A N ALY TICAL CHEMISTRY
508
where
(Y
=
[D
-
4 D 2 - 422]/2(D
- d)
1 .o
But this must be independent of Z, and must be equal to so that we find
B(Z) =
-
T H E O R E T I C A L . NO
10
4 DT.’
(D
- d)3[5 + 18a + 24a2 + 16a3]
(5)
1 CY‘
48 p DT’
(D
TI0 D
Consequently, the longitudinal pressure gradient is grad p =
0 - E X P E R I M E N T A L , LIQUIDS 0 -EXPERIMENTAL, A IR
DV,
- d ) 3 [ 5 + 18a + 24a2 + 1601~1
(6)
and the total pressure drop along the tube, due to the ball, is AP =
grad p dZ = Jr m n
r
[&d]6’2x dE 5
(7)
where, if we call the integral 8 r I / 3 , thereby defining a number I , we have
I =
1 [.\/a 1/2 - + 21’’’ 45
0.398
(7’)
In passing from Equation 6 to 7, we have defined a quantity by the equation $2 = a,and have again neglected terms which contribute only in higher orders of ( D - d ) / D . Thus, for example, the limits on the integrals are not really infinite, but depend upon D/(D - d). This pressure drop must now support the sphere, so that ? r ~ a
(i
-
p l ) g sin 8 =
7r
- ~
4
a 10
2
~
3
u IC
I
.
I
lo5
10-6
I04
10-3
Figure 1. Plot of Theoretical Equation 9’with Experimental Points with the calibration constant K of Hubbard and Brown, in terms of which Equation 9 is
K = -2o 7
[ DT ]- d
512
D-d 7 = 0.0891 [ ] (9’) ‘ “
In Figure 1 has been plotted the expression for K given by Equation 9’, the points measured by Hubbard and Brown, and the points calculated by them from other experimental data. The fit, considering the simplifications in the calculation, is satisfactory. I t can serve for the design of rolling ball viscometers, although, for precision work, the usual calibration in terms of fluids of known viscosity should be carried out. Equation 9 should, of course, be used directly, without going through the definition of K .
p
LITERATURE CITED
and
(1) Hubbard, R. M., and Brown, G. G., IND.ENG.CHEM.,ANAL. ED.,15, 212 (1943). (2) Lamb, H., “Hydrodynamics,” New York, Dover Publications, 1945.
which is our final result. It will be convenient to compare this
RECEIVED for review
July 7, 1952.
Accepted October 15, 1952.
Identification of Flavonoid Compounds by Filter Paper Chromatography Additional Rf Values and Color Tests HELEN WARREN CASTEEL AND SIMON H. WENDER University of Oklahoma, Norman, Okla. APER chromatographic techniques applicable to flavonoid Pcompounds have been developed by Bate-Smith and Westall (1, 9) and by Gage, Douglass, and Wender ( 3 ) . Because of the interest indicated by many research workers in these paper chromatographic studies of flavonoids, the present investigation was undertaken to extend the usefulness of this technique by the determination of R f values for a number of flavonoid compounds not yet reported in the seven solvent systems listed. The colors produced by chromogenic sprays when considered in conjunction with the R, value often aid in the tentative classification of an unidentified flavonoid pigment into one of the major subdivisions of flavonoid compounds. Therefore, the colors produced on paper by chromogenic sprays and certain of the newly studied flavonoids were also determined.
