Adsorption Chromatography of Flavonoid Compounds CLARK H. ICE AND SIMON H. WEKDER University of Oklahoma, Norman, Okla.
EI‘ERAL investigators have applied adsorption chromatography to special, limited studies involving flavonoid compounds (1, 3, 5 , 8). No previous worker, however, was able to find the proper combination of adsorbent and solvents which would afford a general method for separation of closely related flavonoid compounds. Failures were due to such factors as chemical interaction of the flavonoid with the adsorbent, as in the case of alumina, lack of adsorption as with talc, or inability to find proper adsorbents and solvents for separation of closely related compounds. This paper reports a successful combination of solvents and adsorbents for chromatography of flavonoid compounds. The flavonoid compounds are adsorbed onto Magnesol from anhydrous acetone solution, and elution is usually achieved n i t h a solution of ethyl acetate saturated with water. By use of this method, several closely related flavonoids have been separated from each other. The method has also proved t o be very useful in the isolation of pure flavonoids from plant sources, for their detection when present in extremely Ion- concentration in natural products, and for use as a criterion of purity. Recently, Pearl and Dickey have reported the use of acidwashed Magnesol to separate vanillin and syringaldehyde (6) from their benzene solution and to separate a chalkone from lignin oxidation mixtures ( 7 ) . EXPERIMENTAL
The adsorbent used in these studies is Magnesol, industrial grade, regular (Food Machinery and Chemical Corp., Westvaco Chemical Division, S e w Pork, N. Y.). Columns were prepared without pretreatment or sizing of the adsorbent. Samples, unless otherwise noted, were adsorbed from anhydrous acetone sohtions, and elution was usually carried out with a solution of ethyl acetate saturated with water. The glass adsorbent columns used ranged in diameter from 8 to 60 mm. Preparation and Operation of Column. The procedure is illustrated by the preparation of a column 60 mm. in diameter containing a 160-mm. adsorption bed. Six hundred milliliters of anhydrous acetone was added to 150 grams of Magnesol and the mixture was stirred to give a thin slurry. The slurry was added a t once to the column, and the sides of the column were rinsed down with 200 ml. of acetone. When the adsorbent had settled, leaving a layer of acetone above the surface, an acetone solution containing 0.5 gram of a flavonoid mixture was added. After the solution has passed onto the column, a filter paper circle was placed on top of the adsorbent, and the ethyl acetate solution was carefully added. The passage of ethyl acetate through:the column \+-ascontinued until elution of the flavonoid compounds was complete. The individual bands, which were visible under ultraviolet light, were collected as separate fractions. Separations. Of a number of flavonoid mixtures, riot easily separated in most cases by other techniques, but readily separated on the Magnesol column, the following are illustrative: QUERCETIN-MORIS. An acetone solution containing 3 mg. of quercetin (3,3’,4’,5,7-pentahydroxyflavone)and 3 mg. of morin (2’,3,4’,5,7-pentahydroxyflavone) was added to a column 18 mm. in diameter packed to a depth of SO mm. After passage of acetone through the column and adsorption of the flavonoids on the Magnesol, elution with the ethyl acetate solution gave two bands, both yellow in visible light, and showing a bright yellow fluorescence under ultraviolet light. The bands were several centimeters apart and were easily collected as two fractions. Paper chromatography showed that the first fraction was pure quercetin while the second contained pure morin (8). QUERCITRIN-RUTIN-QUERCETIN. An acetone solution containing 5 mg. each of rutin (quercetin-3-rhamnoglucoside) and quercitrin (quercetin-3-rhamnoside), containing quercetin as im urity, was added to a column 18 mm. in diameter packed to a b)epth of 40 mm. with the Magnesol. After adsorption of the three compounds on the LIagnesol, elution with the ethyl acetate
solution produced three widely se arated bands. The band nearest the bottom was quercetin, t i e middle band was quercitrin, and the upper band TYas rutin, After complete elution of all three bands, examination of the three fractions by paper chromatography showed that complete separation had been achieved. XANTHORH.4USIN-&UERCITRIiX-QUERCETIN. An acetone SOlUtion containing 5 mg. each of xanthorhamnin and quercitrin, containing quercetin as impurity, was added to a column 18 mm. in diameter packed to a depth of 40 mm. The flavonoids were adsorbed in a narrow band a t the top of the column. Upon elution a i t h the ethyl acetate solution, quercetin, then quercitrin, moved off as t s o separate bands. Santhorhamnin was not moved even on continued elution q-ith the ethyl acetate. Therefore, follom-ing elution of the quercetin and quercitrin, the column was eluted with a 30% isopropyl alcohol-nater solution. The xanthorhamnin moved rapidly off the column in a narrow band. Examination of the three fractions by paper chromatography indicated that complete separation had been accomplished. XANTHORHAVSIK-RCTIN-QUERCETIN. A complete separation was obtained by the method described in the preceding paragraph. Alternatively, the adsorbent wa8 extruded after elution of the quercetin. The xanthorhamnin (top) band and rutin band a-ere cut out and individually leached from the adsorbent with 95% ethyl alcohol. APIGESIK-QUERCETIX. Elution with the ethyl acetate solution gave tn o bands, xith the apigenin (4’,5,i-trihydroxyflavone) moving ahead of the quercetin. The eluate was collected in three fractions: The first contained pure apigenin, the second was a mixture of the t\i 0 , n hile the third was pure quercetin. ~ ~ P I G E S I S - ~ - R H , ~ \ l K O G L T C O S I D E - ~ . 