was made with the lower electrode a cathode. Note in particular that there are two cathode maximums in both series. The enhancement factor a t a point is the ratio of line to background ratios at that point relative to the center of the gap region. The values plotted were an average for the 13 elements found. The average enhancement, measuring line to background ratios, was approximately 10 times for the following element lines read: Mg 2852.1, A1 3082.2, V 3185.4, Cr 4254.4, Ca 4302.5, Ti 3372.8, B 2497.7, Si 2881.6 Fe 3020.6, Na 3302.3, M n 2576.1, Cu 3274.0, Sr 3464.5. The most interesting use of the supragap and sub-gap regions is with regular sized samples. Samples containing 0.1% each of 42 elements were made by mixing 1 mg. of Spex Mix (Spex Industries, N. J.) with 9 mg. of spectrographic purity GeO and 10 mg. of graphite powder. These were burned in graphite cup electrodes with flat graphite counters (Nat. Carbon Co., N.Y., sfL3903, and #L3960) at 300 volts and 13 amps,
exposed to background. Figure 3 is a photograph of a portion of the spectrum near Ca 3179.3 at each of three positions, shown from top to bottom; supracathode region with anode-excitation, center of gap region with cathode-excitation, and sub-cathode region with cathode-excitation. The % transmission readings obtained were: supracathode, 92/9.1 or 29 times background; center of gap, 67/41 or 1.2 times background; and sub-cathode, 99/87 or 2.1 times background. A summary of the values found for the 42 elements measured in the supra-, center-, and sub-gap regions were as follows: Best in the supra-cathode region with enhancement shown in parentheses: A1 2575.1 (5X), 13a 2335.3 ( l o x ) , Ca 3179.3 (20X), Cr 2677.2 (12X), Fe 2973.2 ( 2 X ) , M g 2783.0 (4X), Mn 2576.1 (3X), Sr 3464.5 (11X). Best in the sub-cathode region because of freedom from band spectra and continuum interference: Ba 4554.0, Cs 4555.4, K 4047.2, R b 4201.9.
Twenty-six elements (Sb, As, Be, Bi,
B, Cd, Co, Cu, Pb, Hg, Mo, Nil Nb, P, Si, Ag, Xa, Ta, Te, T1, Sn, Ti, W, V, Zn, Zr) were better in the center gap region. Ce, Ge, Li, T h , U, aIso in the sample were not determined. N o correlation has been found yet between the obvious factors; ionization potential, degree of ionization, boiling points, or arc reactions, and the element lines favored in the three groups studied. LITERATURE CITED
(I) Ahrens, L. H., Liebenberg, W. R.,
Trans. Geol. SOC.S. Afr. 49, 133 (1946). (2) Mannkopff, R., Peters, C., Z . Phyzik 70, 444 (1931). ( 3 ) Mitchell, R. L., J . Trans. SOC.Chem. Ind. 5 9 , 210 (1940). (4)Oertel, A . C., “Spectrographic ,4nalysis of Mineral Powders,” Comm. Sci. & Ind. Res. Org., Adelaide, Australia
(1961).
GEORGE A.
UM.4N
Los Angeles City Dept. of Water & Power. Los Angeles, Calif.
RECEIVED for review November 9, 1964. Accepted December 9, 1964.
Sugar Analysis of Flavonoid Glycosides SIR: We describe a general procedure for the determination of the sugar components of naturally occurring flavonoid glycosides using, in part, the elegant gas chromatographic technique of Sweeley and coworkers (3). Because often only small amounts of glycosides are isolated from natural sources, a sensitive and reliable method for sugar analysis was desirable. Paper chromatography has been the most commonly used analytical technique, but it suffers from uncertainty since a number of sugars have quite similar R, values in any solvent system. Furthermore, the two anomeric forms, a and p, of a sugar, are represented by only one spot. The discovery (3) that the gas chromatographic analysis of the easily prepared trimethylsilyl ethers of anomeric sugars yielded distinct peaks and that most sugar derivatives were well separated increased considerably the ease and reliability of the sugar analysis, We have modified this technique slightly and developed a general procedure for the analysis of the sugar components of flavonoid glycosides. The method should also be suitable for other classes of glycosides. 288
ANALYTICAL CHEMISTRY
trimethylchlorosilane according to the previously reported procedure ( 3 ) . After a few minutes, the reaction mixture Hydrolysis and Trimethylsilylation. can be injected directly into the gas I n a typical experiment, 2 mg. of chromatograph. This would be the hesperidin, 3,5,3’ - trihydroxy - 4’methoxyflavanone-7-~-rhamnoglucoside, method of choice for quantitative work. For qualitative analysis, we found it was hydrolyzed in 25 ml. of 21%‘ HCl preferable to use a technique already containing about 5 ml. of methanol on a described for the preparation of samples steam bath for 30 minutes. Occato be analyzed by NMR spectroscopy sionally, the complete hydrolysis of other (8). After the excess solvents and compounds, such as flavone-7-glucoreagents were removed under high sides, required up to several hours. vacuum, a few drops of dry heptane were The reaction mixture was passed added. The white, insoluble material through a 2- X 1-cm. column of polywas decanted or filtered off and the amide powder (Polypenco 66D from clear solution was concentrated to a the Polymer Corp., Reading, Pa.), suitable volume. For best results a packed in water. Flavonoids and other final solution as concentrated as possible phenolic compounds are strongly adis recommended. Upon injection into sorbed by hydrogen bonding to this the gas chromatograph of this concensupport. The column was washed with trated clear solution, a straight base water until the eluate was neutral, line was displayed almost immediately This solution contained the sugars, after the solvent peaks in contrast to rhamnose and glucose. Subsequent eluthe original reaction mixture. This tion of the column with methanol modification provided an easier analafforded the aglycone, hesperitin. ysis of the more volatile sugar derivaThe acidic aqueous sugar solution was tives such as those of rhamnose. I n taken to dryness at room temperature addition, no solid material was introunder high vacuum. The residue was duced into the injection chamber. dissolved in 0.5 ml. of dry pyridine Gas Chromatography. A Research and the trimethylsilyl ethers were preSpecialties model 600 dual-injection pared by the addition of 0.2 ml. of instrument with an argon ionization hexamethyldisilazane and 0.1 ml. of EXPERIMENTAL
A.
