ANALYTICAL CHEMISTRY
814
7
Table 11. Comparison of the New and the SchoenheimerSperry Methods hlg. Cholesterol per 100 111.
S e w methoda 253 195 22: 25a 222 221 267 216 257 251 a
S.-S. method 258 193 229 260 204 220 250 220 25s 243
Kew methoda 224 227 218 162 130 193 308 256 262 194
S.-S. method
223 241 229 153 133 190
282 245 242 198
-x8 500 0
Average of two determinations
Comparison with Accepted Method for Determination of Total Cholesterol. Tn-enty different serum specimens were analyzed in duplicate, and the results were compared with analyses of samples of the same serums by the Schoenheimer Sperry method ( 2 ) . Results are shown in Table 11. With the new method, values were obtained M hich had an average deviation of &3.5y0 from the values found with the SchoenheimerSperry procedure.
01
CALCULATIONS
The milligrams of cholesterol in 100 ml. of serum is calculated as follons: Optical density of unknown - optical density of blank X Optical density of standard mg. of cholesterol in standard X lo3 = mg. of cholesterol in 100 nil. of serum
All serums and standards are run in duplicate, and the average optical densities of the duplicates are used to calculate the final results. Duplicates have been found to show an average deviation of less than 5%.
400
"
450
'
500
"
550
'
I
600
'
I
650
'
I
700
A ,MU Figure 2. Absorption Curves of the Reaction Products of Cholesterol, Cholesteryl Acetate, or Serum A, serum; X, cholesteryl acetate; and 0 , free cholesterol Schoenheimer-Sperry method shows that the same holds true for cholesterol and cholesteryl esters in serum. ACKNOWLEDGMENT
The authors wish to thank L. Le& ;ibell of Columbia University for the analyses by the Schoenheimer-Sperry method.
DISCUSSION
I t has been stated that methods based upon direct colorimetry without saponification are inaccurate because cholesteryl esters give higher color densities than cholesterol (1). I n the new method, equimolar quantities of cholesterol and cholesteryl acetate give equal color densities, and the agreement between results obtained by the new method and thope obtained by the
LITERATURE CITED
Soyons, E. C., Biochem. Z . , 303, 415 (1938). (2) Schoenheimer, R., and Sperry, W. M.,J . B i d . Chem., 106, 745 (1)
(1934).
(3) Zuckerman, J. L., and Natelson, S., J . Lab. Clin. .\led., 33, 1322 (1948).
RECEIVED for review August 6, 1952. Accepted December 13, 1952.
Colorimetric Estimation of lsonicotinic Acid Hydrazide E. L. PRATT, Winthrop-Stearns Inc., Rensselaer, N . Y . application of isonicotinic acid hydrazide in the treatRE""" ment of tuberculosis has brought forth the need of a specific T
analytical procedure for control testing this compound. The method dcscribed herein is based on the absorbancy measurement of the chromophore resulting \Then isonicotinic acid hydrazide is reacted with sodium P-naphthoquinone-4-sulfonate and 2 N sodium hydroxide. All steps are carried out in a complete aqueous system a t room temperature. Absorbancy maximum, 480 mfi. Subsequent to the utilization of isonicotinic acid hydrazide in the treatment of tuberculosis analytical procedures for the estimation of this compound appeared. One of the early methods was based on the titration of the hydrazide substituent with nitrous acid (9). An iodometric procedure was proposed by Canback ( I ) , and a similar method, carried out in acid solution, was described by Alicino (1). A second method in the publication of Alicino (1) was based on the titration of the basic ring nitrogen and end amino nitrogen of the hydrazide m-ith perchloric acid in nonaqueous media. The substituted pyridine ring has been
characterized by ultraviolet spectrophotometry (9). and the effect of various solvents and aqueous acidic pH levels in producing both bathochroniic and hyperchromic spectrum shifts has been observed by Auerbach ( 2 ) . A colorimetric procedure has recently been reported by Ballard and Scott ( 3 ) based on the chromophore produced when the T acid, l-chloro-2,4-dinitrobenzene, combines with the basic nitrogen of r-picoline and isonicotinic acid hydrazide. The procedure described herein is based on the early work of Ehrlich and Herter ( 5 ) and the later writings of Feigl (6). I n Feigl's text the identification of reactive CH2 and NH, groups by reaction with alkaline sodium ~-naphthoquinone-4-sulfonate is discussed. I n this paper the use of the aforementioned reagent in estimating the hydrazide substituent of isonicotinic acid hydrazide in microgram quantities is considered. Subsequent to the preparation of this article, Scott (10) has described the qualitative reaction of 1,2-naphthoquinone-4-sulfonate with isonicotinic acid hydrazide.
