ANALYTICAL CHEMISTRY
1582
graphic separation. Essentially, the spreading factor is an attempt to take into account the finite band %-idth of a given amount of material under predetermined conditions. A considerat,ion of both Rf and S has been shown to be far superior to a consideration of R,falone. By plotting S against aeight of cation it is possible that even the limiting proportions of two cations can be predetermined. The phenomenon of double spotting has been observed in some experiments. This objectionable drawback can be eliminated by the proper choice of solvents and bj- closely regulating the drying time and temperature. The two fundamental modes of behavior in the alcohol series lead to the obvious conclusion that at least two different types of 8-quinolinolate are formed. Pro1)at)ly tlioae cations sho\ring diminishing Rf values through the alcohol aeries form 8-quinolinolate that are fundamentally salt like, while the 8-quinolinolate with increasing Rfvalues are true chelates. Feigl(3) has recently arrived a t the Same conclusion from chloroform extractability data. Feigl has postulated the esisterice of two tJ-pes of metal 8-quinolinolate as follows:
I = I s (solid) (solution)
I I (solution)
The continuous conversion of I to I1 in solution then accounts for the extractability of the complexes. Feigl classifies c:ilciuni and magnesium as nonestractable and these cations show decreasing Rf values in the alcohol series. .illurninurn, cobalt, :itid nickel, which rho\\. increasing Rf values are classified by Feigl :is extractable. In addition, Feigl classifies cadmium, copper, iron, and lead tis extractable, while the authors’ data show that c:til-niiuni and copper show diminishing R, v a l u e . This appirent discrepancy can be readily reconciled on the basis of the solidsolution type of equilibrium postulated by Feigl. Further work is being carried out in an attempt to substantiate this hypothesis concerning the structure of the two type? of 8quiitolinolate 11ymeaus of infrared data. The scope of the work is also being extended to cover t l i r rest of the inorganic cations which form 8-quinolinolate and to cover mixed solvents including dioxane-Clark and Lubs buffer mixtures, dioxarie-py~itliiiemixtures, etc. LITERATURE CITED
He tabulates the various metal 8-quinoliiiolate and shows which are extractable and which are riot extractable with chloroform. He further states that substances which exhibit solubility in chloroform would be expected to have forni I1 in solution, but not necessarily in the solid state; solubility is accounted for by the equilibria.
(1) Consden. R . , Gordon, -1.I T . , : ~ n dMartin. ..J.II-’.. , Hiodiou. J., 38,224 (1944). ( 2 ) Evlenmeyei., H., sttd D a h t i , 11.. Hili,. ( * h i m , . l d u , 22 1:W) (1939); 24, 878 (1941). ( 3 ) Feigl, I.’.. “Cheniistt,y of Sgecific., Srlective, s l i d Seitsitive Reactions.” S e w Yo1.k. .\i,aderiiic. I’reai. 1940. (4) Lnnge, “Hatidhook of (‘hemistry.” 6 t h etl., Satirludiy, Ohio, Handbook I’uhtishers, 1946. RECEIVED Deceiiiber 7, 1950. Presented before t h e Ili\-isiori of AnaIytical Cbeiiiistry at the 119th Ueeting of the . \ u h K I c . k x C H E V I C ASOCIETY, L Cleveland. Ohio.
Identification of Flavonoid Compounds by Filter Paper Chromatography ?‘1IO!WiS 1%. GAGE, C.4RL D. DOUGLASS,
AND
SIMOS €1. WENDEK
Cnicersity of Oklahornu, Norman, Okla.
