H. G. Thode, J. Monster, and H. B. Dunford, Geochim. Cosmochim. Acta, 25, 159 (1961). (23) J. Forrest and L. Newman, Atmos. Environ., 7 , 561 (1973). (24) R. L. Tanner, R. Cederwall, R. Garber, D. Leahy, W. Marlow, R. Meyers, M. Phillips, and L. Newman, Atmos. Environ., in press. (25) H. D. Axelrod, J. H. Caw, J. E. Bonelll, and J. P. Lodge, Jr., Anal. Chem., 41, 1856 (1969). (26) N. L. Craig, A. B. Harker, and T. Novakov, Atmos. Environ., 8, 15 (1974). (27) L. D. Hansen, L. Whitlng, D. E. Eatough, T. E. Jensen, and R. M. Izatt, Anal. Chem., 48, 634 (1976). (28) R. B. King, J. S. Fordyce. A. C. Antoine. H. F. Lelbecki, H. E. Neustadter, and S . M. Sidik, NASA Tech. Note, TN D-8110 (1976). (29) “Sulfate Method VIb V i Turbdlmetry”, Technicon Corp., Tarrytown, N.Y. (1959). (30) D. Leahy and R. Garber, Brookhaven Natlonal Laboratory, Upton, N.Y., personal communication, 1977.
RECEIVED for review April 14,1977. Accepted June 27,1977.
Determination of Acid POH Groups of Hydrolysis-Susceptible Esters of Phosphorous Acid Robert Siegfried Federal Research Centre for Nutrition, 0-75Karlsruhe, West Germany
Quantitative determlnation of acid POH groups of hydrolysiesusceptible compounds of phosphorous acld, based on the conductivity in water-free medium is described. Water-free methanol ( 1 ) serves as solvent. I t Is very similar to water and particularly well suited because of Its good dissolving property, low conducting power, and lonlring power. Phosphorous acld Is applied as indicator. The limit of detection for POH groups Is 0.01 mmol.
As was demonstrated by more than 200 publications during the years of 1974 and 1975 (2), titration in nonaqueous solutions is a method widely used to solve, in an elegant way, individual problems such as the determination of acid groups in samples sensitive t o hydrolysis, for instance. Our problem was to determine the acid groups during the polycondensation of ethyl-(P-hydroxethy1)phosphite(I). /a
Cz
O-CH
Hg O-P'
“ 0 - C- H , 4
I
I
2
c
Acid POH-groups are present only as free phosphorous acid or as end groups ROPH(+O)OH, since the two-proton 1584
ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
phosphorous acid cannot contain any more acid POH groups within a diester chain. Titration using lyes in aqueous solutions, however, leads more or less rapidly to the formation of another acid POH group and is therefore not applicable. In a titration using alcoholates (for instance, sodium methylate) in a water-free medium (for instance, methanol), the following reactions are supposed to take place in the presence of free phosphorous acid and its monoesters (diesters do not react). H,PO, + NaOR = NaH,PO, + HOR NaH,PO, t NaOR = Na,HPO, t HOR
(1) (2)
[R,OPH(-tO)O]‘ t H’ + R,ONa= [R,OPH(-O)O]t Na’ + R,OH (3) The reactions can be followed by means of a conductometer. If the conductivity (Q-l) is plotted vs. the quantity (mL) of alcoholate added, a bend can be expected at the point where acid H+ is completely exchanged by Na+. Partly hydrolyzed dialkyl phosphite, however, results in a continuous increase of the conductivity, and it can therefore be assumed that in methanol all participating electrolytes of Equation 3 have about the same ionic mobility. Determination is possible, however, by means of an indicator (in our case: phosphorous acid), as will be shown below under the paragraph Results and Discussion.
EXPERIMENTAL Apparatus. The conductivity was measured at 3 KHz using a conductometer by F. Bauer, Frankfurt/Main, FRG. A glass
h
5 t
/i
ip-7
-Q
u " ! A
B
C
Nu- methylate --t
Figure 2. Conductometric tiitration curve of a monoaikyl phosphlte In water-free methanol with NaOCH3 after addltion of H3P03 Figure 1. (a) Conductometric titration curve of H3P03 In water-free methanol. Titration with sodium methylate. (b) Conductometric titration curve of H3P03 in water-free methanol. Titrated with lithium-methylate cell with 2 platinum electrodes of about 0.5 cm2each at a distance of about 0.7 cm from each other served as measuring cell. The microtitration apparatus Metrohm that we used consisted of (a) a stand, (b) a microburet (accuracy 0.001 mL), (c) a receptacle of 5 mL with heat regulator and cover (the latter with 5 openings for thermometer, measuring cell, nitrogen inlet, nitrogen outlet, and one opening to fill in samples, and (d) a stirring motor. Reagents. Methanol of the highest quality (Merck) was dried over molecular sieves and distilled. Its water content after this procedure was less than (Karl Fischer titration). The alcoholates were made out of this methanol and oxide-freemetals. The titration solutions were: NaOCH3 1.7 N, LiOCH3 1.0 N. Diethyl phosphite (Fluka) of the highest quality was distilled twice over Vigreux columns under nitrogen. Phosphorous acid (Fluka) of the highest quality was found to have a purity degree higher than 99% after aqueous neutralization titration. Procedures. The samples (oligomeric compounds of the phosphorous acid esters) 0.5-1.2 g were weighed into the titration receptacle under nitrogen and 5 mL of methanol were added. The receptacle temperature was kept constant at 25 & 0.1 "C. The platinum measuring cell, the thermometer, and the nitrogen supply lead were connected. By using the nitrogen stream, an access of atmospheric moisture to the apparatus was avoided. Then 17.3mg of phosphorous acid were added (1-mL solution: made of 1.730g phosphorous acid filled up with methanol to 100 mL). The content was vigorously stirred using a Teflon-coated stirring rod. For the titration, the above alcoholates were used.
