Table III. Comparison of Accuracy of Spectrophotometric and Iodometric Methods of Determining Iodides and Bromides in Brines Iodides, Bromides,
Mg./Liter
Mg./Liter
Spectrophoto- Iodometric metric 8.9 7.8 10.4 9.0 14.3 12.5 23.4 19.4 29.4 31.3 32.4 31.5 32.3 33.5 35.6 44.2 39.7
46.9 60.1
44.5 60.9
Spectrophotometric 85.4 89.4 254.4 250.4 345.8 406.9 467.4 560.3 584.4 654.3 687.3 710.4 1560.1 1619.4
Std. dev. 2 .4
Iodometric 82.5 91.6 244.6 247.5 339.6 374.6 451.8 557.6 612.9 650.1 669.4 699.5 1539.5 1612.6
16.
1
extractions, constant conditions were maintained to minimize analytical error. The standard curves w'ere checked daily.
Calculations. Iodide and bromide equivalents were read from the standard curves.
dilute solutions can be concentrated by evaporation after being made alkaline. An excess of silver nitrate can be added before evaporation to prevent loss of halides. A preliminary ether extraction will remove any hydrocarbons that might interfere. The precision of the method was determined by analyzing duplicate samples. The data given in Table I were used to calculate standard deviations of 0.5 mg. per liter for iodides and 4.6 mg. per liter for bromides. The reproducibility of the method for iodides was found good. For bromides the precision was acceptable. The accuracy of the method was determined by analyzing synthetic brine samples to which had been added known concentrations of iodides and bromides (Table II). Comparison with Table I indicates that the accuracy of the method for both iodides and bromides, although subject to a somewhat greater error than that caused by the techniques employed, is adequate for a rapid, analytical procedure in the concentration ranges tested.
To provide an additional check of the accuracy of the experimental method,
Volume of CCL X 100 X mg. I or Br from 6 X sample volume DISCUSSION
The sensitivity of the experimental method was established as being a minimum of 0.2 mg. per liter for iodides and 5 mg. per liter for bromides. More
curve
=
mg. per liter I
or
Br
several samples were analyzed for iodides and bromides by both the iodometric method of Kainrath (4) and by the spectrophotometric method. Table III gives the results of comparing the two
methods and the calculated standard deviations. The indicated differences between analyses by the two methods are somewhat greater than the demonstrated accuracy of the spectrophotometric method. Although the trend is toward lower results for both iodides and bromides by the iodometric method, no conclusions may be drawn as to whether this is a consistent and predictable trend or the reasons for it. As the standard deviations among results obtained by the two methods are greater than those calculated in determining the accuracy of the spectrophotometric method alone, it is concluded that the iodometric method is not more reliable than the spectrophotometric method. LITERATURE CITED
(1) Buckles, R. E., Mills, J. F., J. Am. Chem. Soc. 72, 552 (1953). (2) D'Ans, J., Hofer, P., Z. angew. Chem. 47, 73 (1934). (3) Doering, H., Z. anal. Chem. 108, 255
(1937).
Kainrath, P.,Ibid., 125, 1 (1942). Kolthoff, I. M., Yutzy, H., Ind. Eng. Chem., Anal. Ed. 9, 75 (1937). (6) Laitinen, H. A., Jennings, W. P., Parks, T. D., Ind. Eng. Chem., Anal. (4) (5)
Ed. 18, 358 (1946). (7) Muelen, J. H. van der, Chem. Weekblad 28, 82 (1931). (8) Shiner, V. J., Smith, M. L., Anal. Chem. 28, 1043 (1956). (9) Szabo, Z., Z. anal. Chem. 84, 24 (1931).
Received for review November 21, 1958. Accepted March 2, 1959. Division of Water, Sewage, and Sanitation Chemistry, 134th Meeting, ACS, Chicago, 111., September 1958.
Spectrophotometric Determination of Quercetin L. E.
