mination of zinc in mixtures containing varying amounts of lanthanum. Separation of 0.25 mmole of zinc from an equal or fivefold lanthanum conccntration is satisfactory on an 8-cm. column, but a 10 to 1 lanthanum to zinc ratio requires a 16-cm. column. Many other bivalent metal ions behave in a manner similar to zinc on elution with an ethylenediammonium solution. Results indicate that 125 ml. or less of 0.1M ethylenediammonium chloride will completely elute any of the following divalent ions from an 8-em. column: calcium, cupric, ferrous, mercuric, magnesium, manganous, nickelom, plumbous, or uranyl. A slight modification of conditions may be required for quantitative separation of some of these ions from trivalent metals. For example, copper(I1) elutes like
zinc using p H 2 eluent, but gives a badly tailed curve when eluted with p H 3 ethylenediammonium chloride solution. Other rare earth ions are eluted more rapidly than lanthanum, but a separation from bivalent ions should still be feasible. Thorium elutes a t a slower rate than lanthanum; therefore, separation of a bivalent metal from thorium is quite easj'. K i t h proper choice of conditions, it should be possible to separate lanthanum and other trivalent metals from thorium. ACKNOWLEDGMENT
The authors wish to express their appreciation to Gerald R. Umbreit who originally suggested the use of the cthylenediammonium ion as an eluting agent.
LITERATURE CITED
(1) Dyrssen, David, Svensk Kern. Tidskr. 62, 153
(1950).
( 2 ) Fritz, J. S., Lane, W. J., Richard, 31. J., ANAL. CHEM.29, 821 (1957). (3) Fritz, J. S., Oliver, R. T., Pietrzyk, D. J., Zbid., 30, 1021 (1958). (4) Hering, H., Anal. Chim. Acta 6, 340
(1052).
( 5 ) Hettel, J. H., R9.S. thesis, Iowa State College, 1056.' ( -, 6 ) Kakihana. Hidetake. J. Cheni. Soc. ~~Japan, Pure Chem. Sect. 72,200 (1951). ( i )Schubert, Jack, Russell, E. R., Farabee, L. B., Science 109, 316 (1949). ['8 ) Sweet, R. C., Rieman, William, 111, ~
\
Beukenkama. John, AKAL.CHEM.24,
RECEIVED for review September 20, 1958. -2ccepted December 22, 1958. Contribution S o . 667 of the Ames Laboratory of the U.S. iltomic Energy Commission.
Acetylacetone as Analytical Extraction Reagent Calculation of Successive Formation Constants from Extraction Data A N O O P KRISHEN' and HENRY FREISER' Department o f Chemistry, University of Pittsburgh, Pittsburgh 7 3, Pa.
b Acetylacetone has been employed as both a chelating agent and solvent in solvent extraction studies. To determine the formation constants, the extraction behavior of seven acetylacetonates was studied. Allowing for the presence of charged intermediates, the successive formation constants for nine acetylacetonates were calculated. These show close agreement with values obtained potentiometrically. In the case of copper(l1) acetylacetonate, theoretical calculation of the extraction curve over the p H range 0 to 5 has produced a curve that is duplicated in an experimentally obtained curve.
S
extraction as a tool of analytical chemistry enjoys a favored position because of its ease, simplicity, speed, and wide scope (14). Acetylacetone (2,4pentanedione) has been the subject of a number of systematic studies (12, IS, 17, 18, 21, 22). In most cases attempts have been made to understand the system by applying theoOLVEKT
Present address, Research Center, The B. F. Goodrich Co., Brecksville, Ohio. Present address, Department of Chemistry, University of Arizon:i, Tucson, Ariz.
