ANALYTICAL CHEMISTRY
1190 Table IV.
Microbiological and Chemical Sulfur Assays of Various Substances
(All results reported ae yo sulfur on dry basis) Alkaline Microbiological Magnesium DL-Methionine NasSO, % Protein Nitrate standard standard Sample Soybean flour ( N X 6.25) (defatted) 54.10 0,519 0.417 0.522 Boybean flour ( N X 6.25) (whole) 53.06 0.443 0.338 0,436 Vitamin-free ( X X 6.38) casein 98 18 0.711 0,679 , . .
that cystine and sodium sulfate produce very similar yeast growth response. This observation coupled with data given by Evans ( 7 ) and Chang and Murray (b), showing that the sulfur in soybean is predominantly from cystine, adequately explains this apparently anomalous behavior. Admittedly these authors’ data are a t a variance with a considerable portion of the literature, which shows soybeans to contain a greater relative amount of methionine. However, it is the authors’ belief that Evans (7) presents sufficient justification for his results. Microbiological results on the total sulfur content of casein using Dkmethionine as the standard are in good agreement with those obtained with the chemical method. This is in accord with data cited by Cohri and Edsall (6), Evans ( 7 ) , and Block
and Bolling (9,4 ) , who report casein to contain 85 to 90% methionine sulfur and 10 to 15% Cystine sulfur. CONCLUSIONS
I t is believed that this microbiological assay method, using Dkmethionine as a standard, will yield reliable total sulfur values when used on yeast However, care must be taken in the application of the method to substances other than yeast. An understanding of the composition of the sample to be assayed is essentirl for the selection of a standard which will produce a growth response corresponding to that of the sulfur in the sample. LITERATURE CITED
( 1 ) Assoc. Offic. Agr. Chemists, “Official and Tentative Methods of Apalysia,” 6th ed., p. 127, 1945. (2) Ibid., p. 383. (3) Block, R. J., Advances in Protein Chem., 2, 119 (1945). (4) Block, R. J., and Bollina. D., Arch. Biochem.. 7, 313 (1945). ( 5 ) Chang, I. C. L., and Murray, H. C., CereaE Chem., 26, 297 (1949). (6) Cohn, E. J., and Edsall, J. T., “Proteins, Amino Acids, and Peptides,” p. 348, New York, Reinhold Publishing Corp., 1943. (7) EvanElR. J., Arch. Bzochem., 7, 439 (1945). (8) Fels, I. G., and Cheldelin, V. H., Z&id., 22, 402 (1949). ( 9 ) Fink, H., and Just, F., Biochem. Z.,303, 234 (1939). (10) Latshsw, W. L., J. Assoc. Ofic. B g r . Chemists, 6 , 4 1 4 (1923). (11) Schultz, A. S., and McManus, D. K., Arch. Biochem., 25, 401 (1 950). RECEIVED Maroh 10, 1950.
Indicators for Titration of Fluoride with Thorium HOBART H. WILLARD AND CHARLES A. HORTON’ University of Michigan, Ann Arbor, Mich. In a study of colorimetric and fluorometricindicators for the titration of fluoride with thorium, the best colorimetric indicators found were, in order of decreasing effectiveness, the two-color indicators purpurin sulfonate, Alizarin Red S, Eriochromcyanin R, dicyanoquinizarin, and Chrome Azurol S; and the best fluorescent indicators were pure sublimed morin and quercetin. Optimum conditions for the use of the better indicators are presented.
T
H E need for a better indicator for the titration of fluoride with thorium nitrate has been a matter of study for some time. Yoe, Salsbury, and Cole (8, 9) studied the color change of some 54 dyes in admixture with Alizarin Red S in the hope of enhancing the color change of the latter. Shevdov ( 7 ) studied several zirconium-hydroxyanthraquinone lakes as indicators for the same purpose. Others have compared the effectiveness of only two or three indicators. KomaroviskiI ( 4 ) and Frers and Lauchner (3) employed a airconium-quinalizarin lake as indicator; Zakhar’evskif (fO),methyl red; Elsworth and Barritt ( d ) , a mixture of Alizarin Red S and 1,2,5,&tetfahydroxyanthraquinone; Clifford (1), a zirconium-purpurin lake; Milton ( 5 ) ,Sollochrome Brilliant Blue BS and Chrome Azurol S; and Shevdov ( 7 ) ,an Alizarin Cyanin R-zirconium lake. A large number of compounds for use in the titration of fluoride with thorium nitrate were studied to determine the most sensitive and effective indicator for this method. Compounds that might form soluble colored or fluorescent complexes or lakes with the thorium ion as well as the compounds tested by Yoe and co-workers (8, 9) were tested. The testing procedure consisted of a selection of the most 1 Present address, Carbide and Carbon Chemicals Division, Union Carbide a n d Carbon Corporation, Oak Ridge, Tenn.