RESULTS AND DISCUSSION Whether or not a titration of the acid protons of phosphorous acid in water-free medium is generally feasible, was tested by titration of water-free H3P03 in methanol. This titration yielded a curve with 2 bends, shown in Figures l a and l b . The consumption of methylate until the first bend corresponds precisely to the molar quantity of H3P03applied (neutralization of the first acid POH group). The methylate consumption recorded from the first to the second bend corresponds as well to the applied molar quantity (neutralization of the second acid POH group). The different gradients of the curve sections facilitate the determination of intersection points. When lithium methylate is used as titrating solution, Li2HP03precipitates after the neutralization of the first acid POH group and after further addition of lithium methylate. Frequently, however, a supersaturation occurs in the solution, and in these cases the bend is not sharp or delayed particularly when the phosphorous acid concentration is very low. Sodium methylate is preferable in this case. It is possible therefore to obtain, by addition of H3P03to a monoalkyl phosphite, a titration curve with two bends, which can be interpreted in the following way. The distance A-C (until the first bend) corresponds to the s u m of the acid POH groups of the monoalkyl phosphite and the first neutralization phase of the added phosphorous acid. The distance C-D corresponds to the second neutralization phase of the added phosphorous acid. When C-D is subtracted from A-C, one
I /I
1 0
92
0.4
96
0.8
LiOCH31ml) j
Figure 3. Conductometric tltration curve of a distillate residue of 2thOxy-l,2,3dk~aphospholane.Indicator H3P03. Titer solutlon LiOCH3
0
4
12
0
NaOCH l m l l 3
I
lo2
I4
+
Figure 4. Conductometric titration curve of a distillate of 2-ethoxy1,2,3dloxaphosphoiane. Curve I = 0.5361 g weighed portion, curve I1 = 0.501 1 g weighed portion. Indicator H3PO3. Titer solution NaOCH3 obtains A-B which indicates the share of POH groups (monoalkyl phosphite) in the partly hydrolyzed dialkyl phosphite. The accuracy of the method is demonstrated by the following examples. Five determinations using 17.3 mg of H3P03 were made. The deviations were 0.1 mg H3P03 at the maximum per determination, i.e., less than 1%.Also, multiple determinations of the products obtained by means of polycondensation resulted in deviations of less than 1% in samples of about 500 mg. A sample of monomethyl phosphite (for which we thank Pfluger) was found to be 99.5% pure. This finding could be verified by gas-chromatography, 2-Ethoxy-1,3,2-dioxyphospholane(I) was prepared according to Lucas (3). This compound hydrolyzes rapidly and quantitatively with H20. Increasing (up to molar) quantities of HzO were added, and the POH groups formed were determined. The POH groups formed out of the added water corresponded to the quantity calculated theoretically. The accuracy was better than 170also in this case. If an excess amount of HzO ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
1585
is added to hydrolysis-susceptible esters, complete hydrolysis takes place. In this case no water-free titration is necessary since all POH-groups in aqueous solution can be titrated with lye. The described method is also suitable to determine hydrolysis-susceptible samples with a high degree of accuracy. Figures 3 and 4 show conductometric titration curves. The method allows one to examine, besides phosphorous acid and its monoesters, other acids and esters also for the presence of POH groups.
LITERATURE CITED (1) J. Jander-Ch. Lafrenz, “Wasserahnllche Losungsrnlttel”, Verlag Chernle GrnbH, Weinheirn/Bergstr., 1968. (2) 8 . Kratochvll, Anal. Chern., 48, 355R (1976). (3) H. J. Lucas, F. W. Mltchell, Jr., and C. N. Scully, J . Am. Chem. Soc., 72, 5491 (1950).
RECEIVEDfor review January 10,1977. Accepted June 6,1977. Part of the author’s thesis submitted for his diploma, D.-65 Mainz, West Germany, 1970.