The recent use of flavonoid compounds in the treatment of radiation injury and frostbite has lent new emphasis to the need for improved methods of ideni particular need for tifying these pigments. micromethods of separation and identification of flavonoid pigments in plant extracts has prompted the application of paper partition chromatography methods to this problem. The paper chromatographic behavior of thirty -eight flavonoid-t>pe compounds has been determined in eleven solvent SJ s-
ESDER and Gage (3) and Bate-Smith and Westall (1, 2) have reported studies on the descending filter paper chromatography of flavonoid-type compounds. The present paper reports ext,ensions of these studies to a large number of flavonoids commonly encountered in nature. Eleven different solvent systems are included. I n addition t o the use of the usual two-phase solvent systems, the use of single-phase solvent systems has been found applicable. Many of the flavonoid compounds exhibit characteristic fluorescent colors on the developed chromatograms when examined under ultraviolet light. I n addition, the original color of these compounds in visible and ultraviolet light may be altered or enhanced when a solution of a metal salt, ammonium hydroxide, or Benedict’s reagent is sprayed ont,o the paper strip. RenPdict’s
tems. The visible and fluorescent colors prodirccd by eight chromogenic sprays when applied to thc developed chroniatogranis have been tabulated. The use of paper chromatography coupled with chromogenic sprays presents an additional tool for the classification and identification of flavonoids obtained from natural sources or by synthesis. The method is of particular advantage in evaluating the efficiency of various separation or purification procedures.
solution has been found especial11 suitable for the flavonol glycosides and aglycones. A brilliant yellow pigment zone is outlined against the blue background color of the filter paper strip after spraying with Benedict’s reagent, This reagent is suitable for the location of the sugar as \vel1 as the aglycone zones on the chromatogram of a glycoside hydrolyzate; to develop the sugar zone, it is necessary to heat the paper strip for a few minutes after spra>-ing with Benedict,’s solution. The colors produced by the chromogenic sprays when ronsidered in conjunction with the Rr value often make possible the tentative classification of an unident,ified flavonoid pigment into one of the major groups listed in Tables I and 11. A flavonoid may be tentatively ident,ified by this method, but too great, a reliance should not. be placed upon a color or R, value alonc. The
V O L U M E 2 3 , NO. 1 1 , N O V E M B E R 1 9 5 1 Table I. EthJI acetate Phenol saturated saturated with with water water
( 'ompound
Flavonol aglycone Go3syprtin Kaeni pie rol Morin Sortangcretin PatiiletinY Qiierrrtagr t in Quercetin Rhaninetin Robineriii Flavonol glycoGirlr. Gosqypin Gossylitrin Iso-. A hand-tJ-pe hair dryer wts use 1 to evapo~rtc. the spotting solvent. The sti,ips were then placed in the chromatographic chamber (Chromatocab, Cniversity Apparatus Co., Berkeley, Calif.) by inserting the end of the strip nearest the pigment zone under the glnss rod provided with each trough. .I portion of the solvent syPteni was placed in borosilicate glass pie plates in the bottom of the chamber t o ensure a sufficient supply of solvent vapor and Chu prevent excessive evaporaLioii of solvent from the paper strip during the chromatographic process. The troughs were then filled with the solvent system and t h e chamber was closed. Development of the chromatograms was allowed to proceed until the solvent had traveled :35 t o 40 cni. beyond the starting line. This required 8 to 22 hours, depending upon the rate of movement of the individual solvent system. The strips were then removed from the chamber and allowed to air-dry. The majority of the flavonoids eshibited some color in visible light and appreciable fluorescence in ultraviolet "black" light. The forward boundary of the solvent front could also be located by
ANALYTICAL CHEMISTRY