promising compounds by preliminary Procedures I and 11, followed by a more complete examination of the more promising materials. PRELIMINARY PROCEDURE I
Solutions of the indicator compounds containing 0.5 mg. per ml. were prepared using water or ethyl alcohol as the solvent wherever possible. Each compound was examined in the vicinity of the titration equivalence point for its color change a t three or more pH ranges (pH 2, 3, and 4.7), and a t pH 3.0 in ultraviolet light for fluorescence changes. Sufficient buffer from 0.5 M to 2 M stock solutions was added so that each test solution contained in about 50 ml. 1 millimole of dichloroacetate buffer for pH 2.0, monochloroacetate for pH 3.0 to 3.5, and acetate for pH 4.1 to 4.7. Sodium fluoride samples containing either 45 or 300 micrograms of fluoride were titrated in a volume of about 50 ml. using tall-form Nessler tubes. In cases where alcohol concen’ration might have an effect, titrations were carried out a t several concentrations of ethyl alcohol. The colors in visible light were compared in a Nessler rack, equipped with an opal-glass reflecting mirror with illumination from a Mazda %watt Daylight tubular fluorescent light mounted 45 cm. (18 inches) from the mirror. For fluorescence studies a Hanovia A H 4 lamp covered by a Wood’s filter (Corning 5850) was mounted a t the side of the Nessler tubes and the fluorescence was observed from the to in a darkened room. Each sample was titrated well be ond t i e calculated end point with standard thorium nitrate, a n i the color or fluorescent change, if any, was
V O L U M E 22, NO. 9, S E P T E M B E R 1 9 5 0 Table I.
1191
Compounds Showing No Visual or Fluorescent Change of Indicators for Titration of Fluoride with Thorium Alkaloids
Atropine sulfate Quinine bisulfate
Fluorescein-Type Compounds
Brucine Strychnine
Dichlorofluorescein Fluorescein Tetrahromosul~onphthalein
Amines, Hydroxyamines, a n d Related Compounds
-
-
AceWlamino 4 - aminonaphthalene-7-sulfonic acid 2-Amino-2-ethyl-l,3-propanediol 2 Amino 8 - naphthol 6 - sulfonic acid 1,3-(Bisdiethyl)-diaminoisopropyI alcchol 1,3-DianiinoisopropyI alcohol 1.3-Diarninopropionic acid.HBr 1.1-(DiniethyoO-1-aminoethane Ethylenediamine Ethylenediamine tetraacetic acid m-Phenylenediamine 1,5,7-TrisuIfonamido-2-naphthol rn-Toluenediamine.HC1 1
-
-
-
2-Amino-1-butanol 2-Ethylaminoethanol 2-Amino-2-methyl-1-propanol 2-Amino-2(hydroxymethyl)l,3-proanediol 6-Eromoethy1aniine.HBr 2,j-Diamino-1-naphtho1,HCl 2-Diethylaminoethyl chloride N-Ethylaniline 2-Aminoethanol 1-Amino-2-propanol p-Phen lenediamine sulfonate
.V,N,NY,N'-Tetrakis(2-hydrox."ethyl)-ethylenediamine
-
-
-
- -
- - -
-
-
-
-
-
Azo, Disaro, and Related Compounds Acid Alizarin Black RA Alizarol Orange R Renzoazodiphenylaiiiine Benzoazririn 3 R 1.3 - Dihydroxyhenzo 4 - azo - 3'(1'-hydroxy~~nzenei-sulfonic acid Erie F a d Yellow WB E'lavazin 1Iethy-l red Pontacyl Siilfon Violet R Pontarnine Sky Blue 6 B X Pontaniine Green B X Purpririne 4B Ruperchroiiie Blue B extra Tartrazine
-
Quinoline Derivatives Bromoxine 8 Hydroxyquinoline - 5 - azo - 4'nitrobenzene-2-sulfonic acid ~ - " ~ ~ , o , ~ - h y s d ' . " I ' , ~ ~ ~ ~ ~ ~ ~ ~ n-o~l pi n, Slilfophenyla~o) -e - - hydroxy5-sulfonic acid quinoline (sulfenazoxine) z ( p Sulfophenylazo) 8 hydrexyl-(o-Hydroxyphenyl)-2-benzazine quinoline-5-sulfonic acid 2-(o-Hydroxyphenyll-quinoline 5,6-Benzoquinoline oxalate 8-Hydroxyquinaldine
- -
-
- -
Triphenylmethane-Type Compounds Alkali blue 4 G P Alphazurine A Aniline blue Aiirine Crystal violet Fast Acid Green R Fuchsin Giiinea Green B alachite green Methyl violet Methyl Violet 5B Rosaniline Triphenylchloro inethane Victoria Blue B Wool Violet 4BN
R;
Anthraquinones and Related Compounds Alizarin blue Alizarin orange Anthraflavin Anthrarufin Chrysazin Corhineal Diaminoanthrarufin Dinitroanthrarufin 1,5 Dihydroxy 4,s - diamino1 , 4 Dihydroxy 2 phenoxyanthraanthraquinone-2,6-disulfonic arid quinone 1 - Hydroxy 2 carboxy 4 bromoFrangula extracts anthraquinone 1-Hydroxy-4-chloroanthraquinone 1 Hydroxy 2,4 dichloroanthra1 Hydroxy 2,4 - dianilinoanthraquinone l-&%~y-4-toiuidoanthraquinone Leucoquinizarin Rufigallic acid 1,4.R.8-Tetrahydroxyanthraq1iinone 1,4,5.8 Tetrahydroxyanthraqrrinone'-2-aulfonic acid
