For hydroquinone the mole ratio was found to be 2.70 j= 0.15 (for six determinations), and for NDGA a variable ratio was found, ranging from 3 to 5 . These obsgrvations indicate that one must exercise extreme caution when attempting to titrate with antioxidants with T T B P radicals unless the reaction has been studied in advance. Interferences. The major interference encountered in the use of the TT13P radical as a titrant arose from diffusion of atmospheric oxygen into the titration vessel. Extraneous okidizing and reducing agents present could cause interference. However, the latter problem may not be as difficult as one might anticipate-for example, ferrous iron and iodide ions were not reactive wlth the TTUP radical.
ACKNOWLEDGMENT
The authors gratefully acknowledge the technical assistance of Richard L. Hollinger in some aspects of this work.
(8) Klerk, A. D., Boot-Meurs, B., Anal. Chim. Acta 16,296 (1957). (9) Kolthoff, I. XI., Lingane, J. J., “Polarography,” Vol. 11, p. 552, Interscience, S e w York, 1952. (10) Kuwana, T., ANAL. CHEM.35, 1358
(1963). (11) Lintner,
LITERATURE CITED
(1) Blois, M. S., Nature 181, 1199 (1958). (2) Buchoff, L. S., Ingber, N. M., U. S. Patent 2,967,092 (Jan. 3, 1961)
( 3 ) Cook, C. D., Depatie, C. B., English, E. S., J . Org. Chem. 24, 1356 (1959). ( 4 ) Cook, C. D., Norcross, D . E., Zbzd., 81, 1176 (1959). ( 5 ) Cook, C. D., Woodworth, R . C., J . Am. Chem. SOC.75,6242 (1953). (6) Furman, N . H., ed., “Scott’s Standard
Methods of Chemical Analvsis.” Vol. I. 6th ed., pp. 774-97, Vai postrand: Princeton, iY.J., 1961. ( 7 ) Keidel, F. A , I n d . Eng. Chem. 52, 490 (1960).
C. J., Schleif, R. H., Higuchi, T . , Zbid., 22, 534 (1950). (12) Lundberg, W. O., ed., “Autoxidation and Antioxidants,” Vol. I, Interscience, New York, 1961. (13) hfcGowan, J. C.. Powell. T.. J . Chem. SOC.1960.238. (14) Richter, S . G., Gillespie, A. S., Jr., ASAL. CHEM.34, 1116 (1962). (15) Steele, E. L., Meinke, W.W., Ibid., 34, 185 (1962).
RECEIVEDfor review January 13, 1964. Accepted March 11, 1964. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 19, 1963. Work supported under Public Health Service Grant No. GM-09193 from the National Institutes of Health.
Derivatives of GIyoxa I Bis( 2-hydroxyanil) as Direct Calcium Reagents FREDERICK LINDSTROM and CARL W. MlLLlGAN Clernson College, Clernson, S . C .
b Six derivatives of the direct calcium reagent, glyoxal bis-(2-hydroxyanil), have been synthesized to study the effects of ring substituents and their positions on the performance of the derivatives as calcium reagents. Substitution of methyl groups in the 4 position of each ring produced a reagent of much greater sensitivity than the parent compound; substitution in the 5 position had little effect on sensitivity but greatly increased the stability of the reagent and the calcium complex. Substitution of nitro groups in either the 4 or 5 position yielded stable compounds having no value as reagents for calcium. The direction of future work has been established.