retical principles of extraction and chelate formation. In acetylacetoiie-water s p t e m , the formation constants obtained from the extraction data when the existence of charged intermediates in the aqueous phase has been neglected have shown only an approximate correlation with potentiometric measurements. With a better understanding of the nature of species present in the aqueous phase, it has been possible to calculate the successive formation constants for the acetylacetonates. REAGENTS
Commercial acetylacetone \vas purified as described bv Steinbach and Freiser (22). Standard Metal Ion Solutions. The stock solution of comer sulfate was standardized by elecirbdeposition and diluted for use. Pure hafnium metal (loaned by the Stomic Energy Commission) n-as dissolved in aqua regia and the weight of the metal in solution was found by difference. The solution was evaporated t o dryness and the residue taken up in concentrated sulfuric acid. The solution was made up to a suitable volume to give a solution containing i.00 mg. of hafnium per nil. A weighed quantity of uranium oxide, U,Os, n-as dissolved in nitric acid, fumed
n-ith sulfuric acid, and made up to 7-olunie to give a stock solution containing 5.00 mg. of uranium per nil. -4neighed quantity of reagent grade zinc oxide n-as carefully dissolved in dilute sulfuric acid and made up to volume to give a solution containing 10.00 mg. of zinc per ml. A weighed quantity of zirconium oxychloride tetrahydrate was dissolved in water to give a solution containing 2.50 nig. of zirconium per ml. Preparation of Acetylacetonates. Copper, lead, and uranyl acetylacetonates u-ere prepared as described by Iirishen and Freiser (12). A solution of hafnium, prepared by dissolving hafnium metal in aqua regia, was mixed with 10% sodium carbonate solution and acetylacetone a t 15' C. The precipitate that formed on standing was filtered and recrystallized from hot benzene. Melting point, reported value. 194-6' C. ( 7 ) ; found, 194-6" C. Technical iron(II1) acetylacetonnte as obtained from the Union Carbide and Carbon Corp. \vas recrystallized from 95% alcohol and dried for 1 hour at 120'' 'C. Melting point, reported value. 179' C. (6): found, 178-9" C. Technical zinc ' 'acetylacetonate as obtained from the Union Carbide Corp. was recrystallized from hot 95% alcohol. B sample was dissolved in nitric acid and zinc -determined polarographically. Per cent zinc, theoretical. 24.80; found, 24.8. -411 aqueous solution of zirconium VOL. 31, NO. 5, M A Y 1959
923
oxychloride tetrahydrate was mixed with a mixture of 10% sodium carbonate solution and acetylacetone a t 15' C. The resulting precipitate was recrystallized from absolute alcohol. Melting point, reported value, 194.5-6" C. ( 2 ) ; found, 194-6' C.
KC Table 1. [KI = 108.81(8), Ki = 10-8.95, and ( H R ) , = 1.7MI
PH 0 1
2 3
EXTRACTION PROCEDURE
For the equilibrium studies, the organic phase was prepared by dissolving weighed amounts of recrystallized copper(II), iron(III), lead(II), and zinc(I1) acetylacetonates in water-saturated acetylacetone to give about 10-3M solutions in each case. The aqueous phase was prepared by adjusting the pH with sulfuric acid and sodium hydroxide and saturating with acetylacetone. In the case of hafnium, uranium, and zirconium, the metal ion was originally in the aqueous phase and the volume was made up to 25 ml. finally. Ten milliliters of each of the two phases was pipetted into a dry 50-ml. volumetric flask and shaken for 2 hours on a mechanical shaker and a water bath thermostatted a t 25" C. When the extraction was complete, the p H of the aqueous phase was measured with a Beckman Model G pH meter. A suitable aliquot of either of the phases was pipetted out for determination of metal concentration. ANALYTICAL METHODS
The methods for determination of copper, lead. and uranium have been described (19). HAFNIUMAND ZIRCONIUM. A suitable aliquot of the acetylacetone layer was kept over a few milligrams of sodium carbonate to remove traces of acid, then 2 ml. of 1% solution of 8quinolinol in 95% alcohol was added and the absorbance read a t 395 mp against a blank of acetylacetone treated similarly. The amount of the metal was read from standard curves prepared by taking various amounts of the standard solutions of the acetylacetonates in water-saturated acetylacetone. IRO~Y;. The organic phase was filtered through a filter paper and made up to a suitable volume and the absorbance read against water-saturated acetylacetone a t 440 mfi. The concentration was obtained from the standard curve prepared by reading the absorbance of similar solutions containing weighed amounts of iron(II1) acetylacetonate. ZINC. One milliliter of the acetylacetone layer, after extraction, was dissolved in n-ater, its p H adjusted to between 5 and 9, and the volume made up to 100 nil. The solution was shaken with three successive 10-ml. portions of carbon tetrachloride to remove acetylacetone. Ten milliliters of the solution was shaken with 10 ml. of 0.001% diphenylthiocarbazone in carbon tetrachloride and the absorbance of the carbon tetrachloride layer read a t 520 mp using the dithizone solution as the blank. The amount of zinc was determined using the standard curve prepared by taking 1 ml. of various 924