-
Diiodofluoresrein Phenolphthalein Thymolphthalein
.Acid .\lioarin black SEA Alizarol Verdon S Bemoazophenol Chrysoidine 1.3 Dihydroxybenzo - 4 benzoic acid Evans hlue Methyl orange Pontacyl Carmine 6 B Pontamine Blue B B F Pontamine Violet S Pontamine Brown CG Rainbow Orange G Superchrome Black PI' Superchrome Garnet Y Superchrome Violet B
-
-
azo
-
2'-
rompared to the change given by Alizarin Red S a t pH 3.0. The amounts of the indicator solutions (0.2 to 2.0 ml.) used were selected by trial to'permit the maximum change a t the end point. PRELIMINARY PHOCEUURE I1
The compounds available iri small amounts only were tested by this procedure, primarily for fluorescent effects. Solutions containing 0.5 mg. per rnl., or in a few cases 0.2 nig. per ml., were prepared in 10% ethyl alcohol if possible; 95% ethyl alcohol, propylene glycol, or pyridine if necessary. For each compound a series of eight Klett KO. 802 test tubes was set up, using 10 ml. of water in four tubes and 10 ml. of 50% ethyl alcohol in the second four tubes. The proper pH in each series of four tubes was maintained by the addition of 1 millimole of pH 2.0, 3.0, or 4.15 buffer to the first three, respectively, and water of pH 7 to the last tube. To each tube was added 0.15 mg. of indicator, usually in solution, and the total volume was brought to 11 ml. The initial color under visible light and the fluorescence of each of these solutions were noted. Fluorescence intensities were also measured with a Klett fluorometer equipped with a Corning 5850 primary and a Wratten S o . 4 secondary filter with the t d a n c e diaphragm set at 14 in most cases. T o each of the 8 tubes were added 0.3, 0.3, 0.4, and 0.6 ml. of 0.0157 M thorium nitrate, and the fluorescent reading was noted for each addition. The color under ultraviolet light was also finally observed after introduction of an additional Corning 5790 filter in the primary ultraviolet beam to cut out blue light passing the Corning 5850 filter. If a turbidity or precipitate formed, its color was noted.
Flavonoid-Type Compounds D-Catechol xaringin Phloretin
I-Epicatechin 3,5,7,3',4'-PentahydroxyHavanonc Rutin Miscellaneous Dyes and Compounds
Acridine Acridone ~ ~ ~ ~ i l i ~ Chlorogenic acid o-Dianisidine 2.4-Dihydroxybenzene sulfinic acid Diphenyl carbazide Fluorene Hematein Indigo disulfonate Indophenol B.G. Litmus Luminol @-Mettiylumbelliferone Methylene blue Nordihydroguaiaretic arid Phenosafranine hydrochloride Primuline S A C Quinaldine Resazurin Riboflavin Salicylaldoxirne Thiamine chloride Vitamin C
Acridine orange Auralnine Cacotheline Cupferron Anthraquinonyl sulfide a-Dinitrodiphenylarnine iiilfoxida Dithio-oxamide Gallic acid Hemoglobin Indole Leuco Indol,henol K Logwood extracts Methane trisulfonic acid, Nllr salt 1.lethyl-8-ptienylethylnialonic arid dihydrazide o-Phenanthroline Phosphine Printing Violet R Re.ioriifin Rhodamine €3 Safranine A Sodium diethyl dithiurarhainate Violamine R R Xanthone
RESULTS O F PRELI\IIhAHY PROCEDUHES
The compounds tested 'which showed no color or fluorescent change by Procedure I or I1 are listed in Table I . In addition, 30 compounds of unknown structure previously investigated by Yoe and co-workers (8, 9) were tested. The compounds which showed color or fluorescent change by Procedure I are listed in Table 11, and the indicating compounds tested by procedure I1 are listed in Table IJI. DISCUSSION OF PRELIMIYARY PROCEDURE
Samples of the same indicator compound from different suppliers generally gave comparable results by either procedure. The agreement was especially notable for samples of Alizarin Red S. However, two samples of 2',6'-dichlorohydrox).dimethylfuchsondicarboxylic acid, Alizurol Azurine ECA, anti Basic Color MD-1293, differed in sensitivity as did two samples of 0'sulfohydroxydimethylfuchsondicwboxylic acid, Eriochromcyanin R and Pontachrome Blue ERC (see Table 11). Differencw in sensitivity wer? especially pronounced for various samples of quercetin and morin. Quercetin obtained by the hydrolysis of rutin supplied by S. B. Penick & Company and sublimed Schuchardt's morin recrystallized from acetic acid proved the most sensit.ive fluorescent indicaators. SELECTION OF OPTIMUIM INDICATOR 4 Y D 0 P T I . M A STUDIES