V O L U M E 25, NO. 5, M A Y 1 9 5 3
815
Sodium ~-naphthoquinone-4-sulfonate combines xvith the hydrazide substituent of isonicotinic acid hydrazide producing an orange-red chromophore having an absorption maximum a t 480 mp. With semicarbazide a similar spectrum is obtained which is qualitatively identical but has a considerably lesser absorption when compared to an equimolar quantity of isonicotinic acid hydrazide (Figure 1). Hydroxylamine displaye a strong absorption curve when reacted with the naphthoquinone reagent, but its maximum is found a t 440 mp. This may be explainable on the basis of extended conjugation afforded by the tautomerism in those substances having a carbamyl or substituted carbamyl grouping
OH (R=s--s==L--n/)
H O ( R = s - - ? jI - ~ - R ’ t)
K i t h hydroxylamine such an extension of conjugation is not possible. Table I presents several compounds, their quinone reaction product absorption maxima, and their observed molecular extinction values a t specified maxima. The wave length of maximum absorption has been found to be either at 440 to 450 nip or a t 480 nip in those cases where a spectrum has been observed. These maxima are consistent with the above explanation. Furthermore, it is apparent that, of the compounds tabulated, the 480 mp molecular extinction values for sernicarbazide and isonicotinic acid hydrazide indicate high specificity for the hydrazide grouping. Quantities of 6 micrograms per ml. of the latter substance are the optimum concentration for minimum error colorimetry when the procedure described is used. This sensitivity suggests application in the analysis of body fluids as well as use in routine control analysis.
A 2 A- sodium hydroxide solution has been used exclusively for all color developments discussed herein. Higher concentrations of hydroxyl ion tend to increase the reagent contribution to the gross absorption a t 480 mp. REFERENCE STANDARD
Isonicotinic acid hydrazide was recrystallized from 80% methanol. A constant melting point of l i l ’ C. corrected n’as obtained after one recrystallization. REAGESTS
Sodiuni sulfite, C.P. (Baker’s analyzed) Sodium hydroxide, 2 AAcetic acid, 6y0(v./v.) Sodium p-naphthoquinone-4-sulfonate(purchased from Eastman Kodak and recrystallized by procedure of Folin, 8 ) PROCEDURE!
Transfer 40 mg. of recrystallized sodium 3-naphthoquinone-4sulfonate and 250 mg. of sodium sulfite to a 200-nil. volumetric flask. Add approximately 100 ml. of distilled Tyater, and when solution is complete, transfer 3.0 ml. of 6% acetic acid to the flask. Make the reagent solution up to volume m-ith distilled water. (This solution is colorless; it should be prepared on the day the assay is to be run.)
Z
a
I_.-..-.
9
0 10.0
semicarbazide hydroxylamine
30 40 50 60 70 80 TIME, (minutes) Figure 2. Rate Curve for Sodium P-Saphthoquinone-4sulfonate-Isonicotinic Acid Hydrazide Chromophore Formation 10
X
t-
X W
a
a
-J
3 0 W
A 0
z
-
\
1
I
9
I1Y.L 440 460 480 500 W A V E LENGTH
I 420
I
I
I
I
mu
Figure ,l. Reaction Product Spectra with Sodium P-Xaphthoquinone-4-sulfonate
The relationship between absorbancy and time is illustrated in Figure 2. These rate data were obtained on a 90-microgram sample of isonicotinic acid hydrazide in a total volume of 15.0 nil. relative to a reagent blank. A stable color is observed after 15 minutes and remains stable for approximately 1 hour. Figure 3 illustrates the absorbancy versus concentration relationship. These data show both conformation to Beer’s Law and the concentration range over which this procedure of assaying isonicotinic acid hydrazide is applicable.
20
Dissolve 75.0 mg. of the reference standard in 250 nil. of distilled water. Dilute this solution, 10 ml. to 100 ml., Yith distilled water. (Each ml. of the final dilution contains 30 micrograms of isonicotinic acid hydrazide.) I n the manner described for the reference standard, prepare a diluted solution of the material to be assayed. T o each of three glass-stoppered tubes transfer 3.0 ml. of the diluted reference solution standard and transfer to each of a second set of three tubes 3.0 ml. of the solution to be assayed. T o a seventh tube add 3.0 nil. of distilled i n t e r for a blank. Add to each tube 10.0 ml. of the hydroquinone reagent followed by 2.0 ml. of 2 LV sodium hydroxide solution. Invert each tube to ensure thorough mixing, and allow all tubes to stand for a period of 15 minutes. Transfer each of the solutions to a cell or cuvette, and determine the absoi bancy relative to the reagent blank at 480 mp. (The use of a Beckman Model B spectrophotometer or an equivalent narron band filter colorim-
Table I.
Chromophores Obtained with Sodium /3-Naphthoquinone-4-sulfonate
Sodium 8-naphthoquinone-4-sulfonate with Hydrazine sulfate Phenylhydrazine hydrochloride Aniline hydrochloride Hydroxylamine hydrochloride Acetamide Urea
X 11~1 ~ a r . (Visible Spectra) (440-450) 440
445 440 Sone 480 Sone 480 480 Sone Sone ?;one
Nolecular Extinction at llaximum 673 315
203 3580
i6o ... ’
6130 10100
...
... ...