the calcium compound and the reagent gradually decompose and darken in the alkaline alcoholic solution needed for color development. Even the reagent itself will not keep well in a neutral alcohol stock solution. The reagent and its calcium complex are not watersoluble, so organic solvents must be used to obtain homogeneous test solutions. The parent compound and six derivatives selected t,o contrast different types of substit’uents on the parent compound were prepared and studied to determine the effect of ring substituents and their position on the properties of the reagents and their calcium complexes. The structural formulas of t,he seven compounds are shown in Figure 1. The polarity of the imino nitrogens and LYOXAL bis(2-hydroxyanil) is imthe phenolic oxygen atoms should be portant as a colorimetric reagent changed by the presence of methyl for calcium because in a basic solution groups on the ring. This electronthe reagent itself is yellow and the donating group should increase the calcium complex is bright red, thereby electron density in the chelation area permitting direct spect~rophot~ometric and thereby increase the stability of the determination of calcium. -1lthough the compleses with calcium. Xitro groups reagent reacts with numerous metal should have the opposite effect but to a ions t,o produce color, t,he calcium much larger extent. Substitution in the complex is formed above pH l l ! so few 4 position would place the groups para other metal ions remain in solut’ipn to to the nitrogens and should influence interfere. The reagent mas first them most; the 5 positions are para to synthesized by Bayer (2) and was used the hydroxyl groups, so substitution for the colorimetric determination of there should influence the acidity of calcium by Kerr (8) and later by those groups. The purpose of the Killiams and Wilson ( 1 4 ) . investigation was to see how ring subYnfortunately, glyoxal bis(2-hydroxystitution could be used to minimize the anil) (GBH.1) has several disadvandefects of the parent compound. t a g e as a reagent for calcium. Both
G
1334
ANALYTICAL CHEMISTRY
EXPERIMENTAL
Apparatus and Materials. All absorption spectra were obtained using a Perkin-Elmer Model 4000.1 recording spectrophotometer equipped with a P-E Model 3800 repetitive scanning attachment. A Beckman Model G pH meter was used for all p H measurement,s. It was standardized prior to each measurement against the phthalate (4.01) and boras (9.18) buffers recommended by the Kational Bureau of Standards. The following chemicals were used : practical grade o-nitrophenol, p-cresol, and N,S-dimethylformamide, Eastman Kodak Co.; purified (95 to 98%) mcresol, 30% aqueous glyoxal, and reagent grade methanol, Fisher Scientific Co. ; 2-amino-4-nitropheno1, 2-amino5-nitrophenol, and 3,4-dimethylphenolJ Aldrich Chemical Co.; tetrahydrofuran, Matheson, Coleman and Bell; sodium dithionite, Baker and Adamson; and reagent grade calcium carbonate, Mallinckrodt Chemical Co. All water used was deionized with a column charged with Fisher Scientific Co. Rexyn IRG 501, mixed-bed cationanion exchange resin. dl1 elemental analyses were performed by Galbraith Laboratories, Inc., Knoxville, Tenn. GLYOXAL BIS(2-HYDROXYANIL). It was much simpler to prepare the 0- , aminophenol required for this synthesis t>hanto purify the commercial product. Seventy-three grams of steam-distilled o-nitrophenol were added to 150 grams of sodium hydroxide in 1500 ml. of water. The misture was heated until the phenol just dissolved, and sodium
\ \
1
600
650
.20
il
H
.oo G O
400
450
500
550
w Figure 2. Apparent p H 1 2 . 2 5 . 1 0-cm. cells
H
Figure 1 .
\
14
CompolJnds studied
dithionate was atitied to the red solution until it wah completely decolorized. The solution was boiled with Sorite h, filtered. and neutra1izc:d with concentrated hydrochloric avid. The pure, colorless amine precipitated. l'siiig l3ayer'F i)roc:etlure exactly ( 2 ) , 57 grarns of o-aniinophenol and 40 ml. of 30% aqueous glyoxal yielded 55 grains (817.5%) of product [in.p. 207" d . : 204" t i . @)I. GLYOXALI ~ l s ( 2 - H T I I R O X Y - 4 - ~ ~ ~ : T H T L A X I L ) . A s the 2-arninc1-5-methylphenol rithesis was not available. it was prepared by nitration of mcresol as outlined b:; Schultz ( l e ) , followed by reduction to the amine. Two hundred grams oj' m-cresol in 400 ml. of bmzrne were nitrated a t 25" C. with 600 nil. of 1 to 1 nitric acid for 2 hours. The l o w r phaw was separated and discarded. The benzene solution, containing three nitration products, was >teani-distilled zfter treatment with sodium bicarbonate to remove