DOWD
Central Research and Development, Weyerhaeuser Timber Co., Longview, Wash. The colored complex formed on reaction of quercetin with aluminum chloride is used as the basis for a precise and reproducible spectrophotometric method for quercetin. The complex, consisting of mole of quercetin per mole of aluminum chloride, exhibits maximum absorption at 430 m/x in 0.01 M aluminum chloride solution adjusted to pH 4.0 with Beer's law is folpotassium acetate. lowed over the range of 2 to 15 7 of quercetin per ml. of solution. Phlobaphenes and tannins present as impurities do not interfere. Standard deviation is ±0.4% and the 95% confidence limit for triplicate determinations is ± 1.0%. 1
1184
•
ANALYTICAL CHEMISTRY
of quercetin from bark, an accurate and dependable determination was needed for process control and measurement of purity of the final product. Initial studies employed a modification of the method of Porter et al. (11) involving spectrophotometric determination of quercetin in alcoholic solution. This method was abandoned primarily because a linear Beer’s law relation did not exist and because of instability of quercetin in the final dilute alcoholic solution. Other methods usually involved a time-consuming isolation of quercetin from interfering flavonoids or flavanol glycosides by paper chromatography. These methods were not strictly quantiproduction
InDouglas fir
tative, up to 10% of the quercetin being adsorbed by the paper (8, 10). Flavonoids and flavanol glycosides are almost entirely absent in quercetin obtained from Douglas fir bark by water extraction and oxidation of the resulting solution of dihydroquercetin (5), suggesting that quercetin could be determined without a preliminary separation. When direct methods based on the formation of a quercetin-aluminum chloride complex were used (8, 6, 8,10), the tannins and phlobaphenes interfered in alcoholic solutions, and results were inconsistent in aqueous solutions of aluminum chloride. In the latter case, critical examination of the variables and optimizing of conditions led to de-
pH is adjusted to 4.00
velopment of a rapid, accurate, and reproducible analytical procedure. The method, though most useful for the determination of quercetin from bark, is generally applicable to the determination of quercetin from any source. When interfering flavonoids are apt to be present, the quercetin must first be isolated chromatographically ($1-4, IQ). This requires an empirical correction for adsorption of quercetin by the paper, but good results have been obtained in the determination of quercetin aglycone in foods.
± 0.02 by addition of 20 to 30 grams of potassium acetate crystals. The pH of the hot solution is checked by cooling small aliquots to room temperature. After the pH is adjusted, the solution is transferred immediately to a 20-liter bottle and allowed to stand 24 hours. The bottle is sealed with a stopper contain-
Figure 1.
ing a siphon arrangement and a breathing tube filled with soda lime. A reagent so prepared and stored is stable for several weeks. The absorbance of a known quantity of reagent grade quercetin is determined in the aluminum chloride stock solution by the method described here. The absorptivity of the quercetin in the aluminum chloride is calculated on the basis of the concentration of quercetin in grams per liter. Each stock solution of aluminum chloride reagent must be so standardized.
Quercetin crystal
PROCEDURE
Figure 2. Absorption spectra of aluminum chloride complex of quercetin and dihydroquercetin
_
0.01M aluminum chloride solutions at pH 4.0 • x
Quercetin Dihydroquercetin
APPARATUS AND REAGENTS
Figure 3. Dependence of absorbance of quercetin-aluminum chloride complex on pH at different levels of ionic strength and aluminum chloride concentration Quercetin, 6.04 mg. per liter Aluminum chloride, 0.01 M; ionic strength, 0.10 x Aluminum chloride, 0.20M; ionic strength, 1.89 •
Beckman Model DU spectrophotometer with photomultiplier attachment. Quercetin, reagent grade (3,3',4',5,7pentahydroxyflavone). This grade of quercetin is not commercially available, but it may be synthesized from dihydro(3,3',4',5,7-pentahydroxyquercetin flavanone) by a modification of Kurth’s method (9). Ten grams of pure dihydroquercetin (available from Central Research and Development, Weyerhaeuser Timber Co., Longview, Wash.) is dissolved in 1 liter of distilled water, and 300 grams of sodium metabisuifite added. The solution is refluxed 1 hour, cooled, filtered, and washed with water and IN hydrochloric acid. The quercetin is then dissolved in 500 ml. of hot reagent grade ethyl alcohol, and water is added to the point of incipient crystallization. On cooling, the quercetin crystallizes as_ rosettes (Figure 1). After the crystallization is repeated three additional times, the quercetin is dried to the dihydrate by heating at 40° C, for 2 hours under 28 inches of vacuum.
Aluminum chloride stock reagent. Twenty liters of distilled water is boiled for 10 minutes, then cooled to 90° C., and 48.28 grams of aluminum chloride hexahydrate is added.