Reaction-Rate Method for the Determination of Hydrocortisone R. M. Oteiza, D. L. Krottinger, M. S, McCracken, and H. V. Maimstadt* School of Chemical Sciences, Universiv of Illinois at Urbana-Champaign, Urbana, Iliinois 6 180 1
A reactlon-rate method for the determination of hydrocortisone Is described. The method is based upon a modification of the widely accepted blue tetrazolium reaction. An analysis tlme of only 30 s Is required. Reiatlve standard deviatlons of about 1% or less are obtained, and the analytical worklng curves are Ilnear. Analysls of pharmaceutical skln preparations by the new rate method gave results which correlate well wlth the time-consuming standard equilibrium method.
Table I. Reaction-Rate Result.for Different Measurement Timesu Rate, AmA/sb Measurement time, s 1.0 5.0 10.0 15.0 30.0 45.0
26.5 25.6 25.2 24.8 23.7 23.8
RSD, % 3.7 1.0 0.8 0.4 0.5 1.0
Analysis of 2.5 mg/dL standard with 15-s delay time. Average of 5 determinations on a single sample. The quantitative determination of corticosteroids by various spectrophotometric methods has been previously discussed (1). One of these is based on the reduction of blue tetrazolium in an alcoholic solution of a strong base by the a-keto1 group on the CI7side chain of the corticosteroid to form a chromagen which has an absorbance maximum a t 525 nm. This absorbance, measured 90 min after mixing the sample with blue tetrazolium and the base, is then compared to that of a standard and blank solution to obtain quantitative information concerning the steroid concentration in the sample (2,3).This is the basis for the official method of the National Formulary ( 4 ) and the United States Pharmacopeia (5). Graham et al. have studied the blue tetrazolium procedure and have noted a first-order dependence of the corticosteroid concentration on the rate of the reaction (6). By employing the time-saving advantage of reaction-rate methods (71, we have developed a new procedure which decreases the analysis time considerably. Results obtained by the reaction-rate procedure are compared with the official method of the USP for pharmaceutical skin preparations.
EXPERIMENTAL Apparatus. The apparatus used for the reaction-rate method was the automated system described by Malmstadt et al. (8). This system provides for automatic aliquoting and mixing of sample and reagent and delivery of the mixed solution into the measurement cuvet (2-cm pathlength, 60-wL volume) by means of a stopped-flowunit incorporated in a modular spectrophotometer. A ratio-recording spectrophotometer (Model 721, GCA/ McPherson, Acton, Mass. 01720) was used for the equilibrium measurements. Reagents. A single 10 mg/dL hydrocortisone stock solution was prepared weekly by dissolving 10 mg of hydrocortisone (Sigma Chemical Co., St. Louis, Mo. 63178) in 100 mL of 95% ethanol. A 0.5% blue tetrazolium (Sigma Chemical Company) solution was prepared by dissolving 0.5 g of blue tetrazolium in 100 mL of absolute methanol. A 5 % solution of tetramethylammonium 1586
ANALYTICAL CHEMISTRY, VOL. 49, NO. 11, SEPTEMBER 1977
hydroxide was prepared by dissolving 5 g of tetramethylammonium hydroxide pentahydrate (Sigma Chemical Company) in 50 mL of USP, reagent quality, absolute ethanol (US. Industrial Chemicals Company, Tuscola, Ill. 61953). Different base concentrations were prepared from the 5% solution by appropriate dilution with absolute ethanol. The standard hydrocortisone solutions were prepared daily by adding 2 mL of the blue tetrazolium solution to an appropriate volume of the stock hydrocortisone solution and diluting to 10 mL with 95% ethanol. Sample Preparation. Samples were prepared from the pharmaceutical preparations-creams, gels, and ointments-by the column chromatographic procedure of Graham et al. (9) in which the corticosteroid is trapped in the column while interferences are removed by n-heptane. The corticosteroid is then removed from the column with chloroform. The eluate obtained from the column is carefully evaporated to dryness. The residue from the chloroform eluate is then dissolved in 95% ethanol and diluted to 25 mL. A 5-mL aliquot is then added to 2 mL of the blue tetrazolium solution and diluted to 10 mL with 95% ethanol. This 10-mL solution will be referred to in subsequent discussions as the sample. Approximately 30 minutes are routinely required for the sample preparation which provides an interference-free sample for analysis. Equilibrium Procedure. The equilibrium procedure was the official procedure given in the USP XIX (5) with the absorbance measured 90 min after mixing the standard with the two reagents. Reaction-Rate Procedure. One hundred p L each of a tetramethylammonium hydroxide solution and the appropriate standard or sample are sampled by the automatic syringes of the stopped-flowmodule (8). The syringes in the module then drive the solutions through the mixer and transfer the mixed solution to the observation cell. The change in absorbance is automatically monitored at 525 nm during the measurement time and used to construct a rate curve, working curve, or provide quantitative concentration information for the pharmaceutical skin preparations. For the results presented, the solutions and spectrophotometer were at ambient temperature in a temperature-controlled lab-