1584 ultraviolet light, because of the fluorescence of impurities in the paper (or degradation products of cellulose produced by solvent interaction with the paper) which moved with the solvent front. The pigment zones were encircled lightly with a pencil while under the ultraviolet lamp. The distance traveled by the flavonoid was then determined by measuring from the starting line to the forward edge of the pigment zone. The distance traveled by the solvent front was determined in a similar manner and the Rj values listed in Table I were calculated from the resulting ratio. The use of chromogenic sprays was especially helpful for the location of the pigment zones of the less highly fluorescent flavonoids such as oroxylin A, naringin, neohesperidin, hesperidin methylchalcone, phloretin, and pomiferin. The strips were suspended in an improvised rack and the area between the starting line and the solvent boundary was lightly sprayed with an atomizer (Devilbis No. 251) containing the appropriate reagent. After air-drying, the colored or fluorescent zones were located and the Rf values determined as previously described. Table 11 lists the visible and fluorescent colors produced by the various sprays. Traces of creso1, remaining on the paper after chromatography in this solvent system, interfered markedly with ammoniacal silver nitrate and ferric chloride tests. This could be avoided by allowing the strips to air-dry considerably longer and heating the strips in the oven for a short time prior to spraying. DISCUSSION
The Rj values listed in Table I represent average values for each pigment. The capacity of the cabinet used for chromatography was sufficient to run 96 strips at one time. Thus, at least two sam-
ples of the same pigment., as well as samples of many different pigments, were chromat.ographed Pimult8aneously. Some variation in Rj of a given pigment occurred from time to time, but the variation was usually less than 1 0 . 0 4 R, value and in most cases less than 10.02 R/ value. The insulated chromatographic chamber was protected from temperature changes due to radiation sources such as sunlight, open flames, ovens, etc. The temperature varied slightly from day to day, but comparison Rj values for several samples obtained in a constant temperature room a t 20" C. indicated that reproducibility of results was not much greater by the latter method. The lid of the chamber vias weighted during the runs in order to prevent leaks of solvent yapor. This was especially important with the more volatile solvent systems. Some of the flavonoid samples used in this study were found by 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 very great compared with that of the smaller pigment zone of trace contaminant. For this study, no effort has been made to identify every contaminant. I n the case of conimerc4al samples of rutin, however, quercetin was shown to be present to the extent of 1 to 375. Quercetin obtained from American dyer's oak or lemon flavin preparations contained a small quantity of a
Table 11. Colors Produced by Chromogenic Sprays
_I'ntreated-
Conipound
Y
Y Y
Y Y PY Y Y
Y
e
PY
PY P'L:
Pk
PY
PY
Y
.. , .
.. , .
Pir PY
.. ..
Orox ylin-A
Wogonin Flavanone a lycones Homoerio&tyol 3,3',4',5,7-Pentahydroxyflaranone Flavanone glycosides Hesperidin Xaringin Neohesperidin Flavane aglycones &Catechin l-Epicatechin Chalcones Hesperidin methylchalcone Phloretin Z', 3,4-Trihydroxychalcone Others Esculetin Pomiferin Brown B. Bk. Black B1. Blue Dark D. 0
Visible light.
6
~ 1% v uv
UVb
Flavonol aglycone Gossypetin Iiaempferol Slorin Yortangeretin Patuletin Quercetagetin Quercetin Rhamnetin Robinetin Flavonol glycosides Gosawin Gosskpitrin Isoquercitrin Quercimeritrin Quercitrin Robinin Rutin Xanthorhamnin Flavone aglycones Acacetin Apigenin Auranetin Chrysin Genkwanin Isowogonin Xoruogonin
-
Alcoholic Aluminum Chloride
P1GY Y
Y
GY
Y Y
YB GY OR Y
Ei
Y GY
T Y T
P 1B
I'
RB B YO OB B
Y T Y 1 ' Y
RB RB B1W RB RB B RB
GY
PY
B
'I-
..
PY
..
T
B PY Y
..
B
..
..
..
..
PY
BlM
..
..
.. ..
..
..
..
.. ..
.. ..
..
..
Y
..
..
..
..
..
..
G. I. 0. P.
S
GI'
GY PY
Y Y
Y
Y Y OY Y GY GY BW PY Y B PB RB B
..
uv
Y GY GY B GY GY GY GY GY
Y Y
Y Y
I
Y Y GY
PY
BY
PY Y GP PY Y
Y
.. ..
Y
..
..
Y
PY LY DY
Basic Lead Acetate
1% --v uv
B Y
Y
Y DB YB B B RO
I
Y
0 0
Y
Y
Y
0
Y Y
Y Y
Y GY
Y
Y
Y
Y
Y
Y
Y
BlW PY
PY
GY
Y
I
BIW
Y
0
..
.. ..
BlW BIW
I
..
Y
GY Y
0
Y V
OY
Y
Y
O
RO
..
GY I
..
PY
BlW
B
YB
RB GY PY RB RB
..
YB Y Y
B DB OY OY OY Y OB OY
DB
Y Y
..
I
YB YB Y
PY
..