RHAMNOSE AND GLUCOSE EOUILIBRATED
SUGAR PRODUCTS FROM HESPERIDIN
1
4
10
I
Figure 1.
Chromatograms of sugar derivatives
A and B were obtained under identical conditions: SE-52 columns a t 180’ C. and 7 and 8 represent a- and 6-rhamnose and glucose, respectively
detector was equipped with 6-foot X 0.25-inch U-shaped stainless steel columns. T h e results reported in this paper were obtained with a column packed with acid washed silanized chromosorb W coated with 3y0 SE-52; (Applied Science Labs, State College, Pa.) with inlet pressure of 15 p.s.i. Peaks 2, 4, 7, and 8 of the chromatogram of the sugar derivatives from hesperidin (Figure 1A) corresponded to those observed with the derivatives of authentic rhamnose and glucose (Figure lB), previously equilibrated in hot pyridine ( 3 ) . All eight peaks were present when the authentic rhamnose and glucose mixture was treated in a manner analogous to that used for the hydrolysis of hesperidin. DISCUSSION
Most natural flavonoid glycosides contain glucose, rhamnose, or combinations thereof. The retention times of the trimethylsilyl ethers of these sugars are very different. As indicated by Sweeley et al. (3),a single peak is observed for the derivative of crystalline CY-glucose or any other anomerically pure sugar. Most anomerically pure sugars equilibrate rapidly in hot pyridine and give two peaks, assigned to the CY- and p-forms (Figure 1B). Often, a third peak is observed. Since most flavonoid glycosides have a low solubility in water, some methanol is usually added at the start of the hydrolysis reaction. Under these conditions, besides the expected CY- and p-sugar derivatives, additional peaks were always present. An authentic sample of the suspected sugar was treated under conditions identical to those used in the hydrolysis of the glycoside. The number and retention times of the peaks in the two chromatograms were identical; thus, the matching of the retention times of the
15
1IMI.MlNUlIf
TINE MINUTIS
Peaks 1 to 4 a r e assigned to rhamnose and 5 to 8 to glucose.
peaks of the standard and of the unknown, separately and in mixture, is a reasonable proof of their identity. We found that the number of additional peaks and their relative intensities depended on the experimental condition. For instance, after the treatment of a mixture of glucose and rhamnose for a few minutes in hot 2 N HCl containing some methanol, four additional peaks 1, 3, 5, and 6 were observed (Figure 1A). Two of them, 3 and 5, could be observed even in the absence of methanol. On continued evaporation of the solvent, peaks 1, 3, 5, and 6 disappeared. However, advantage should be taken of the presence of the additional peaks since they increase the reliability of the method. Comparison of the unknown with a standard w g a r treated in the conditions of the hydrolysis is therefore a necessity. Furthermore, we have refluxed glucorhamnosides for one to two hours in 2 5 HC1 without methanol. Only four peaks, assigned to the CY- and p-forms of glucose and rhamnose, were observed. Flavonoids often occur as polyglycosides and the order of attachment can be established in some cases by comparing the relative proportion of the sugars hydrolyzed, as a function of time. For example, mild hydrolysis of a luteolin-7diglycoside yielded a small ratio of glucose to rhamnose as determined by the relative peak areas. Longer hydrolysis increased this ratio, indicating that glucose was linked to the 7-position of the flavone. This result was confirmed by the examination of the flavonoid intermediate which was identical with luteolin-7-glucoside, known to be quite resistant to hydrolysis ( I ) . The order of attachment of the sugars was also independently deduced from KRIR
Peaks 2 and 4
analysis of the trimethylsilyl ether of the diglycoside ( 2 ) . Because of the rapid mutarotation of sugars in acid solution t,his method does not allow the determination of the configuration of the sugar linkage in the glycosides. This informat>ioncan, however, often be obtained from rotation values or from XMR analysis of t’he trimethylsilyl ethers of the original glycosides ( 2 ) . The lat’ter technique also gives the total number of sugars in the molecule. The use of specific hydrolytic enzymes is also informative although of limited application because of the low solubilit’y of many flavonoids in aqueous solutions. The limit of sensitivity of the method herein described is less than 0.5 pg. of the sugar. This value was determined with glucose but the method is even more sensitive for sugars having more volatile derivatives, such as rhamnose, because of the increased sharlmess of the peaks. Beside being more reliable, this technique is therefore appreciably more sensitive than paper chromatography. LITERATURE CITED
(1) Hattory,
S., “The Chemistry of Flavonoid Compounds,” T. A . Geissman, ed., p. 325, RIacmillan, New York, 1962. (2) Mabry, T. J., Kagan, J., Rosler, H., Phytochemistry in press. ( 3 ) Sweeley, C. C., Bentley, R., Makita, R I . , Wells, W. W., J . A m . Chem. SOC. 85, 2497 (1963). JACQUES KAGAN T. J . MABRY De t. of Botany and 8ell Research Institute The University of Texas Austin, Texas INVESTIGATION supported by the National Institutes of Health Grant GSI-11111-02 from the Division of General hledical Sciences. VOL. 37, NO. 2, FEBRUARY 1965
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