Filter Paper Chromatography of Inorganic Cations with 8-Quinolinol DONALD E. LASKOWSKI AND W. C. 3Ic:CROKE
Armour Research Foundation, Illinois Institute of Terhnology, Chicugo, 111. This work was undertalien to determine the applicability of 8-quinolinol-impregnated filter paper to the separation of inorganic cations, and to determine the Ry values of these cations in a large number of solvents and solvent pairs. The results obtained show t h a t 8-quinolinol-impregnated filter paper is applicable to the separation of many of the cations investigated. The tabulation of the RJ values of the cations in a large number of pure solvents resulted in data t h a t proved useful in predicting the outcome of a given Separation. The main result of this preliminary investigation, however, was to point the way to further, more exhaustive research. From the practical point of view, the fundamental data
having been determined, i t is now possible to determine whether or not a desired separation can he achieved hy chroniatographinp on 8-quinolinolimpregnated filter paper. .\lost of the conditions of separation have ahead? been predetermined. The theoretical implications are perhaps more important t h a n t h e practical. Feigl's conception of the structure of the metallic. 8-h?drox?quinolates has been, to a large extent, substantiated. This work may result in the separation of t h e possible geometrical and optical isomers of the metallic 8-hydroxyquinolates. By the proper correlation of t h e chromatographic data with other types of data, the structure of these compounrls can he more clearly elucidated.
P
REVIOUY investigators have used 8-quinolinol as a colorforming reagent in the column chromatography of inorganic cation? ( 2 ) . This investigation extends the use of 8-quinolinol to include filter paper chromatography.
n
TERMINOLOGY
Throughout this paper, the classical li, value, defined ( I ) as the ratio of the distance moved by a giver1 hand to the distance moved by the solvent front, is used. In addition, S,the spreading factor, may he defined. Let d he the initial band width B and B the final band width; then Si = -I. The subscript i d denotes a given cation.
1
n
REAGENTS
LThatman S o . 1 fi1tt.r papei, untreated. Solutions of Cations. XI1 reagents were analytiral giadr. S b + + + , B a + + , C d + + , Ca+', Co++, and F e + + + qolutions were made from the chlorides, AI++-, Cu'-, My'+, anti N i + + from the sulfates; and P b - + iiom the nitiate Solutions were prepared to contain 5 mg. of cation per ml. and then diluted onefifth to ield solutions containing 1 mg. of cation per ml. In the case of J b + + + ,it was necessary to add concentrated hydrochloric acid drop by drop until the solution remained clear. Filter Paper.
A
-
DIRECTION
8.
B,
SOLVE1JT FRONT
O F SOLVENT
Figure 1. Two-Component C:hroniatograni
Developing Solvents. -111 solvents ivere c.P., used without further puxification. 8-Quinolino1, reagent grade, used without further purification. Buffer Solutions. Clark and Lubs buffer, prepared according to Lange ( 4 ) . The pH of these solutions was measured by a Becknian Model G p H meter. EQUIPMENT
Figure 2 shows the type of column used for developing the chromatograms. A shows the completely disassembled column and all its component parts. B and C ehow the column in various stages of
C
Figure 2.