Moisture in the sample of quercetin is first determined by drying 1.5-gram portions for 2 hours at 105° C. under 28 An amount of saminches of vacuum. ple equivalent to 0.10 to 0.13 gram of anhydrous quercetin is then weighed to the nearest 0.1 mg. in a tared weighing bottle. The sample is washed quantitatively into a calibrated 250-ml. volumetric flask, using 125 ml. of isopropyl alcohol. The quercetin is dissolved by heating the alcohol to 50° C. on a hot water bath and swirling the flask gently from time to time over a period of about 10 minutes. The solution is cooled to 20° C., diluted to volume with distilled water, and mixed thoroughly. The solution is set aside for about 15 minutes to allow any insoluble solids to settle. With a calibrated pipet, 1 ml. of quercetin solution is transferred to a calibrated 100-ml. volumetric flask. The quercetin solution is diluted to volume with the standardized aluminum ehloride reagent and the dilution factor
The
calculated.
After 10 minutes the absorbance of the bright yellow, fluorescent solution of the complex is determined at 430 nut against an aluminum chloride reagent blank, using 1-cm, silica or Corex cells. Cell corrections are applied and the per cent quercetin is calculated. 100 (absorbance ± *
_
(1
—
cell
correc-
tion) (dilution factor) % moisture) (sample 100 weight) (ab'
sorptivity of Q in
AIC1,)
EXPERIMENTAL
Adherence to Beer’s Law. A linear Beer’s law relationship exists over the range of 2 to 15 y of quercetin per ml. of aluminum chloride reagent. The departure from linearity is slight below 2 y per ml, and such low concentrations can be used with only a small loss in accuracy. Development and Stability of Color. The colored complex forms immediately when quercetin is added to VOL. 31, NO. 7, JULY 1959
•
11
85
0.01M aluminum chloride. After 10 minutes, the absorbance remains nearly constant for at least 90 minutes. If the concentration of aluminum chloride is reduced, the time to reach maximum color intensity is increased to a maximum of 45 minutes as the concentration is decreased from about 1 X 10-3tol X 10 ~*M. Drying of Quercetin. In drying quercetin the heating time is critical. Up to 2 hours at 105° C. is required to dehydrate the quercetin. An additional drying time of up to 16 hours causes no further decrease in weight, but any heating beyond 2 hours tends to reduce purity as measured by this test. To avoid this critical drying step, the weight of the quercetin to be
440
Figure 4. Effect of pH and ionic length of strength on wave peak absorbance of quercetinaluminum chloride complex •
x
samples analyzed.
With alcoholic aluminum chloride the
phlobaphenes exhibited considerable interference. For example, a sample which contained 72% quercetin gave a value of 82% in alcoholic aluminum chloride reagent. Effect of Acidity, Aluminum Chloride Concentration, and Ionic Strength. These three variables are discussed together because a change in any one influences the effect of the other two, and the combination of the three controls the degree of light absorption. The pH is the most critical. As the pH is decreased below 5, absorbance rises sharply to a maximum at pH 4
tannins
1186
or
•
ANALYTICAL CHEMISTRY
0.01M/
ionic
0.2M;
ionic
Figure 5. Effect of ionic strength absorbance of quercetinaluminum chloride complex at different aluminum chloride concentrations on
analyzed is corrected to an anhydrous basis by determining the moisture content on a separate portion of the sample. Interference of Impurities. Small amounts of impurities, such as tannins and phlobaphenes, are sometimes present in quercetin from Douglas fir bark, but these do not interfere when aqueous aluminum chloride reagent is used. This was shown by using two methods to analyze a number of products from experimental
preparations with quercetin contents ranging from 70 to 98%. In the first group the samples of quercetin were analyzed as obtained. In the second group the quercetin was selectively extracted with ethyl acetate and determined in the ethyl acetate solubles. Douglas fir tannins and phlobaphenes, being largely insoluble in anhydrous ethyl acetate, are left as a residue. Any dihydroquercetin present is carried along with the quercetin, but the aluminum chloride complex of dihydroquercetin is colorless and does not absorb at 430 m/j, (Figure 2). The quercetin content of 10 samples determined by the two methods did not differ by more than 0.7% in any case. As a check, the absorbance of the ethyl acetate-insoluble impurities was measured in aluminum chloride and found to be negligible at the concentrations at which the impurities occurred in the