EXPERIMENTAL
Experimental apparatus, materials. and procedures used correspond BB nearly as possible to those of Gage et al. (3). A newer
model “Chromatocab” chamber (Chromatography Division, University Apparatus Co., Berkeley, Calif.) was used in the present study, however. This chamber was much better sealed and better insulated than the previous model used. Thus, uniform saturation, indicated by movement of solvent fronts through equal distances for all strips within the chamber, was obtained if sufficient time (usually 24 hours) was allowed for saturation. Also, rate of movement on the paper was usually much more rapid in the newer chamber. A 250-ml., all-glass spraying flask (University Apparatus Co.) operated by compressed air a t 5 pounds pressure delivered an even mist of chromogenic reagent. The spray was controlled by an air hole covered by the thumb during delivery. RESULTS AND DISCUSSION
Table I lists the R / values obtained for twenty-one flavonoid compounds in seven different solvent systems. These listed values represent average R f values for each compound. Some variation in Rt of a pigment occurred from time to time, but the variation was usually less than 1 0 . 0 4 Rf value and, in most cases, was less than 50.02 Rr value. Some of the flavonoid samples used in this study were found by
V O L U M E 2 5 , N O . 3, M A R C H 1 9 5 3 Table I.
509 R / Values of Flavonoid Compounds
Compound Ethyl acetate saturated with water Flavonol aglycones Flavonol Galangin Myricetin Quercetin Flavonol glycosides Kaempferitrin Myricitrin Quercitrin Flavone aglycones Acacetin Baicalein Tec t oc hrysin Flavone glycosides Diosmin Rhoifolin Flavanone aglycones Butin Hesperitin Katsuranin Liquiritigenin Pinobanksin Pinocembrin Pinostrobin 7-Hydroxyflavanone Flavanone glycoside Hesperidin 4
Phenol saturated with water
m- and p-cresols
saturated with water
Solvent Systems Butanolacetic acidwater, 4: 1 : 5
Isopropyl alcoholwater, 3 : 2
Acetic acid-water, 3:17
0.94= 0.92 0.45 0.68
0 : is 0.12 0.05
Acetic acid-water, 3:2 0.86
0.75 0.31 0.40
0.26 0.35
0.82 0.48 0.58
0.63
0.17 0.30
0.79 0.74 0.83
0.89 0.87 0.88
0.77 0.52 0.58
0.86 0.73 0.78
0.96 0.97 0.98
0.96 0:99
0.98 0.99 0.98
0.96 0.90 0.97
0.90 0.83 0.94
0.00 0.24 0.27a
0.79 0.79 0.89
0 07 0 08
0.77a 0.85
0.62 0.61
0.54 0.69
0.67 0.88
..
0.98 0.97 0.97 0.97 0.98 0.99 0.99 0.98
0.88a 0.96 0.87 0.92 0.97
0.91 0.94 0.93 0.95 0.96 0.97 0.97 0.95
0.93Q 0.94
..
0.79a 0.97 0.76 0.91= 0.94 0.98 0.98 0.97
0 90
0 66
0 63
0.54
0.98
0.99
0 12
..
0:87 0.81a
0.95 0.95 0.97 0.96 0.96
0.55 0.50 0.63 0.68 0.58 0.52
..
0.87 0.88 0.92 0.89a
0 86
0 80
0 88
G YG OB DB
Sormal Lead Acetate, 1% \-is. UV Tan PY 0 BK DV B
Benedict’s Solution Vis. UV G YG Tan G Ochre DB
YG YG PO
PY PY
PY
0,93
0.85 0.82
0.6Za
0.86a
hforr than one spot appeared; doubt as t o the identity of the selected spot is possible.
