of chloroform. The extract was placed on a strip of Whatman KO. 1 c*hromatograph paper as described above. The extract-inoculated strips were developed for 10 hours with a sol1.ent composed of 15 ml. of concentrated hydrochloric acid and 100 ml. of 1-butanol saturated with distilled water. This solvent is similar to that proposed by Munier (7, 8). The position of the separated alkaloids was determined by comparing the strips to a control strip treated with Dragendorff’s reagent (1). The alkaloid sections (3 to 4 em.) of the chromatograms were cut out and placed in the elution apparatus. Each section was eluted for approximately 3.5 hours with 30 ml. of a solvent composed of 3% (w./v.) hydrochloric acid in 10% (v./v.) ethyl alcohol. Then the exhausted paper segments were removed, air-dried, and tested for complete removal of alkaloids by DragendoriT’s reagent. Elution was complete in all tests performed. The eluates were evaporated to dryness under a stream of air and assayed spectrophotometrically by the T’itali-Norin
LITERATURE CITED
1111.
(1) Block, R. J., LeStrange, R., Zweig, G., “Paper Chromatography,
a Laboratory hianual,” p. 136, Academic Press, Sew York, 1952. ( 2 ) Brindle, H., Corless, J. E., Woodhead, H. B., J . Pharni. and Pharniacol.
3,793 (1951). (3) Colby, A. R., Beal, J. S.,J . A m . Pharin. Assoc., Sci. Ed. 41, 351
(1952). E. h.,Foster, G. E., J . Pharni. and Ph,arrnacol. 5 , 839 (1953). (5) Grpgory, G. F., Science 121, lti9 (4) Drey, 12.
(1955).
Figure 3. Paper holder (detail of Figure 2, F)
( 6 ) Moore, A. hl., Boylen, J. B., Zbid., 118, 19 (1953). ( 7 ) Munier, R., Bull. SOC. chim. biol.
reaction as reported by Colby and Beal (3).
( 8 ) Zbid., p. 862.
33, 857 (1951).
The elution apparatus was found to be 99.6 =k 0.4% efficient in eluting known concentrations (ranging from 0.05 to 0.2 irg.) of hyoscyamine.
RECEIVED for review October 22, 1!)56. ilccepted February 5,1957. Investigation supported in part by funds provided for biological and medical research by the State of IVashington Initiative Measure NO. 171.
Determination of Phenolic Glycosides and Aglycones on Paper Chromatograms JOHN B. PRIDHAM’ The Institute o f Paper Chemistry, Appleton, Wis
b The method was developed in order that small amounts of phenols and phenolic glycosides, present in enzymic digests, could b e accurately determined. The technique has been used for the determination of catechol and arbutin over a range of 0 to 100 y , and saligenin from 0 to 70 y. The errors in all cases were within *4%. (p-Hydroxyphenyl)-P-gentiobioside has also been determined in enzymic digests after separation of the components of the digests by paper partition chromatography. Quantitative analysis of mixtures of phenols and phenolic glycosides may b e rapidly carried out by the simple procedure described. Only a few microliters of the solutions to b e analyzed are required, and application of these solutions to the paper as single spots ensures good chromatographic separations of the components. The completeness of separations may be readily observed in view of the fact that the colorimetric reactions are carried out on the chromatograms.
T
methods available for the determination of phenols and their derivatives have recently been reviewed by Clarke and Nord ( 5 ) ,Trim (IS), and Bray and Thorpe ( 3 ) . The analytical principles involved are many and varied, but the majority of techniques have been devisrd for specific phenolic compounds and little attention has been paid to the quantitative separation and determination of mixtures of phenols. One of the few exceptions, however, is Stone and Blundell’s method, where the phenolic aldehydes obtained by alkaline nitrobenzene oxidation of lignin are separated by paper partition chromatography and then determined spectrophotometrically after elution from the paper with ethyl alcohol (9). This method has recently been utilized for the determination of phenolic carboxylic acids ( I ) . A good chromatographic spray reagent for phenols is diazotized p-nitroaniline ( d ) , which has also been used for the determination of p-hydroxybenzoic acid and p-hydroxyberizaniitle in the ether extracts of urine ( 2 ) . HE
In the procedure described herein the author has utilized this reagent, in conjunction with paper partition chromatography, to determine phenolic coinpounds in mixtures. The technique used is similar t o that described for determination of sugars with p-anisidine hydrochloride (6). The solution containing the phenols is applied to the chromatographic paper ns small discrete spots, and, after development with a suitable solvent, the rhroniatogram is dried and sprayed with n solution of diazotized p-nitroaniline buffered with sodium acetate. The excess moisture is then allowed to evaporate, the spots are cut from the chromnbogram, and the color is eluted from the paper with a solution of potassium hydroxide in aqueous methanol. Finall>-3 the absorbance of the resulting solution is nieaaured in a spectrophotometer. The intensity of color bears a linear rcllationship t o the weight of phenolic coniPresent address, Chemistry Dcpartnieiit, West Mains Road, The I-niversity, Edinburgh 9, Scotland. VOL 2 9 .