Aluminum chloride, strength, 0.063 Aluminum chloride, strength, 1.94
Quercetin, 5.96 mg. per liter • Aluminum chloride, 0.01 M/ pH 4.0 x Aluminum chloride, 0.1 M; pH 4.0
Figure 6. Effect of aluminum chloride concentration on absorbof quercetin-aluminum ance chloride complex Quercetin, 6.04 mg. per liter (2 X 10-8 mole per liter) pH 4.0; ionic strength, 0.24
and drops sharply as the pH is lowered further. The effect of pH is somewhat less
at high aluminum chloride
concen-
tration and high ionic strength, but this difference becomes minimal at pH 4.0 (Figure 3). At low pH’s, the wave
length of peak absorption shifts to lower wave lengths (Figure 4). Although ionic strength has a lesser effect on absorbance than does pH, the absorbance is lowered significantly as the ionic strength is increased. Increasing ionic strength by addition of sodium sulfate or potassium chloride has a greater effect at low than at relatively high aluminum chloride concentration (Figure 5). At pH 4.0 and at a constant ionic strength, the absorbance of quercetin is nearly constant over the range of aluminum chloride concentration between 0.004 and 0.03ilf. At concentrations below 0.004A7 the absorbance decreases
linearly with the log of the concentration (Figure 6). An excess of about 200 moles of aluminum per mole of quercetin is required for complete complexing of the quercetin.
DETERMINATION OF QUERCETIN FROM OTHER SOURCES
Modifications of the aluminum chloride method have been used to determine quercetin aglycone in several foods such as onions, strawberries, apricots, and applesauce. The following analysis of onions is a typical example. were slurried with Waring Blendor; the pulp was dried at 65° C. Then 10 vacuum grams of dry pulp were Soxhlet extracted with methanol for 6 hours to remove quercetin. The methanol extract was concentrated to 5 ml. and diluted to 100 ml. with water. The aqueous phase was extracted three times
Several onions
water in
a
with equal volumes of diethyl ether to separate quercetin from any quercetin glycosides. The ether extract was evaporated to dryness in a 40-ml.
centrifuge tube, and the solids were extracted three times with 10 ml. of hexane and three times with 25 ml. of ethylene dichloride to remove fats and oils. This was followed by three extractions with 10 ml. of water to remove water-soluble material extracted with the ether. The remaining solids were dissolved in isopropyl alcohol and transferred to a 50-ml. volumetric flask. Then 25 ml. of this solution was concentrated to 5 ml., and 640 *tl. of concentrate was spotted on one paper and a known amount of pure quercetin on another. The papers wTere irrigated with isopropyl alcohol-water (22 to 78, v./v.) (quercetin Rf 0.06). The quercetin spot was extracted with ethyl acetateethyl alcohol (30 to 70, v./v.) and the eluate made up to 5 ml. A 2-ml. portion was transferred to a 10-ml. volumetric flask and the solution was evaporated to dryness. The solids were dissolved in a few drops of isopropyl alcohol and the solution was diluted to volume with aluminum chloride reagent. The absorbance was determined as previously described. After appropriate corrections were made for adsorption of quercetin by the paper, the quercetin content was calculated. The amount of quercetin aglycone in the onions was 500 p.p.m. To check the accuracy of the analytical technique, 10.0 mg. of pure quercetin was added to one sample of onions. Subtraction of the quercetin found in the onions from the total quercetin isolated from this sample indicated a 98% recovery of the added quercetin. DETERMINATION OF EMPIRICAL FORMULA
To meet the requirements of the slope ratio method (7), two series of solutions were prepared. In the first series, the aluminum chloride concentration was held constant and the quercetin concentration varied; and in the second series, the quercetin concentration was held constant and the aluminum chloride concentration was varied, the concentrations of the constant component being identical in each The absorbance of each solution case. in the series was measured and plotted against the concentration of the variable component. The ratio of the slopes is equal to the combining ratio of the components (Figure 7). In this case, the ratio of the slopes was found to be 1.15 to 1. Statistical analysis showed that a departure from one of 15% was not significant and the probability of the ratio’s being other than 1 to 1 was very small. If a is the degree of dissociation, C