D
Apparatus for Running Chromatograms
assembly; 1 is a length of borosilicate glass tubing 85 mm. in outside diameter and 18 inches (45 cm.) high; 2 is a 100-ml. crystallizing dish used to hold the developing solvent; 3 is a No. 14 rubber stopper s i t h a small hole drilled through its center and a plug to fit the hole; 4'is a 6-inch square of rubber dam with a hole approuimately 2 inches in diameter cut out of the center. I n assembling the column, 10 to 12 paper strips are fastened by means of thumbtacks to the underside of the rubber stopper. These are placed about 0.25 inch apart and form a complete circle. The stopper is then inserted firmly into one end of the column, the paper hanging down inside the column. -4 rubber 1579
1580
ANALYTICAL CHEMISTRY
band previously extracted by the developing solvent to be used ie placed around the opposite end of the column to hold the strips in place. The strips should be spaced evenly around the bottom of the column, and should not touch each other. As the diameter of the base of the stopper is less than the diameter of the column, the aper strips run diagonally from the base of the column to the Ease of the stopper, the only point of contact between the strip and the glass being a t the place where the paper is drawn over the column. When the strips are wetted with solvent, they expand and draw inward, so that care must be taken in order not to place too many strips in the column a t one time. SI
pipetted directly onto the spot formed by the cation solution. After air drying for 1 hour, the spots were lightly outlined in pencil; ultraviolet light was used to detect barium, calcium, and magnesium. Treatment of Chromatograms. In general, the chromatograms were allowed to develop approximately 18 hours a t room temperature. With most of the solvents tested, this gave a total ength of solvent travel of between 35 and 40 cm. After development, the columns were disassembled and the stoppers, with the strips still attached, were hung in a ventilated place until the solvent evaporated. I n most cases, it was not necessary to mark the point to which the solvent front had risen, as there was a visible excess of 8-quinolinol a t the solvent front. As a matter of course, all chromatograms were vieTed under both white and ultraviolet light, The data necessary to calculate R/ and S factors were obtained by measurement with a scale and the strips were then either filed away or discarded. THEORETICAL IMPORTAhCE OF SPREADING FACTOR
0.1
’
0’
0
The spreading factor, as defined, can be shown to exert as great an influence on the outcome of a chromatographic separation as does the &value. The variables which affect the spreading factor are (a) the total length of travel, ( b ) the total amount of cation present, (c) the initial band width, ( d ) the nature of the solvent, ( e ) the strip width, and (f)the type of paper. Keeping ( b ) , ( d ) , ( e ) , and ( f ) constant and making certain assumptions, it is possible to predict the outcome of a given attempt a t a chromatographic separation, providing Rj and S factors for the substances to be separated have been previously obtained. It must be assumed that these are the same for the mixture as for the pure components. The following general discussion illustrates how to predict whether two substances will be separated.
V
/
I
I
I
I
l
I
0.1
0.2
0.3
0.4
l
I
1
0.1 0.6
I
I
0.7 0.8
l
l
0.9
1.0
12
Figure 3. C h a r t for D e t e r m i n i n g Rj Value at Which Separation C a n Be Achieved
Assume a total length of solvent travel L; component 1 having and an Rj value Rj1, while component 2 has a spreading factor SI, an Rj value of Rj2. Assume further an initial band width A and that R,,>R,x. Referring to Figure 1, the length of travel of the front of component 1 is LR,, anti that of component 2 is L&. The final band width of component 1 is S I ~ ~ ( S ,=A
2.d B
= BI).
Therefore, for complete separation of components 1 and 2,
f
About 50 ml. of the desired solvent are now placed in the crystallizing dish and a small wire hook is inserted underneath the rubber band. The column is then set upright in the crystallizing dish and a t the same time the rubber band is gently drawn upward and out of the solvent. The weight of the glass column is sufficient to hold the paper strips in place. The rubber dam is now stretched so that the column will fit into the hole in its center and the dam is pulled down until it overlaps the crystallizing dish. A rubber band is used to hold the rubber dam to the crystallizing dish, the natural elasticity of the rubber dam holding it to the column. When all the wrinkles are straightened out, the system is almost airtight, exce t for the hole through the rubber stopper. The purpose of the Role is to allow the solvent level to equalize inside and outside of the column. After a few minutes this hole is stoppered with its plug. In this work, five such columns were used simultaneously, giving a total capacity of 50 to 60 chromatograms per day. By this means, a large amount of data could be obtained in a short period of time. Impregnation of Filter Paper. Two sheets of Whatman KO.1 18.5 X 20 inch filter paper were cut to form ten 18.5 X 4 inch strips. These strips were rolled to form a cylinder and placed in a glass cylinder 11.5 em. high having an internal diameter of 5.5 cm. and a smooth top. A solution of 5 2= 0.1 grams of 8quinolinol in 200 ml. of 95% ethyl alcohol was then added to the vessel. The strips were allowed to stand completely immersed for 1 hour a t room temperature, then removed and shaken to remove most of the ethanolic solution. The strips were then hung up and allowed to dry 4 hours a t room temperature. After drying, these large strips were cut into 0.25 X 18.5 inch strips and subsequently used to chromatograph the various cations. Spotting the Filter Paper. About 0.01 ml. (0.02 ml. in the case of copper, nickel, and lead) of cation solution was placed about 2 inches from the end of the impregnated strip, a 0.1-ml. graduated Kahn serological pipet being used to measure the volume. In the case of antimony, barium, calcium, and magnesium, 0.02 ml. of Clark and Lubs buffer solution a t p H 9.28 was immediately
SIA LRp 5 LR,I - SIA or Rj2 5 R,, - 7. If it is assumed that L = 35 cm., this equation reduces to the form Rj, 5 Rj1 0.043S1. Figure 3 shows a chart constructed from this inequality.