The indicators which showed a color or fluorescent change in this titration were studied further to determine the best indicator
ANALYTICAL CHEMISTRY
1192
Table 11. Compounds Showing Some Change in Visible or Ultraviolet Light as Indicators for Titration of Fluoride (Procedure I) 0 no change; VS very slight change; S slight change; M moderate change; G good change (comparable t o Alizarin Red S a t p H 3.0 in water); X very good change; X X excellent change in the color or fluorescence; ? doubtful result Best Compound PH Changes in Visible Light .4nthraquinone Types Alizarin Alizarin Red S
3.0,3.5 3.0 3.2 Alizarin sapphire 2.0 Alizarin Y 4.7 Anthragallol 4.2 Anthrapurpurin 4.7 Cascara sagrada (50.% alcohol extract) 4.7 2-Chloroquinisarin 3.0 Dicyanoquinizarin 4.2 Leucotetra4 3.0.3.5 Purpurin 4.7 Purpurin sulfonate 3.5,4.2 Quinalizarin 3 5 Quinizarin 3.0 Synthracene Blue WRSb 3.5 Azo-Type Compounds Diamine Pink B D ('2.1. 128) Nitrophenylazoresorcinol ontachrome Blue R Pontamine Light Yellow 5 G X ('2.1.
4.2,4.7 3.0,4.7 3.0 3.0.4.7
Poiizmine Yellow 5 X (C.I. 620)
3.0.4.7
6-
246)
Class
G G
X
vs
G
EM M vs G S
EG Y S vs
VS S
vs vs
7%
hlcohol
0 0 0 0 10
20 0-20 0 0-20 0
0-20 0-25 0 0-20 10-25 0 0 0 0 0 0
Azo Quinoline Compdund 5 (or 7)-(4-nitro-l~phenylazo-2-sulfonic 4 . 7
RI-G acid) -8-hydroxyquinoline Triphenylmethane-Type Compound5 .4lizurol Azurine E C A c (C.I. 720) 2,O-4.7 M Aluminon 4.2.4.7 M-G Basic ColLr M D 12934 ('2.1. 720) 4.7 G-X Chrome Azurol S (C.I. 723) 3.0 G Eriochromcyanin R (C.I. 722) 4.2,4.7 G Pontachrome Blue E R C I 4.7 M Miscellaneous Compoun'ds Alkali Fast Green 2G ((3.1. 735) 2.0 S Alkannin (C.I. 1240) 4.7 vs 6.0 S Chrome Blue G D ((2.1. 878) 4.2.4.7 s-hl p-Dimethylaminobenzilidine rhodanine 4 . 7 M 2-Nitroso-1-naphthol 4.7 S Pontacyl Fast Violet 10B ('2.1. 696) 4.7 S Rose petal powder, 50% alc. extract 4.2 M-G PPt. Polyporic acid 4.2 " 1 4,5 8-tetrahydroxyanthraquinol. 1~2,4~5,6,&hexahydro~yanthraquinone-3(or 7)-sulfonic acid, From National Amline Co. Obtained through Chemical CorD. C From German source.
0
0 0 0 0 0 0
0 0 10 0
0
0-20 0 0
0-50
*
in each claw. The best indicators are listed in Table I V with a rank of effectiveness assigned to each compound and the optimum aonditions for titration as determined in the studies outlined below. Both 35- and 300-microgram aliquots, of standard fluoride were titrated visually a t the optimum p H and alcohol content previously approximately determined ,with varying indicator concentrations to select the best indicator concentration for a volume of 50 ml. The illumination conditions specified in Procedure I were used for both visible and ultraviolet studies. In addition, fluorescence changes were followed using a modified Klett fluorometer equipped with a 140-ml. rectangular cell and the filters mentioned previously. It ww generally observed that the smallest amount of indicator which would give a light color with the buffer in the presence of fluoride in the case of two-color indicators, or a light color and moderate fluorescence in the presence of a slight excess of thorium for the fluorescent indicators, was more sensitive in change a t the equivalence point than larger amounts of indicator. Using the optimum indicator concentration, variations in the color or fluorescence change were studied a t several pH values. The proper proportion of alcohol was added where necessary. The optimum pH for the best color change of the better indicators is given in Table IV. The studies confirmed previous conclusions (6) that pH 3.0 to 3.5 is the optimum for the titration
Rest Compound pH Class Compounds Also Showing Fluorescent Change Changes in Visible Light 5 Congo Corinth ((2.1. 375) 3.0 Pontachrome 'Blue-Black R M (C.I. 3.5 5
202)
Superchrome Blue-Black 6 B P (C.I.