NO. 8. AUGUST 1957
1167
pound over a range of 0 to 100 y for catechol and arbutin, and from 0 to i o y for saligenin. The method has found application for the determination of (p-hydroxyphenyl)p-gentiobioside ( 7 ) in enzymic digests, which in addition contain arbutin and quinol. The method might also be used for the determination of many other phenols and derivatives, if they are capable of coupling with diazotized p-nitroaniline, and if no decomposition occurs during the extraction R-ith metlianolic potassium hydroxide solution. REAGENTS
Diazotized p-Kitroaniline Solution. This spray reagent is made up as described by Swain (IO). To a solution of p-nitroaniline in 2-Y hydrochloric acid (0.5% IT., v . ; 5 nil.) is added aqueous sodium nitrite solution (5.0% w./v.; 0.5 nil.) followed by sodiurnacetatebuffer (20’351~.\.; 15ml.). These reagents are stable, but the diazotized p-nitroaniline is unstable and should be freshly prepared just prior to use, although it may h e stored in a refrigerator for 1 to 2 hours after preparation. Eluting Reagent. The solution used contains uotassium hvdroxide (0.2% w./v.) in iqueous methanol (95% r;./v.’$ For the determination of catechol the coniposition of eluent is modified by decreasing the amount of methanol in the aqueous solution from 95 to 80%.
The absorbances of the colored solutions are measured in a Beckman spectrophotometer (Model DU, 1-cni. cell), the red color given by saligenin being diluted ten tinies before measurement , Absorbance nieasurements are made a t the following wave lengths: arbutin, 458 mp; catechol, 470 nip; and saligenin, 495 nip, The values obtained for the standard determinations are plotted against the weights of phenols and the resulting graphs are used to determine the unknowns. The rate of forniation of (p-hydroxyphenyl)-p-gentiobioside \vas studied by withdrawing 1-nil. saniples froni the enzymic. digests at vwying time intervals. Each sample, after withdrawal, ITas immediately rleproteinized liy shaking with Sevag’s re:igent (chlorofornin-aniyl :ilcohol; 5 : 2 v./v.) (8) and suit-
able volumes of the top layer were then spotted onto paper chromatograms. These were developed with the 1butanol-pyridine-water solvent and the (p-hydroxyphenyl)-p-gentiobioside was then determined by the procedure described, using arbutin as the standard for comparison. The weights of (p-hydroxyphenyl)-@gentiobioside present were calculated on the assumption that the stoichiometry of the reactions of diazotized p-nitroaniline with arbutin and (p-hydroxyphenyl)-p-gentiobioside, respectively, vere similar, and that equivalents of the azo dyes thus formed gave the same color intensity. RESULTS A N D DISCUSSION
Azo dyes behave as indicators, and, in general, those formed by coupling diazotized p-nitroaniline with phenols
1.0
o.9[
t
o.O
0.71
Yz
0.6-
Arbutin 450 my. .Saligenin
495
mp
PROCEDURE
Suitable volumes of the phenol solutions are spotted onto paper chroniatogranis (\\-hatman No. 1 paper) with a n ultramicroburet. Three or four spots of standard phenol solutions. the components of which correspond qualitatively to those in the unknown, are also applied to the chromatograms. The standard solutions are spotted in amounts ranging from 0 to 100 y for arbutin and catechol and from 0 to 70 y for saligenin. Development of the clirornatograma is effected with a suitahle solvent. such as 1-butanol-pyridine-n ater (6:4:3 v./v.), and the papers are then allowed to dry in the air. The chroniatogranis are next sprayed, as uniformly as possible. \vith the diazotized p-nitroaniline solution and the excess moisture is allowed to evaporate from the paper. The resulting orange-yellow spots. together nith suitable blanks, are cut out with scissors. the areas of the pieces of paper being kept constant and as sinal1 as possible. Elution of the colored compounds from the paper is carried out by mechanical shaking in test tubes with the aqueous niethanolic potassium hydroxide solution. the volume used varying with the phenol being determined. For arbutin and catechol 3 ml. are used, and for saligenin, 5 ml. The blanks are eluted in a similar manner. 1 168
ANALYTICAL CHEMISTRY
7
OF PHENOL
Figure 1. Increase in absorbance of azo dyes with increase in weight of catechol, arbutin, and saligenin
y
OF PHENOL
Figure 2. Stability of azo dye formed by reaction of diazotized p-nitroaniline with arbutin A. 6.
At zero time After 2 hours and 72 hours
applied to the determination of sugars with p-anisidine hydrochloride (6) and amino acids with ninhydrin ( 2 1 , l a ) . It is rapid, and a useful procedure for the quantitative study of mixtures of compounds of biochemical interest. This is well illustrated in Figure 3, based on a study of the rate of formation of (p-hydroxyphenyl)-p-gentiobioside and the simultaneous liberation of glucose when arbutin is incubated with a p-glucosidase preparation from the cambial region of Populus grandidentata.