If the R, and S values are tabulated for the various cations, as in Table I, it is possible to predict whether a given separation is possible. Assume a two-component mixture whose Rj and S values are I the chart tabulated. The highest R/ value is taken as R ~ on (Figure 3) and a ruler is placed through this value parallel to the Rlz axis. At the point where the ruler intersects an S line just higher than the S value tabulated for component 1, a perpendicular is dropped to the Rjx axis, giving the RI value a t which separation can be achieved. The spreading factor of the second component is inconsequential to the calculation. In order to use this chart for all concentration ranges of the mixtures, a plot of S,as a function of micrograms of component i would be necessary. This refinement has not yet been made. EXPERIMEI’TA L RESULTS
Table I shows the Rf and S values for the various cations in different solvents. The R, valuaf: listed are the mean of several determinations run on strips from different sheets of paper impregnated a t different times. In most cases the Rf value was reproducible to f 0.05; the S values varied by a factor of about 2 for values between 1 and 2. Besides the solvents shown in Table I, the following solvents were also tried: petroleum ether, benzene, ethyl acetate, butyl Cellosolve, trichloroethylene, propylene glycol, carbon tetrachloride, and ether. None of the cations moved in petroleum ether, trichloroethylene, or carbon tetrachloride. Bluminum moved in benzene and ethyl acetate with an R, of 0.84 and 0.82, respectively; however, a large
V O L U M E 23, NO. 11, N O V E M B E R 1 9 5 1
1581
plausible to assume that the spreading factors of these compounds in the alcohols DiPyriChloroAce121-Buare so large as to render the compounds oxane dine form tone Methanol Ethanol Propanol Propanol tanol invisible on the chromatogram. This ex1 1 A1 0 . 6 5 0 . 8 8 0 . 6 5 0 . 9 7 0.79 0.96 planation is partially substantiated by (11.6) (4.5) (19) (9.2) (1.1) 25 69 ‘203:: 57 0.34 8b 0 .. 0 0.90 m i x e d s o l v e n t d a t a . W h e n alcohol(2.6) (2.2) (0.2) 0 0 0 .. 0 Ba 0.68 pyridine mixtures were used as the de8) (8.2) veloping solvent, a t low concentrations of 0.01 0 0.14 0.91 0 0 Cd );( (2.1) pyridine, the iron and lead bands were very 0 0 0.08 42 0.08 0.11 .. Ca (2.0) (9.8) (1.4) broad; but as the concentration of pyri1 0.86 0.70 1 0.99 co 0.94 dine increased, the bands consistently nar(12.5) (11.9) (13.8) (13.6) (4.8) (2,9) (0.3) (7.7) 0 0 0.65 0.68 0.62 0 0.91 0.81 cu rowed down. (3.8) (2.5) (3.2) (2.1) .. .. .. 0.92 .. .. 0.99 Fe Some difficulty was encountered in pre(5.5) (2.4) cisely defining the limits of the lead, cal0 0.55 .. .. .. .. 0.83 Pb (1.6) cium, and barium bands, because of the 0.1 0 0 0 0.6 0 .. h.1 g (12.9) (2.1) (7.3) pale colors of these complexes. Horn ever, 0 . 7 5 0 . 7 1 1 1 0 . 7 9 0 . 8 4 0.93 0 . 7 9 Ni when viewed under ultraviolet light, it nas (3.5) (1.5) (2.5) (1.6) (3.7) (1) (3.2) (2.6) easy to define the limits of the calcium and 5 Decimal values are R f , numbers in parentbesea are spreading factors, S. barium bands. It was found helpful to expose lead chromatograms briefly to hydroTable 11. Separations Rased on R f - S Chart gen sulfide vapors. Rfa R f z from from When 10 micrograms of copper and SI Figure3 Table I Resulta Cations Solvent Rii nickel were spotted on the strips, the Complete separation Fe a n d P b Pyridine 0 , 9 2 (Fe) 0.83 0 . 8 3 (Pb) 2 , 4 (Fe) 0.92 J u s t separated A1 a n d Co Pyridine 1 . 1 (AI) 0.87 (Co) 0.96 (hl) bands had a tendency to form long tails a t Dioxane One band 0 . 9 4 (Co) 0.59 0 . 7 9 (Al) A1 a n d Co 7 . 