3.0,4.2
5
Ferroh Isoquinoline Oxine Oxine sulfonate
4.7 2.0-4.2 4.7 4.7
S-M
201)
Flavonoid-Type Compounds C y a h d i n chloride (crude, impure) 4.7 Quercetin (9. B. Penick & Co.) 3.5 Quercetin (9. B. Wender) 3.2-3.5 Quercetin (Am. Dyewood Corp.) 3.5 Quercetin, quercetin mixture0 3.0-4.2 Quercetin-6 -sulfonic acid, Na salth 3 .O-4.2 Quercetin-6'-suifonic acid, Na salt % 3.0.3.5 Morin, tech. (Eastman Kodak Co.) 3.5 Morin (Schuchardt, Germany) 3.5 Morin ,(sublimed and recryst. from 3.0.3.5 above) Morindin chloride (crude, impure) 4.2 Morin-t-sulfonic acid 2.0-4,7 Kaempferol (S. B. Wender) 3.0-4.2 Gossypetin (W. Clark) 4.2 Changes in Ultra! ric)let Light Congo Corinth 3.5 Pontachrome Blue-Black R M 4.7 SuDerchrome Blue-Black 6 B P 4.7 Feiron 3 0-4.7 Isoquinoline 2 0 Oxine 4.7 Oxine sulfonate 4 7 Flavonoid-Type Compounds Cyanidin chloride (crude, impure) 2 .O-4.2 Gossypetin (W. Clark) 3.0 Kaempferol ( S . B. Wender) 3 .O Quercetin (9. B. Penick & Co.) 3.0,3.5 Quercetin (Am. Dyewood Corp.) 3.0,4.7 Quercetin (S. B. Wender) 3.0.3.5 Quercetin, quercetin mixture0 3.0 Quercetin-6'-sulfonic acid, S a salth 3 0 Quercetin-6'-sulfonic acid, Na salt t 3 0 Morin, tech. (Eastman) 3 5 .Moron (Schuchardt) 3 5
56
Alcohol
0 0
0 0
s PPt.
35 30 0
$ X
0 50 50 50 50 20
M PPt. M
G M X
vs
0 50
M G-X M-X
50
50
5
0-v5 G G
vs vs vs
0. 0-50 50 50
0 0 0 0
M-G VS
40 30 0-29
G"
S-M 5
X
x-xx M-G
x-xx G
X
0
M X
0 50 50 50 50 50 50 50 50 50 50
I From Jackson Lab., D u Pont. Sample from Schuchardt, Germany, ratio unknown. h From W. Clark freshly prepared solution. i From WeClark: solution after standing 12 days, oxidation apparent.
of fluoride ion with thorium nitrate. Because the optimum color change of Alizurol Azurine ECA, Basic Color MD-1293, and 5(or 7)-(4-nitro-l-phenylazo-2-sulfonicacid)-&hydroxyquinoline does not occur in this range, they were eliminated from further consideration. The most suitable alcohol content was redetermined for indicators which required alcohol. Alizarin Red Alizurol Azurine, Chrome Azurol S, Eriochromcyanin R, and purpurin sulfonate do not require alcohol. .4 high alcohol content with the flavonoid fluorescent indicators, quercetin and morin, is accompanied by greater intensity of fluorescence and sensitivity in the titration. For these a limiting alcoholic concentration of 50y0 was selected to avoid interference of foreign ions at greater concentrations, even though greater fluorescence intensities might be obtained with higher alcohol concentrations. Using the best conditions det,ermined above, the responses in the titration of 5 to 50 micrograms, 250 to 500 micrograms, and 1.0 to 5.0 mg. of fluoride in a total volume of 50 ml. were determined. The color changes observed for the better indicators are given in Table V. From the results of these studies the best two-color indicators in visible light in order of decreasing effectiveness are:
s,
1. Purpurin sulfonate 2. Alizarin Red S 3. Eriochromcyanin R
V O L U M E 2 2 , NO. 9, S E P T E M B E R 1 9 5 0
1193
Table 111. Compounds Showing Change in Visible or Ultraviolet Light When Used for Titration of Fluoride (Procedure 11) Class Symbols. Symbols used in Table 11: max maximum change for intsrmediate amount of thorium: min minimum change for intermediate amount of thorium: neg decrease in color or fluorescence of class as indicated: ? doubtful fluorescence. vis visual. fluor fluorescent Color Symbols. b k Alack; b k h biackish’: bl blue; dlh bluish: b r brown: bri bright. buf buff or buffish; d dark or dull: g green:, gh greenish: 1light: Iav lavender: mag magenta: no none: or orange: pk ink: p p t precipitate: pu purple: r red; r h reddish: SI slight: t u r turbi&y: T Tyndall blue scattering by a turbidity; v very; vi0 violet: y yellow: y h yellowish; Color or Type Best % Compound Observed p H .4lcohol Class Fluorescence Flavonoid Types 3’-Carbethoxyhesperidin vis. 2 . 0 . 3 . 0 50 VS I y-g bl-g 0, 50 S-hl fluor. 3 . 0
0, 50
. . . not determined. For color changes, if original solution is practically colorless or nonfluorescent, only color formed after addition of thorium i, mven. I n other cases initial color is given, followed by final color. D a t a Presented. T o conserve space. visual a n d fluorescent changes are given only for p H and alcohol content, if any, a t which best change occurred or where some unusual effect was noted. Additional details are given in thesis from which this paper is abstracted. Tests in neutral solution are not considered. Compound Pomiferin
Type Best % Observed p H Alcohol Class Flavonoid Types (Contd.) vis. 2 . 0 - 4 . 1 0, 50 0 fluor. 4 . 1 0 S neg
Color or Fluoresence no T bl
0 V S max
no bl
Quercetagetin
vis. fluor.