ANALYTICAL CHEMISTRY
816
Table 11. Per Cent Recovery of Isanicotinic Acid Hydrazide Operator I , Beckman Model B Operator 11, Beckman XIodel B Operator 11, Lunietron Model 402E Operator 111, Beckman Model D T
Sample A Sample B Sample C 98.3 100.8 102.8 98.8 103.2 103.7 97.i 102.4 100.2 99.5 101.0 95.7 99.7 100.0 98.4 98.0 98.4 98.2 99,2 101.5 101,s 99.2 100.9 101.5 98.8 101.1 103.5 96.1 96.3 96 9
101 9 101.3 101.3
95.3 94.9 97.3
I-
Table 111. Analysis of Data
41 Standard
/
Deviation
Sinxle determination Av. duplicate determinations A r . triplicate determinations
1
eter is recommended.) The per cent of isonicotinic acid hydrazide is calculated as follo\vs: Av. absorbancy of unknown (90) Av. absorbancy of standard (microgram unknown in 3.0 ml. of final dilution)
loo
=
% Isonico-
tinic acid hydrazide
E V 4 L U 4 T I O N OF METHOD
The results obtained in the analyses of three commercial lots of isonicotinic acid hydrazide when carried out by different operators using different instruments are given in Table 11. These data indicate triplicate measurements of standard and unknown aliquots will provide 95% assurance that the average unknown value ~villbe reproducible within a range of =k2.70%. A gain of 42% in reproducibility is realized when triplicates are run in comparison to a single measurement. Analysis of data is given in Table 111. .411 compounds listed in Table I are reagent grade chemicals as received from various suppliers with the exception of picolinic acid, ethylnicotinate, and acetamide. The latter substancr was prepared by the method of Fisher ( 7 ) and recrystallized three times from chloroform (m.p. 79.5’ C.). Spectra were obtained (Figure 1) with the Beckman Model DU spectrophotometer. ACKNOWLEDGMENT
Acknowledgment and thanks are accorded to bI. E. Auerbach and H. TIT. Eckert, Sterling-Kinthrop Research Institute, J. F.
Figure 3.
1
1
1
1
1
l
1
1
1
60 80 100 C 0N C E NT R AT I0N(mcgm)
1
Absorbance us. Isonicotinic Acid Hydrazide Concentration
Scully and B. B. Spiegel, Kinthrop-Steams Inc., for their assistance in obtaining the collaborative data tabulated in Table 11; and to E. L. Bauer and J. K. Borland for statistical evaluation of data. Thanks are also extended to Hugh B. Corbitt and Irwin S. Shupe, Winthrop-Stearns Inc., for their helpful criticism and suggestions in the preparation of this paper. LITERATURE CITED
(1) dlicino, J. F., J . Am. Pharm. Assoc., Sci. Ed., 41, 401 (1952).
(2) Auerbach, M. E., Sterling-Winthrop Research Institute, unpublished report, April 1952. (3) Ballard, C. W., and Scott, P. G. IT.,Chemi8try R. Industry, 29, 715 (1952). (4) Canbjck, T., J . Pharm. and Pharmacol., 4, 407 (1952). (5) Ehrlich, P., and Herter, C. A , , Z . physiol. Chem., 41, 379 11904). (6) Feigl, F., “Spot Tests,” p. 291-3, Sew Tork, Sordeniann Publishing Co., 1937. (7) Fisher, H. L., “Laboratory Manual of Organic Chemistry,” p. 115-16, Kew- York, John Wiley and Sons, Inc., 1920. (8) Folin, 0.. J . BioZ. Chem., 51, 389 (1922). (9) Nepera Chemical Co., Yonkers, Kew York, private communications. (10) Scott, P. G. IT., J . Pharm. and Pharmacol., 4, 681 (1952). RECEIVED for review October 16, 1952. Accepted December 17, 1952.
pH Adjustment for Determination of Ammonia Nitrogen CLAIR N. SAWYER Sedgwick Memorial Laboratories of Sanitary Science, Massachusetts Institute of Technology, Cambridge, iMass.
of normal samples of water and s e w g e the addiI-tion ofanalysis the prescribed amount of 0.5 M phosphate buffer will Y THE
produce and maintain proper pH conditions ( 1 ) . However, in the case of many industrial wastes the acidity or alkalinity of the samples is so great that neutralization of the sample in the pH range of 7 to 8 follo\wd by addition of phosphate buffer is accepted practice. I t is the purpose of this memorandum to shon. that, with certain industrial wastes, even though the sample be neutralized to a pH of 7 to 8 and the phosphate buffer added, the resulting pH may be far below the optimum. This is especially true of n-astes containing appreciable concentrations of calcium which will unite TT ith phosphate ions present to form insoluble calcium phosphate
and, thereby, release hydrogen ions from the mono- and dihydrogen phosphate ions, causing the pH to drop. The effect of calcium is shown by the folloPring data: C a t + , P.P.M. 0 100 250 500 1000 2000
PH 7.50 7.40 7.20 6.75 6.10 5 80
In these tests, solutions of calcium nitrate containing the amounts indicated in the first column were neutralized to a pH of 7.4, and 0.5 M phosphate buffer ( p H = 7.5) was added a t the rate of 40