3.0-
-
E 2.0-
.I” 0
HOURS
Figure 3. Formation of (p-hydroxyphenyl)-P-gentiobioside and liberation of glucose
LITERATURE CITED
Arbutin incubated with f?-glucoridare preparation from Populus grandidenfafa
(1) Beper, D. L., Pearl, I. A,, Inetitute of
are orange-colored under acidic conditions. If the p H is increased, however, a color change results, which often aids in the identification of certain phenols. These dyes in alkaline solution usually exhibit maximum absorption peaks at a higher n a v e length than when they are in acid solution and therefore, if neressary, the absorbance of the former can be readily measured in a simple colorimeter. For t1~e.e reasons the quantitative relationship between the absorbance of the azo dyes and the weights of phenols \T as examined in alkaline rather than in acid solution. Attempts to bring about the color change by spraying the chromatograms with alkali were abandoned, as this invariably resulted in the colors streaking down the paper. The alkali IT as therefore incorporated into the eluting reagent. I n the case of catechol, some difficulty was experienced with the elution of the azo dye from the paper. By increasing t h e water content of the eluting reagent, Iiowever, this was overcome.
Standard curves for arbutin. saligenin, and catechol are shown in Figure 1. The relationship between weight of phenol and absorbance was linear from 0 to 100 y for catechol and arbutin and 0 to 70 y for saligenin, and the errors were within 147,. The stability of the azo dyes formed with the above phenols appeared to be high. The absorbance of the blanks, ho\\-ever, gradually increased over a period of 2 to 4 hours and then remained steady. This gave an apparent decrease, with time, in the intensity of the dye solutions, the standard curves remaining parallel but below the original curves, and no longer passing through the origin (Figure 2). This, however, is of little importance as far as the accuracy of the method is concerned, as the determination of standard phenols together with the unknowns on the same paper chromatogram alleviates any error which might arise from this phenomenon. The general technique has also been
Paper Chemistry A4ppleton,Wis., unpublished results, 1956. (2) Bray, H. G., Humphr~s,B. G., Thorpe, W. V., White, K., Kood, 1’. B., Biochem. J . 52, 416 (1952). ( 3 ) Bray, H. G., Thorpe, K. V., in Glick’s “Methods of Biochemical AGlysis,’’ ~ o l .I , p. 27, Interscience, New York, 19.54. (4) Bray, H. G., Thorpe, IT. V., White, K., Biochetn. J . 4 6 , 2 7 1 (1950). ( 5 ) Clarke, D. D., S o r d , F. F., in Paech and Tracey’s ”AIodern Methods of Plant Analysis,” Vol. 3, p. 332, Springer-Vwlag, Berlin, 1955. (6) Pridham, J. B., A s . 4 ~ . CHEX 28,
1967 (1956). ( 7 ) Pridham, J. B., unpublished results, 1955. (8) Sevag, 11. G., Lackman, D. B., Smolens, J., J . Riol. Chem. 124,425. (1938). ( 9 ) Stone, J. E., Blundell, 11,J., h s a ~ . CHEX 23, 771 (1951). (IO) Swain, T., Riochem. J . 5 3 , 2 0 0 (1953). (11) Thompson, J. F., Stewart, F. c., Plant Physiol. 26, 421 (1951). (12) Thompson, J. F., Zacharius, R. 11., Stewart, F. C., Ibid.,26,375(1951). (13) Trim, A . R., in Paech and Tracey’s “Modern llethods of Plant hnslysis,” Tol, 2 , p. 295, SpringerT‘erlag, Berlin, 1955.
RECEIVED for revie17 December 1, 1956. i2ccepted l l n r c h 21, 1957.
Gravimetric Determination of Boron Precipitation as Nitron Tetrafluoborate CLAUDE A. LUCCHESI’ and DONALD D. DeFORD Northwesfern University, Evanston, 111.
b A simple gravimetric method for the determination of boric acid in aqueous solution is based upon the conversion of boric acid to tetrafluoboric acid and the precipitation of the latter with the organic reagent, nitron. Fluoride ion and weak acids and bases do not interfere. Over the range in which the method has been tested, from 125
to 250 mg. of boric acid, the average absolute accuracy for 24 determinations was f 1.1
yo.
T
of almost all boroncontaining materials requires a preliminary treatment which ultimately results in a n aqueous boric acid sample. A t present the most precise, reliable, HE ANALYSIS
and generally useful methods for the quantitative determination of aqueous boric acid are based upon the unique property of boric acid to form with polyols acidic chelates which can be titrated with strong base. These meth1 Present address, Analytical Research Department, The Sherwin-Williams Co., Chicago 28, Ill.
VOL. 2 9 , NO. 8, AUGUST 1957
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