Absorption spectra of reagents and calcium complexes 10 ml. of 1 O-3M reozent, ca. 10 pg. o f colciur, to 5 0 ml. with methanol;
exce- acid. Of the nitration products. only 2-nitro-3-methylphenol and 211itro-5-methylphenol were steam-volatile and the latt,er is insoluble in water. Yield of 2-nitro-5-met~hylphencil was 45 granis (16%) [ m . p 56"; 56" ( l e ) ] . 2-.4mino-5-methyIphenol was prepared by reduction of the 2-nitro-5methylphenol as described for o-aminophenol. Hecause of the gradual decomposition of this amine, the yield was not determined [ni.p. 157-9" d . ; 162" d. (lf)]. Twenty grams of 2-amino-5-methylphenol and 16 ml. of the 307, aqueous glyoxal solution were added to 4000 ml. of water a t 80" C. Nitrogen war bubbled through the soluticm to prevent air oxidation. The product was recrystallized from tetrahydrofuran with difficultv. Yield: 35 prams (80%): in.11. 2i8" d. Calcdatid for C16H1602K2.C, 71.62; H, 6.01; S , 10.44. Found: C. 71.72: H. 6.10. IY, 10.41. l'hr microicopic white rhornbic cryst,als u-ere soluble in S,S-dimethylformaniide and S,.V-dimethylacetamide; slightly ,soluble in ethanol, methanol, and tetrahydrofuran; and insoluhle in ethyl acetate, water, ethyl ether, chloroform, carbon tetrachloride, and 4-niethy1-2pentanone. GLYOXALI 3 I s ( 2 - H r I ~ R o X T - j - h f r : T H Y L AXIL). The amine needed for this synthesis was obtained by the nitration of p-cresol as de,scribed above for mcresol, followed by dithionite reduction of the steam-distilled nitro compound. The 2-nitro-4-methylphenol was the only steam-volatile product. One hundred grams of p-cresol yielded 106 grams (757,) of 2-nitro-4-methylphenol [m.p. 35"; 36" ( I O ) ] . Fifteen grams of the nitro compound yielded 10 grams (837,) of the amine [m.p. 132"; 135" (1)I.
Fourteen grams of 2-amino-4-met,hylphenol, 12.3 ml. of 30y0 aqueous glyoxal, and 300 ml. of ethanol were heated under reflux for 30 minutes. dfter cooling to 0" the precipitated product was filtered and washed with water. Several recrystallizations froin ethanol
gave 7 gram- (46%) (n1.p. 212" d.). Calculated for Cj6Hl60&?. C, 71.62: H, 6.01; IT,10.44. Found: C, 71.51; H. 5.97: S.1D.29. The biayial tabular flakes were soluble in ethanol, benzene, tetrahydrofuran, and acetune, but irisoluble in water, chloroform, and carbon tetrachloride. GLYOXAL
I~IS:(2-HY1)ROXU-4,5-1)1-
Thirty grams of 3,4dimethylphenol were nitrated by the method of Diepolder (5); and the yield for 3,4-dimethyl-6-nitrophenol was 15 grams (36.6T0) [m.p. 85-7"; 87" (5)I. The amine was obtained by dithionite reduction with quantitative yield [m.p. 173-5" d.; 173-5" d. (5)] Xine grams of 3,4-dimethy1-6-aminophenol, 10 ml. of 307, aqueous glyoxal solution, arid 4000 nil. of water were heated to 80" under nitrogen. The product crystallized from solution. Recryst,allization from ethanol gave 8.5 grams (87.7%) (rn.11. 213" d.). CalC, 72.94; H, culated for CI8HZ0O2N2: 6.80; S , 9.45. Found: C, 72.72; H, 6.77; IK, 9.26. The fine needles were soluble in methanol, ethanol, benezene, chloroform, and carbon tetrachloride, and insoluble only in water. GLYOXAL I ~ I S ( ~ - ~ ~ Y I ) R O X Y - ~ , ~ - ~ I MI~:THYLANIL). Because of doubts about the orientation of the methyl groups of the above compound, the other I'ossible derivative from 3,4-xylenol was made. ('sing the riitration procedure of lIueller ( - 9 ) , 2-nitro- rather than 6-nitro-3,4xylenol was obtained and reduced with dithionite to 2-amino-3,4-sylenol in 417, yield over-all [m.p. 138-9"; 138-9" ( 9 ) ] . Three grams of the amine and 2.00 grams of 30% aqueous glyoxal were dissolved in 200 ml. of ethanol arid refluxed under nitrogen for 1.5 hours. The mixture was cooled in an ice bath and 300 mg. of crystalline product were obtained (m.1). 248" d.). Calculated for ClsHroN20e:C, 72.94; H, 6.80; S ,9.45. Found: C , 73.10; H, 6.68; S,9.49. GLYOX.4L 131S(2-HYUROXY-4-SITRO 4SIL). Sixty grams of 2-amino-5-nitrophenol and 100 ml. of 56% aq1,eou.G METHYLANIL).