Table 11. Colors Produced by Chromogenic Sprays
Compound Hesperitin Kaempferitrin Myricetin Myricitrin Pinobanksin Pinocembrin Rboifolin Tectochrysin
Untreated Vis. UV’ .. PG .. POB Y Y DY B PY YB PG
Y
PY
o
PDV
Alcoholic Aluminum Chloride, 1% Vis. UV I G Y YG DY YG PY YO I YG PI G YG f Y
Alcoholic Thorium Chloride, 1% Vis. USf PY Y PY YO Y DG Y OY PY Y Y PY YO PY OB
Basic Lead Acetate, 1% Vis. UV Tan PY OY PY 0 DY B PY YB PY Y PY OY PY Tan
Alcoholic Ferric Chloride,
Aqueous Sodium Carbonate
1%
1%
Vis. Tan Olive Tan Olive Tan Tan Tan Tan
UV BK BK BK BK BK BK BK BK
Vis. BO Y Gray BO Y Cream Y Y
B, Brown; B K , Black; D, D a r k ; G, Green; I , Ivory; 0, Orange; P, Pale; R, Red; Y, Yellow; V, Violet:
paper chromatography to contain as impurities, lesser amounts of other flavonoids in addition to the principal pigment present. In these cases, however, the color density of the principal pigment zone was usually very great compared with that of the smaller pigment zone of trace contaminant. If not, and there could be some possible doubt as to the identity, the value is reported, with a footnote. For this study, - . no effort was made to identify every contaminant. To link the Rt values of Table I (run a t a different season of the vear in a different chamber and on a fresh batch of Whatman No. 1 paper) with those in the previous publication of Gage et al. (S), the reference compounds acacetin. quercetin, and quercitrin were run again a t the same time as the new compounds, and their newly determined Elvalues werelisted. Also new Rjvalues for hesperidin are recorded in Table I. The previous values were obtained when the hesperidin was spotted in basic solution, whereas the present ones represent the values for hesperidin when spotted from acetone solution. Additional experience with the solvent systems involving chloroform-water and chloroform-isopropyl or isobutyl alcoholwater indicates that they are relatively poorer solvent systems, often streaking badly, and are, therefore, not rerommended for general use. Considerable streaking- often occurs also with the ethyl acetate-water system. Table I1 lists the colors Droduced on Whatman No. 1 filter paper by chromogenic sprays on some of the flavonoid compounds, after spraying and drying, as viewed both in the visible and under ultraviolet light. A flavonoid compound may be tentatively identified by R / values in several solvent systems and use of chromogenic sprays, but too great a reliance should not be placed upon a color or RJ value alone. For more dependable information as to possible identification, one also should run mixed chromatograms in several carefully chosen solvent systems, if an authentic sample of t,he pigment in question is available.
UT’
YG
. .. S o
.,
Pn 9” T an Pn PYG Y Y
PRB
G YG YB
appreciable color.
Although the solvent systems listed together with chromogenic sprays were successfully applied to separating and tentatively identifying individual flavonoids of other grou s and are of value in differentiating flavanone aglycones from t i e other groups of flavonoid compounds, they do not afford a good means of separating one individual flavanone from another. Lindstedt et al. have had some success on flavanones with other solvent systems
( 4 . 6). ACKROW LEDGMENT
The authors wish to express their appreciation to the following individuals and organizations for the donation of some of the flavonoid samples used in this investigation: Shizuo Hattori, Botanical Institute, University of Tokyo, Hongo, Tokyo, Japan; Gosta Lindstedt, Kungl. Tekniska Hogskolan, Organisk-kemiska Institutionen, Stockholm, Sweden; Joseph Pew, Forest Products Laboratory, Madison, Wis.; T. R. Seshadri, University of Delhi, Delhi, India. C. E. White, Chemistry Department, University of Maryland, dollege Park, Maryland; and Research Dept., Sunkist, Growers, Ontario, California. This research was supported in part by funds from the Office of Naval Research. LITERATURE C I T E D (1) B a t e - S m i t h , E. C., “Biochemical S y m p o s i u m No. 3,” C a m b r i d g e , C a m b r i d g e U n i v e r s i t y P r e s s , 1950. (2) B a t e - S m i t h , E. C., a n d Westall, R. G., Biochim. el Biophys. Acta., 4. 427 (1950). (3) Gage, T. B., Douglass, C. D., a n d \Tender, S. H., ANAL.CHEM., 23, 1582 (1951). (4) L i n d s t e d t , G., Acta Chem. Scand., 4, 448 (1950). (5) L i n d s t e d t , G., and Misiorny, A,, Ibid., 5 , 1 2 1 (1951). RECEIVEDfor review September 13, 1952. Accepted November 10, 1952.