B1W B Red Y. Yellow V. Violet W. White
uv
,.
..
..
v
B DB Y G Y Y Y Y Y RB RB PB B B OB B Y I GY
YB
.. ..
BIG
1% ___
DB
PBI Y w-
..
170
GY
.. ..
Y
Aqueous Sodium Carbonate
..
GY
Y Y
Normal Lead Acetate
GB DY B1W B 0 Y B DB BY
Y Y Y Y
Pir
Y
BIW BlW
0
B1W DY GY Y BY DB B
..
PY
0
PB BY
Y Y
..
Y
DB Y Y Y B OB B 0 O
BY
B B BIW B
B
Y
OY
v
v
PB
GY
..
B1
GY RB
,.
..
.. ,.
Y YB
I
..
..
I
uv
B Y Y
qka
GB PB OB
Y li Y
PY
*.
I Y
Y
YB
Y Y Y
I'
GY
1
.. .. , _
Y
..
Y
Y Y
,.
B YB
Y
YB
Y OY Y
GB GY B1 W Y GY
T
B PY
Bk Bk DB BBk Bk
PG PG Olive Olive
OY OY
OB
Bk B
Olive GY
OY Y
OB
Bi B
OY
Y OB 1-
Y
OB Y
Y Y
B Y B B B B
.. ..
..
..
1-B B
..
PG GY
G G PG
...
...
PG PG YG B
.. ..
PG' PG
... ...
B B
..
Gray GY G Tan
.. .. .. .. ..
B
B
B
DB
... ...
T Y
Y
Y OY Y OY OY Y
..
Y
Y
YB YB Y
Y
T
...
'I'B OY Y
U'
...
B B
RB RB
RB RB
...
B B
.. B1G .. v
....
R.B
... ...
Y
R Y
I
R
Bk
Bk
BlW
Bk RB
Bk B
DB
C
Prior to heating.
d
After heating 5 minutes a t 110' C.
w
B
RB RB
RB RB
Y
YB
Bk
.. ..
H
OY OY OY B OY OY OY 1-
YB
.. .. ..
B B
GY RB
B
DY
BIW B1 B1G
RB
V
Benedict's Solutlon v IJV
B B B Bk
B
I PY
OY
170
Bk RB Bk YB RB Bk Bk RB RB
B
PY
PY B
Alcoholic Ferric Chloride
BBk RB B OB OB B BBk RB RB
..
PY
.. .. GY I
v
B T Y
.4inmoniacal Silver Xitrate -____ P6 Ad
Y
...
Y
... ...
Y Y
w . . . .
R
R
Y
Y B
..
R.
Green Ivory Orange Pale
Ultraviolet light.
GY G 1YB GY B1 GY
1% ___
Y
..
Y
Y
Alcoholic Thorium Chloride
No appreciable visible (or fluorescent) color.
,
1585
V O L U M E 23, NO. 11, N O V E M B E R 1 9 5 1 second flavonoid aglycone of unknown constitution. Quercetin obtained from buckwheat, by hydrolysis of rutin, was free of this impurity. Purified hesperidin contained a small amount of a second flavonoid glycoside. This could be largely removed by preliminary extraction of t.he relatively insoluble hesperidin with hot water.
California Fruit Growers Exchange, Research Department, Ontario, Calif. This investigation was supported in part by research grants from the Office of Naval Research (Project NR-059-226) and the Division of Research Grants and Fellowships of the National Institutes of Health, U. s. Public Health Service.
ACKNOWLEDGMENT
LITERATURE CITED
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: Charles H. Horton, Plant K-25, Oak Ridge, Tenn.: T . R . Seshadri, University of Delhi, Delhi, India; Joseph Pew, Forest Products Laborator! , Madison, Wis. ; Pharmacology Laboratory, Bureau of Agricultural and Industrial Chemistry, -4lbany 6, Calif.; and The
(1)’ Bate-Smith, E. C., “Biochemical Symposium KO.3,” Cambridge. Cambridge University Press, 1950. ( 2 ) Bate-Smith, E. C., and Westall, R. G., Biochim. Biophys. Acta.. 4, 427 (1950). (3) Wender, S. H., and Gage, T. B., Science, 109,287 (1949). RECEIVED February 1, 1951. Presented before the Division of Biological Chemistry a t the 118th Meeting of the AMERICAN CHEIIICAL SOCIETY, Chicago, Ill.