4.1 2.0
50 0
S-R.1 S max
?d g
2. 0-7 .O 0 , 50 4.1 50
0 VS
no bl
Quercetin (S. B. Penick)
vis. fluor.
3 . 0 , 4 . 1 50 3 . 0 , 4 . 1 50
X-XX XX
bri y-g bri yh-g
vis. fluor. fluor.
3.0,4.150 3.0.4.1 0 3 . 0 , 4 . 1 50
VS
I lav t o rh-lav
6’-Sulfoquercetin, Na salt (fresh solution)
vis. fluor.
2.0-4.1 2.0, 3.0
0, 50 50
G X
V Y
S max S neg
vis. fluor.
2.0-4.1 3.0
vis. fluor.
4.1 3.0
0. 50 0
S
Dianiline gossypol oxime
6’-Sulfoqriercetin, Na salt (old solution)
VS
0, 50
S rnax
$1, bl-g
gh-y, s l g
vis. fluor.
2.0-4.1 2,O-4.1
0. 50 50
M
Quercitrin
3,4’-Dihydroxy-4’-methoxychalcone-4-phosphate, disodium salt
Y-g bl-g
vis. fluor.
3.0 4.1
50 50
S-M S max
VS
1 Y-P 1 Y-g
Rhamnetin
vis. fluor.
2.0, 3 . 0 2.0-4.1
0 50
G ppt X
Y PPt bri g
Eriodictyol
vjs.
0,50 0, 50 50
ppt
W
fluor.
2.0.3.0 4.1 2.0
Gossypetin
vis. fluor.
3.0.4.10 3.0 50
Gossypin
vis. fluor. fluor.
3.0.4.1 4.1 4.1
0. 50 0 50
Gosaypitrin
via. fluor.
2.0-4.1 3.0, 4 . 1
0,50 50
S
S-M
v g
Hesperidin
vis. fluor.
4.1 4.1
50 50
S-hf hl
Y-R
Homoeriodictyol
vis. fluor.
2.0 2.0
0. .5O
M M-G
w pt, w t u r T El
Other Types of Compounds 4-Carboxyesculetin. Na vis. 2.0-4.1 50 6 salt fluor. 2 . 0 , 4 . 1 50 VS
vis. fluor. fluor.
2 . 0 - 4 . 1 50 2 0 50 2.0 0
S-M
S-hl
1 y-g
Chlorogenic acid
W
vis. flqor.
2.0-4.1 2.0,4.1
0. .50 50
0
V S neg
VS
no bl-g
Kaempferol
vis. fluor.
3.0,4.150 2 . 0 , 3 . 0 50
X X
y-g bri g
Curcurnin
vis.
fluor.
2.0-4.1 2.0,3.0
0. 50 50
0
B neg
no change bri g,g
Morin ( S c h u c h a r d t ~
vis. fluor.
3.0 3.0
50
X-XX X-XX
bri y-g bri yh-g
Chloroxine
50
vis. Ruor.
3 .O 2.0-4.1
0 50
S S-M
1g g
hlorin (rublimed and recryst. from Schuchardt)
vis. fluor.
3 .O 3.0
50 50
G
bri y-g bri g
N,.V’-Disalicylidene-ophenylenediamine
vis. fluof.
3 .O, 4 , 1 0, 50 3.0 0
VS S ne6 min
1 Y-g
XX
h’aringin
vis. fluor.
2.0-4.1
0.50 0
0
no bl-g
Esculetin
3.0
vis. fluor.
2.0-4.1 3.0
50 50
S 5-h1
Ig bri 1 bl
Feohesperidin
vis.. fluor. fluor.
2 . 0 , 3 0 0,50 . . . 2.0 50 >I-G 4.1 0 S max
w ppt. PI t u r T bl bl
Xordihydroguaiaretic acid.
vis. fluor. fluor.