7 (Co) the rear; however, when 20 micrograms of Incomplete separation 0.78 0.81 (Cu) FeandCu Dioxane 0 . 9 9 (Fe) 4 . 2 (Fe) the cation were used, this difficulty a a s overcome. When spotted from a neutral solution, aluminum, iron, and magnesium left a considerable amount of amount of material was left a t the starting point. Only calciuni moved in butyl Cellosolve with an Rf of 0.24, leaving a large material a t the starting point. By adjusting the p H of the initial solution to the acid side (about 5), this objectionable phenomenon amount of material a t the starting point. Propylene glycol was was eliminated. When magnesium was applied from an acid solumuch too flow a solvent to use, moving only about 5 cm. in 4 tion, it was necessary to expose the strip to ammonia vapors for a days, and ether was too volatile for the type of column used in short time before developing. The drying time and temperature these experiments. were also found to be important in influencing this tendency to The blank spaces in Table I represent instances in which it was “double spot.” It was empirically determined that a drying time impossible, after repeated attempts, to detect a band on the of 1 hour a t room temperature minimized double spotting. chromatogram, even though most of the original material had Copper double-spotted consistently in methanol, regardless of the moved from the starting point. Lead and iron in the alcohol series are the most striking eyamples of this type of behavior. drying time. This effect was eliminated by the addition of pyridine to the methanol. As the complexes of lead and iron are stable in the alcohols, it is In order to verify the validity of the reasoning embodied in the discussion of the theoretical significance of the S value, several borderline cases were picked from Table I and the separations Kere attempted. Table I1 gives the results obtained. The RJ -S chart predicts that iron and lead would just separate in pyridine if the solvent front were allowed to travel 35 cm., and it was observed that they were separated by about 0.5 cm. (40cm. travel). The chart predicts separation of aluminum and cobalt in pyridine, and the aluminum and cobalt bands just touched. .4 consideration of RJ alone would predict that aluW minum and cobalt would be completely separated in dioxane, but 3 J the Rf-S chart predicts otherwise. The results show the 5 R f - S chart to be right. The same holds true for iron and c o p a per in dioxane. The resulta of this experiment justify the reasoning employed within the limits of expected variation in Rf and S values. Figure 4 is a graphical representation of part of Table I. As the alcohol chain length increases, the Rf values for aluminum, nickel, and cobalt also increase, while the Rf values of all other cations decrease. These represent two fundamentally different modes of behavior which are undoubtedly of major theoretical importance. Table I.
Rj and S Yalues of Cations in Various Solvents5
(2i!
%:23 (A:% (A.
(g)
!:(
%:Pi
(0“: 2l
(::2
(2%
-
DISCUSSION O F RESULTS
Figure 4.
ALCOHOL Dependence of Rf on Alcohol Chain Length
The R1 values of the different cations vary in different solvents; some solvents poesess almost specific activity for certain cations, but in these cases double spotting is a major disadvantage. The concept of the spreading factor has been shown t o be of major importance in predicting the outcome of a chromato-
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 Dahti, 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 identifying these pigments. i particular need for 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