4 R I S GElution Ilj. with ethyl acetate moved naringin (4’,5,7-trihydroxyflavanone-7-rhamnoglucoside) off the column as a sharp band. Apigenin-7-rhamnoglucoside did not move, and was later leached from the Magnesol with 95% ethyl alcohol. SARIKGIS-HESPERIDIN. 4 complete separation TT as obtained using a column packed to a depth of 90 mm. Hesperidin (4’methoxy-3’,5,i-trihydro.;yflavanone-i-rhamnog1ucoside) moved off the column first. Of all those tried, the one mixture which could not readily be separated into its components by the elution with ethyl acetateIYater solution XYas quercetin and dihydroquercetin. Purification of Rutin. A 0.50-gram sample of commercially available rutin (S. B. Penick and Co., Kew- 1-ork, S. I-.) was dissolved in 1250 ml. of anhydrous acetone, and the solution was passed through a column 60 mm. in diameter packed to a depth of 10 cm. with Magnesol. Rutin and its aglycone impurity, quercetin, n-ere adsorbed in a 10-mm. band at the top of the column. Upon elution with ethyl acetate-n ater solution, quercetin moved off as a sharp yellow band, whereas the rutin moved only a few millimeters. When the quercetin had been completely removed, the rutin was eluted with 1 liter of 50% ethyl alcohol. The alcoholic eluate was concentrated to a volume of 150 ml. by distillation a t reduced pressure; the concentrate r a s cooled, and then neutralized by the addition of a few drops of hydrochloric acid. After standing overnight in the refrigerator, the precipitated rutin was filtered off, washed with distilled water. and dried a t 120” C. Yield was 0.41 gram, melting point 193-194” C., uncorrected, A paper chromatogram Phowed that the rutin was now completely free of the quercetin. DISCUSSION
The adsorption chromatographic method described is very versatile and has proved to be useful in the solution of a number of problems encountered in the detection, separation, and purification of flavonoid compounds. A general adsorption chromatographic method using AIagnesol for the isolation of flavonoid rompounds from natural products, such as from plant material, has been successfully applied in this laboratory in studies of the flavonoids present in old fustic, in Yerba Santa, in licorice root, grapes, rose petal jelly, black currants, and leaves of Vuccinium myrtillas. The general method, which may be modified to fit the individual case (the preliminary application of ion exchange resins to separation of the flavonoids has often been of value, 4 ) ,may be summarized as follows:
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V O L U M E 2 4 , NO. 10, O C T O B E R 1 9 5 2 The powdered, dry material is extracted with anhydrous acetone, and after concentration to a suitable volume, the acetone solution is assed through a Magnesol column. On continued washing wit! anhydrous acetone, chlorophyll, if present, is eluted, while flavonoids and some other components remain a t the top of the adsorbent. Elution of the column with ethyl acetate-water solution causes most flavonoid compounds, if present, tomove off in separate bands, usually leaving a zone of brown material on the upper portion of the column. I n this way, i t is possible to collect flavonoid fractions which are relatively pure and suitable for study by paper chromatography. I n addition to detecting and ascertaining the number of flavonoid compounds present, information may be gained as to their nature, both by their appearance under ultraviolet light and by their relative rates of elution. By a second Magnesol chromatographic treatment of the separate bands, it is possible to obtain pure samples suitable for absorption spectrum studies. I t is convenient next to evaporate the individual fractions to dryness under reduced pressure. The solid may then be dissolved in dry acetone for still additional column treatment, if necessary, or it may be recrystallized from a suitable solvent for melting point determination. The usefulness of this adsorption chromatography method using lllagnesol may be extended by the selection of other solvent
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systems, or by suitable pretreatment of the Magnesol. I t has been possible to adsorb the pigments from a methanol solution, in certain cases. Changes in pH of the adsorbent as effected by various treatments cause radical changes in column performance. LITERATURE CITED
(1) Fontaine, T. D., Ma, R., Poole, J. B., Porter, W.L., and Kaghski, J., Brch. Biochem., 15,89 (1947). (2) Gage, T. B., Donglass, C. D., and Wender, S. H., ANAL.CHEM., 23,1582 (1951). (3) Gage, T. B., Gallemore, C., and Wender, S.H., Proc. Okla. d c a d . Sci.. 2 8 . 7 1 (1948). (4) Gage, T. 'B,, kforris, Q. L., Detty, IT. E., and Wender, S. H., Science, 113, 522 (1951). (5) Mager, X.,Z. physiol. Chem., 274, 109 (1942). (6) Pearl, I. A, and Dickey, E. E., J . Am. Chem. Soc., 73, 863 (1951). (7) Ihid., 74, 614 (1962). (8) Robezniecks, I., Z. Vitaminforsch., 8, 27 (1938). RECEIVED for review March 2 5 , 1952. Accepted June 13, 1952. Portions of this article were presented a t the 7 t h Southwest Regional Lfeeting of the - 4 M E R I C A N CHEMICAL SOCIETY, December 6 , 1951, Sustin, Tex. Investigation supported in part by t h e Atomic Energy Commission, a n d Office of K a r a l Resarch.