2.0-4.1 3.0 3.0,4.1
0, 50 0 0 VS 50 VS neg
Erne bl
3,5,7.3’,4’-Pentah)-droxyflavanone
vis. fluor.
2.0-4.1 3.0
0, 50
0
no bl
Polyporic acid
M-max
vis. fluor.
3.0 3.0
50 50
Iav to g t u r blh-g
Phloretin
vis.
2.0-4,l
0. 50 0
0 VS neg
no
n-Catechol
vis. fluor.
2.0-7.0 2.0
L-Epicatechol
vis. fluor.
Cyanin chloride
VIS.
Isoquercitrin
fluor.
a0
0, 50
0.50
50
Table I\-.
Rank5
Compound
Opt. Indicator Concn. for 50 MI., Mg.
2.4
.4lizarin Red S
0.20
4A
2,3-Dicyanoquinizarin Purpurin sulfonate
0.60
IA
0.15
Best pH
M
0
no bl
Robinin
hf-G
vis. fluor.
2.0.3.0 2.0
50 50
S-hl
yh bl-g
G S
gh-bk br ?
Rutin
vis. flupr.
4.1
2 .O. 3 . 0
0, 50
0
VS
no gh-bl
>I
Y-P y-g hl-g
Rutin methylglucamine
vis. fluor.
2.0-4.1 3.0
50 50
VS VS
Y
2’,3.4-Trihydroxychalcone
vis. fluor.
3.0, 4.1 2.0-4.1
50 0.50
S-M
Y-g no
Xanthorhamnetin
vis. fluor.
2.0 2.0,3.0
50 50
S
S
Y-g 1 yh-g
S
S neg
VS
bl-g
a
0
S
0
h1 S-M mal
Y-g
vlg 1 bl
1 y-p gh-bl
bl-g
...
Optimum Conditions for Best Indicators by Procedure I For Range of F-, hlg.
Opt. .Ah. Content,
%
Rank& 8h
Aluminon
7h 5A 3A
Basic Color M D i 2 9 3 Chrome Asurol S Eriochromcyanin R
3.4 3.0 3.8
0.6up Under 0 . 2 All ranges
Kone None 10-20
3.7 3.3
0 . 6 up Under 0 . 2 All ranges
Sone None 30-40
Compound
opt. Indicator Concn. for 50 MI., Mg.
5(or 7)-(4-nitro-l- 0 . 2 0 4.2 phenylaao-2-sulfonic acid1-8-hyQuercetin-6’-sulfodroxyquinoline nate, N a salt (fresh 6.4 Alisurol Azurine 0.15 3.5-3.9 All ranges 0-5 solution) ECA A , two-color indicators: B. one-color visual indicators and also fluorescent indicators.
9.4
bri g
0.4-0.9
Best pH
4.2-4.5 3.6 0.10 3.75 0.15-0.25 3 . 0 - 3 . 2 0.6-0.8 4.2 3.6 0.50 3.5 0.50 3.5-3.8 1 0 3.5-3.8 0.3-0.7 3.2
For R a n e of F-,d g . Best all ranges Satisfactory All ranges All ranges Best all ranges Satisfactory All ranges All ranges 411 ranges All ranges
opt. Ale. Content,
%
None 0-10 None None 50 50 50 50
ANALYTICAL CHEMISTRY
1194 Table V. Color Change of Best Indicators in Visible Light by Procedure I Using Optimum Conditions Listed in Table IV -~
Compound Alizarin Red S Dicyanoqriinizarin Purpurin sulfonate
With excess fluoride Yellowgreen Dull lavender Orangish yellow Yellow
5(or i)-(4-nitro-lphenylazo-2-sulionic acid)-8-hydroxyquinoline ( a t p H 4.7) Alizurol Azurine ECA Pink Aluininon
Pink
Basic color MD1293
Light pink
Erioclironicyanin R
Orangish pink A I mos t colorless
Quercetin (Penick) hlorin (Schuchardt) Morin (sublimed)
Colorless
Quercetin-6’-sulfonic acid, Na salt (fresh solution)
At end point
Pinkish buff Dull purple Light pink Buffish orange
Color With slight With large excess of excess of thorium thorium Light pink Rose
Bluish Lavender blue violet Light Purple lavender Orange Deep orange
Lavender Lavender pink Pinkish Pink-rose rose Light pink- Lavender lavender blue Reddish Oranmsh orange red Light Yellowgreen green Same a8 quercetin Light Yellowgreen green Same as quercetin
Lavender blue
Foreign ions such a3 sulfate, chromate, ralrium, and iron were found to affect all two-rolor indicators to a similar extent. Halide ions and sodium acetate or chloroacetate affect all t,he two-color indicators slightly, when present in high concentrations. The chloride, bromide, iodide, chlorate, perchlorate, acetate, and chloroacetate ion3 did not affect the results with the flavonoid fluorescent indicators. Thus, the. latter would be especially suitable for determination of the fluoride obtained by alkaline decomposition of halogenated organic rompounds. . Of the best two-color indicators, purpurin sulfonate has some advantage over Alizarin Red S, as it lacks the grayish buff color range just before the end point. The last t.hree two-color indicators are inferior to both purpurin sulfonate and Alizarin Red S in the sharpness of their color change at the equivalence point.