MOLAR CONCENTRATION OF VARIABLE COMPONENT X
10«
Figure 7. Plots for application of slope ratio method Quercetin, 2 X 10 “5M Aluminum chloride, 2 X 10 ~5M Ratio of slopes, calculated from least squares
•
x
fit of data,
1.1 5 to
1
is the molar concentration of the reactants, and the molar ratio of the components is 1,
C(1
a)
-
aC +
=
ccC
(1)
or
The value of relationship a
a
=
is obtained from the
Ae -1-
—
Ax
Ae
where Ae is the absorbance when a given amount of quercetin is completely complexed by aluminum chloride and Ax is the absorbance of an identical amount of quercetin in a stoichiometric amount of aluminum chloride. A solution which was 2 X 10-W with respect to quercetin gave values of 0.532 for Ae and 0.121 for Ax, resulting in a value of 0.773 for a. Substituting the values for a and C in Equation 2, the value of K was calculated to be 5.2 X 10~5. The empirical formula and dissociation constant were confirmed by an independent experiment using the method of Bent and French (1). DISCUSSION
In determination of the empirical formula, the slope ratio method and the method of Bent and French suffer from the same disadvantage: Both require the ratio of the constant component to be large in relation to the amount of complex. Because of the low solubility of quercetin, this condition can be met only with very dilute solutions of aluminum chloride. Consequently, the concentration of complex is so low that only a narrow range of absorbance readings can be obtained with reasonable accuracy in 1-cm. cells. Statistically, this results in a rather high error function and relatively wide confidence limits on the slopes or ratio of the slopes.
The variations are, however, of a low enough order to preclude a significant probability that the mole ratio in the complex could be other than 1. The composition of the complex in aqueous aluminum chloride differs from that in alcoholic aluminum chloride where the ratio is 2 moles of quercetin per mole of aluminum chloride (8). Preparation of aluminum chloride solutions which will give constant absorptivities with pure quercetin is made extremely difficult by the pronounced effect of both pH and ionic strength on absorbance. As a result, it is necessary to standardize each batch of aluminum chloride with pure quercetin to assume maximum precision. Pure quercetin in freshly and carefully prepared aluminum chloride has a maximum absorptivity (based on the concentration of quercetin) of 89.6. Where the reagent is made in large quantity and set aside to stabilize, absorptivity falls in the range of 86.5 to 88.5. Reproducibility of the method was established by having 10 replicate determinations made on a single sample by each of two different analysts. Statistical evaluation of the results showed a standard deviation of ±0.4% and 95% confidence limits on the mean of ±1.0%
for triplicate analyses. ACKNOWLEDGMENT
The author thanks R. B. Bowhay for statistical analysis of the data and A. S. Gregory for his valuable advice and counsel in preparation of this paper. LITERATURE CITED
(1) Bent, H. E., French, C. L., J. Am.
Chem. Soc. 63, 568-72 (1941). (2) Casteel, H. W., Wender, S. H., Anal. Chem. 25, 508-9 (1953). (3) Dillaha, Janis, Gage, T. B., Wender, S. H., Proc. Oklahoma Acad. Sci. 31,
102-104 (1951).
(4) Gage,
T. B., Wender,
S.
H., Ibid.,
29, 145-8 (1949). (5) Gregory, A. S., Brink, D. L., Dowd, L. E., Ryan, A. S., Forest Prods. J. 7, 135-40 (1957). (6) Hagedorn, Paul, Neu, Richard, Arch. Pharm. 286, 486-90 (1953). (7) Harvev, A. E., Manning, D. L., J. Am. Chem. Soc. 72, 4488-92 (1950). (8) Horhammer, L., Hansel, R., Arch. Pharm. 285, 438-44 (1952). (9) Kurth, E. F., Ind. Eng. Chem. 45, 2096-7 (1953). (10) Naghski, J., Fenske, C. C., Jr., Couch, J. F., J. Am. Pharm. Assoc. 40, 613-16 (1951). (11) Porter, W. L., Brice, B. A., Copely, J. J., Couch, J. F., U. S. Dept. Agr., Bur. Agr. and Ind. Chem. AIC 159 (1947).
Received for review July 21, 1958. Accepted March 16, 1959. Division of Cellulose Chemistry, 133rd Meeting, ACS, San Francisco, Calif., April 1958.
VOL. 31, NO. 7, JULY 1959
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