I
VOL. 3 6 , NO. 7, JUNE 1 9 6 4
1335
t
ai-
7
$/
/"
Figure 3. parent pH
i
J
Beer's law plots at ap-
12.25
1 ml. of 1N potassium hydroxide, 15 ml. of 1 W 3 M reagent, to 50 ml. with methanol; IO-cm. Dells
just like the 4-nitro derivative. Sixty grams of 2-amino-4-nitrophenol and 40 ml. of 307, aqueous glyoxal yielded 26 grams (41%) (m.p. 238" d.). CalcuC, 50.91; H, lated for C1&00&4: 3.05; S , 16.96. Found: C, 50.71: H, 2.90; X, 16.87. The yellow pleochroic needles had the same solubility pattern as the 4-nitro derivative. Absorption Spectra. An aqueous alcoholic solution of each reagent was treated with a n alkaline solution of calcium to determine if colored compounds could be formed. The parent compound and all methyl derivatives except the 5,6-dimet,hyl derivative reacted to produce red and purple colors with calcium; the hues appeared to change as the calcium concentration was varied. The absorption spectra of the reagents and the calcium cumplexes are shown in Figure 2 . The nitro compounds reacted with calcium to give red complexes which faded before their absorption spertra could be obtained. The color formed and vanished within 10 seconds.
Absorbance US. Concentration. Solutions 0.001M in the reagents were glyoxal were added to 13,600 ml. of prepared in reagent grade methanol. water a t 80" and stirred for 30 minutes The solution of the 4-methyl derivaa t 80". The yellow crystalline derivative (GBH-4-31e.l) was prepared by tive was filtered and recrystallized from dissolving the reagent in the smallest an acetone-water mixt'ure several times, possible amount of .V,AV-dirnethylwhich yielded 28 grams (44%) (m.p. formamide, followed by methanol. 264" d.). Calculated for ClrHloOs~,: The calcium stock solution was preC, 50.91; H, 3.05: N, 16.96. Found: pared by dissolving 0.1000 gram of C, 50.93; H, 3.04; S,17.07. The fine calcium carbonate in 25 ml. of 1 S yellow needles were soluble in acetone hydrochloric acid, evaporating to dryand tetrahydrofuran. slightly soluble in ness, and diluting to 1000 ml. A 50ethanol and methanol, and insoluble in ml. aliquot of this solution was diluted water, benzene, chloroform, and carbon to 1000 ml.; 1 ml. contained 2 fig. of tetrachloride. calcium. From this stock solution, GLYOXAL BIS(2-HYDROXY-5-NITROvarious amounts of calcium ranging from ANIL). This compound was prepared 1 through 7 ml. were added in turn to seven 50-ml. volumetric flash. One milliliter of I S potassium hydroside was added, followed by 15 ml. of the reagent. Methanol was added to the mark and the flasks were set aside for 30 minutes. After each addition, the flasks were shaken to ensure completc mixing. The absorption spectrum of each solution was recorded againqt deionized water as the reference. 0nr.centimeter ctlls were used in thc 1-c' of GHH-4-;\1e.l but 10-em. cell.< wow used for the other reagents, for their solutions were not as intensely colored.
+.60 r""
+.40t
i
t
- I 00 -4 8
-4.6
-44
-42
-40
-38
Figure GBHA
5.
Bent and French plot for
1 ml. of N potassium hydroxide, 10 mi. of 1 0 - 3 M excess component, 1 to 7 ml. of 1 0 - 3 M varied component, to 5 0 ml. with methanol; IO-cm. cells
seven 50-ml. volumetric flasks, followed by 1 ml. of 1 S potassium hydroside solution. Various amounts of the 0.00151 solutions of the colorimetric reagenth ranging from 1 through 7 ml. were added in turn to each flask. The solutions were diluted to the mark with methanol and shaken several times after each addition to ensure complete mixing. The absorbance of each solution \vas obtained as described above. Khether the reagent or the calcium concentration was in escess. the above order of addition had to be followed to ensure reproducible result