X-Ray Photoelectron Spectrometer for Chemical Analysis RALPH G. STEINHARDT, JR., AND EARL J. SERFASS Lehigh University, Bethlehem, P a . Of all the many and diverse known types of charac; teristic spectra, the only one which has not been investigated previously from the analytical viewpoint is the x-ray photoelectron spectrum. Because of the low energies of x-ray photoelectrons, only electrons that originate close to the surface of an x-ray bombarded target contribute to the characteristic spectral edges. A n instrument is described which measures x-ray photoelectron intensities at energies from
I
F A beam of x-radiation is allowed to strike a solid target, two types of radiation will be emitted by the target: fluorescent x-rays and photoelectrons. Both types of radiation exhibit spectra that are characteristic of the material of which the target is composed. Although fluorescent x-rays have been used for chemical analysis ( I O ) , x-ray photoelectrons have not. This communication describes an instrument designed for the utilization of x-ray photoelectron spectra for chemical analysis. THEORY
The emission of photoelectrons and fluorescent x-rays through excitation by x-rays is governed by the equation:
T = hu,
- hu
-
wo
(1)
in R-hivh I’ = kinetic energy of photoelectron h = Planck’s constant L’. = frequency of exciting radiation v = frequency of fluorescent radiation w,, = work function of target material If u0 is such that the exciting radiation lies in the “soft” x-ray region (lo1*to 1019cycles per second), two conditions may be imposed on Equation 1:
(hu, - hv)>>w, hu,
hu
(2) (3)
The condition indicated in Equation 2 alters Equation 1 to:
T = h(u,
- u)
(4)
The condition indicated in Equation 3 implies that the energy of the emitted electron will be relatively small. Several important conclusions may be reached on the basis of the above:
6 to 17.5 k.e.v. and is shown to be capable of yielding qualitative and quantitative data for elements having atomic numbers greater than about 25. The instrument is a 180” magnetic deflection electron energy selector using a high-intensity x-ray tube for excitation and an ultra-thin window Geiger-Muller counter for detection. The potentialities of the instrument for performing atomic surface analyses would appear to be considerable.
If the frequency of the exciting monoenergetic radiation is maintained constant, the initial energy of the photoelectron is characteristic of the tarket material only. The energy of photoelectrons emitted under these conditions will be of the order of 0 to 17.5 k.e.v. Therefore only that portion of the target on or very close to the surface will emit electrons capable of escaping from the target. Furthermore, photoelectrons ejected from atoms lying beneath the surface will be partially absorbed and many of them will escape only after considerable energy loss; only those ejected from surface atoms will experience a virtually undetectable energy loss in escaping. Because of this absorption process, the low energy side of a characteristic photoelectron peak will exhibit an exponential rise in intensity while the high energy side will have an almost vertical descent. The energy defined by this vertical edge will be characterized by the chemical composition of the surface. The energy of the photoelectron will be practically unaffected by changes in the work function of the target, since wo is of the order of several electron volts. Thus it appears that n-ith an instrument capable of measuring the energies and corresponding intensities of x-ray photoelectrons it should be possible to perform a quantitative analysis of the nominal surface of a solid regardless of the physical characteristics of the surface. Furthermore, because the emitted electrons have their origin in the inner energy levels, the state of combination of an atom will usually not affect its x-ray photoelectron spectruni to any appreciable extent. The typical photoelectron spectrum is not as simple as might appear on the basis of Equation 4. The reasons for this are a9 follows: The incident radiation is not perfectly monoenergetic. The use of a crystal x-ray monochromator is not practicable because of the high intensity of incident radiation necessary to excite a sufficient number of photoelectrons to be measured precisely. The space required for a crystal monochromator would reduce seriously the intensity of the incident radiation through the operation of the inverse-square law. Instead, a Hull (11) filter is used, Further reduction of the contribution of the con-