Determination of Coupled Aromatic Nuclei in an Azo Protein C . EDWIN WEILL iVewark College of Arts a n d Sciences, Rutgers T/nirersity, .\-ernark, ,Y. J . R O T E I S S TThich have been joined to an aromatic nucleus Pthrough an azo linkage have been used in many chemical investigations on altered proteins. There is need for a rapid method of determining the number of aromatic nuclei bound to these molecules. Tyrosine is one of the amino acids in the peptide chain which can couple with a diazotized amine (4). There is a possibility that the absorption of light per mole of azo group in a coupled protein might be proportional to the absorption of the same wave length of light by the compound formed when the same aromatic amine is coupled to pure tyrosine. This study !vas undertaken to see if there is any such correlation. Any agreement between the molecular extinction coefficient of the aromatic azotyrosine and the extinetion coefficient per mole of azo group in the protein would be used only as an empirical method of determining the number of bound groups in a sample of protein. S o claim is made that tyrosine is the only amino acid Thich ill react. I n fact, other amino acid residues mill couple under the proper conditions (1, 2, 4). I f the extinction coefficient is to be calculated per mole of azo group in the coupled protein, it is necessary to have some independent method of measuring the number of these bound groups. Follonirig the idea of Boyd and Hooker ( I ) , p-arsanilic acid was used because the presence of arsenic would allow a direct chemical measurement of the number of aromatic groups attached to a unit weight of the coupled protein.
I n the preparation of the arsaniloazotyrosine and arsaniloazo-pcresol the cold diazotized arsanilic acid mas added to cold, alkaline solutions of tyrosine and p-cresol. The diazotized arsanilic arid 15-as the minimum reagent in each case. I n a separate experiment arsaniloazo-p-cresol was isolated and recrystallized. The arsaniloazo proteins were prepared from Armour bovine albumen fraction V. Increasing numbers of azo groups mere coupled by increasing the volume of the diazonium solution, and by raising the pH of the coupling solution. The proteins were all dialyzed, precipitated with acid, washed with acetone, and dried. Colorimetric measurements were all carried out on solutions of pH 7.0 f 0.1 n i t h the instrument set a t 340 mp. A water blank n-as used because it could be shoivn that the absorption of light by excess tyrosine and p-cresol was negligible in the concentrations used. The terms used in the calculation of extinction coefficients are those of Killard, Merritt, and Deane (6). Arsenic determinations were conducted according to the method of Magnuson and Watson (3). RESULTS AND DISCUSSION
The isolation and purification of the azotyrosine proved to be a most difficult step. In experiments with arsaniloazo p-cresol it 1%as found that the molecular extinction coefficient of the recrystallized product n a s 17.3 x 103 as compared with 17.5 x 103 for the product kept in solution and merely diluted and neutralized for the optical measurements. Thus, it was not necessary to isolate the arsaniloazotyrosine; it could be prepared and merely diluted and neutralized. Arsaniloazotyrosine prepared in this manner had a molecular extinction coefficient equal to 10.0 x 103.
REAGERTS ARD APPARATUS
Reagents used n-ere tyrosine, Eastman Kodak 666, p-arsanilic acid, Eastman Kodak 1369, and Armour bovine albumen fraction V. p-Cresol \vas redistilled before use. All other chemicals were of C.P. grade, and were used v-ithout further purification. Apparatus. Beckman Model DU spectrophotometer with Corex cells of 1.001- to 1,003-em. width, Beckman Model G pH meter. PROCEDURES
Diazotized p-arsanilic acid was prepared by the method of Eagle and Vickers (W),except that a pH meter was used to follow the adjustment of acidity.
Table I. Results of Experiments with Arsaniloazo-p-cresol
Sample so.
k (Specific
Extinction Coeff .)
AS,
70
Moles of Bao Group per Gram of Coupled Protein, X 10-1 From arsaniloazoFrom E f &rAs analyses tyrosine