Dull dark rose Medium blue Rose red Bright greenish yellow Bright yellowish green
A C h h O U LEDGMENT
The authors acknowledge the kind supply of various indicators by several firms and individuals, especially William G. Clark and Simon H. Wender who supplied the flavonoids. This work wm done under rontract with the Technical Command, Chemical Corps, I T S. .-lrmy. LIT EH91’1:HE CITED
4. 2,3-Dicyanoquinizarin 5. Chrome Azurol S The best one-color visual indicators similarly are: 1. Quercetin (S. B. Penick & Co.) 2. hlorin (T. Schuchardt) The best fluorescent indicators similarly are: 1. hlorin (sublimed and recrystallized from Schuchardt’s morin) 2. Quercetin (S. B. Penick & Co.) 2. Morin (Schuchardt) 3. Ferron 4. 8-Hydroxyquinoline-5-sulfonic acid 5. Kaempferol
Clifford, P. A., J . Asuoc. Ofic.Agr. Chemists, 23, 303-7 (1940). Elsworth, F. F.. and Barritt, J., Analyst, 68, 298 (1943). Frers, J. N., and Lauchner, H., Z . anal. Chem., 110, 251 (1937). KomaroviskiI, A. S., et al., Ibid.,91, 321 (1934). Milton, R. F.,’et al., Analyst, 72, 43-7 (1947); 74, 54 (1949). Rinck, E., Bull SOC. chim., 1948, 305-24. Shevdov, V. P., J . Applied Cheni. (U.S.S.R.), 10, 94Cb-5 (1937). Yoe, J. H., private communication, 1946. Yoe, J. H., Salsbury, J. M., and Cole, J. W., Office of Scientific Research and Development. OSRD Rept. 3481, 1-19 (March 1944).
Zakhar’evskii, V. .4.,Zaaodskaya Lab., 6, 1019 (1937). RECEIVED February 11. 1950. .4bstrar:ed from a thesis presented by Charles A . Horton for the doctor of philosophy degree in the Horace Rarkham Graduate School, University of Michigan.
Photofluorometric Titration of Fluoride HOBART H. WILLARD A N D CHARLES A. HORTON’ University of Michigan, Ann Arbor, Mich. A procedure is presented for the photofluorometric titration of fluoride in the presence of considerable sodium chloride, using thorium nitrate as the titrant and quercetin as the fluorescent indicator. The method may be used following alkaline decomposition of fluorocarbons or after the Willard-Winter separation.
A
LIZAKIS Red S and Chrome Azurol S are now commonly used m visual colorimetric indicators for the titration of
fluoride with thorium nitrate. These indicators have the disadvantage of a subtle color change a t the end point and interference by semicolloidal thorium nitrate when large amounts of fluoride are titrated. Fluopescent indicators are readily adaptable for the instrumental titration of fluoride with thorium nitrate. The best fluorescent indicators, in order of decreasing effectiveness, have been reported to be ( 1 ) : pure sublimed and recrystallized morin (2’,3,4’,5,i-pentahydroxyflavone),pure quercetin (3,3’,4’,5,7pentahydroxyflavone), ferron (8-hydroxy-7-i~do-5-q~inolinesulfonic acid), kaempferol (3,4’,5,i-tetrahydroxyflavone),and % hydroxyquinoline5sulfonic acid. Because pure morm is at 1 Present address, Carbide and Carbon Chemicals Corporation, K-25 Plant, Oak Ridge, Tenn.
present generally unavailable, the work reported here is restricted to the use of quercetin as an indicator. APPARATUS
Modified Klett Fluorometer. The regular cell compartnic.n t lid, filter support, cell support, and mirror assembly of a KIett fluorometer were removed. A cardboard support was used to hold the secondary filter in front of the photocell. A special high cell house cover was constructed, with a hole for the buret tip located to the rear out of the path of the ultraviolet and fluorescent light paths. The arrangement is shown in Figure 1. Filters. The fluorescence of the thorium-quercetin complex using 50% ethyl alcohol and monochloroacetate buffer of p H 3.0 showed a wide band from 530 to 580 mp with a, maximum at about 575 mp. Thus Corning KO.3385 was chosen as the secondary filter; however, Corning S o . 3384 or Wratten No. 4 or S O .8 is also satisfactory. Optimum sensitivity in the titration of 0.1 mg. of fluoride was obtained using a standard thickness Corning N o . 5850 primary filter.