ANALYTICAL CHEMISTRY
w
1st Term 1 10-1 10-4
10-6
Kf
,!O
10-1.5
10-2.5 10-3.5
3!0
i.0
=
formation constant of the che-
(HR), = concentration of the reagent in aqueous phase n = charge on the metal ion
2nd Term 10-0.5
1 I!,
ionization constant for the re-
late
50
d
=
agent
5!0
PH
'
Figure 1. Comparison of calculated and expermental extraction of copper acetylacetonate
concentrations of zinc acetylacetonate in acetylacetone. In the aqueous phase, zinc was determined polarographically using 1M ammonium hydroxide and 1M ammonium chloride as the supporting electrolyte (1). A 0.002% solution of Triton X-100 (Rohm & Haas) was used as the maximum suppressor. The half-wave potential was - 1.33 volts us. S.C.E. The standard curve was prepared by using standard zinc sulfate solution. DISCUSSION OF RESULTS
Under the experimental conditions employed, similar equilibrium status mas attained irrespective of whether the metal species were originally in the organic or the aqueous phase (11,12). In some solvent extraction systems, the course of the chelate extractions may be described by the following equation :
- -E - D = 100 - E where
E = per cent of metal extracted into the organic phase D = distribution ratio of the metal KD, = distribution coefficient
This simplified equation is a very reasonable approximation and gives correct slopes for the extracton curves in dithizone or 8-quinolinol (oxine) extraction systems. I n those cases the value of (HR),, the concentration of the reagent in the aqueous phase, is rather low (about lO-*M) and the ionization constant is also low for oxine K., K,, = 10-14.83 (16). The effect of the presence of charged chelates of type MR+ can be neglected. I n the acetylacetone-water system, the conditions are not comparable. Thus, the concentration of the reagent inwater isvery much higher, 1.7M, the ionization constant is high, , and the distribution coefficients, K D ~are lower. Under these conditions the effect of the presence of charged intermediates cannot be neglected. The extraction equation must be modified to give:
D =
(2) To get a curve which conforms to Equation 1, the second and the subsequent terms in the denominator of Equation 2 have to be neglected. Considering the case of a simple divalent ion like copper(II), where the denominator has only two terms, the deviation from Equation 1 becomes evident (Table I). The second term becomes progressively more important as pH increases, so that beyond a p H value of 1, it is the first term that may be neglected. I n the case of copper acetylacetonate, the log of the over-all formation constant has a value of 15.16 @), the K D , has been found to be 6.5, so that it is possible to calculate the theoretical value for extraction a t various pH Values. These values in the p H range 0 to 5.0 were calculated on the basis of both Equations 1 and 2. A comparison of these two theoretical curves with the experimentally obtained curve (Figure 1) makes clear the need for considering the complete equation. Using Equation 2 this not only gives the correct slope throughout the pH range, but the theoretical and the experimental curves are found to be identical within the experimental errors. When other metal ions are considered, where more than one intermediate charged chelate can exist, the effect of each succeeding term in the denominator becomes increasingly more important even a t lower pH values. Thus, deviations from the slope calculated from
Equation 1 were found in each case, the limiting value of the slope being attained in only a few cases. The slope was found to be nearer that calculated for a monovalent metal in most cases. This observation is supported by results reported by Steinbach (20-22). In the cases he considered, even where the slope was close to the calculated value, the agreement was reasonable in only a limited range of p H values. Rydberg (16) studied the extraction of thorium(1V) acetylacetonate with benzene using significantly lower concentrations of acetylacetone in water. The decreased reagent concentration should decrease the value of the second and the succeeding terms in the denominator of Equation 2. However, close agreement between the calculated and experimental values of extraction was possible only when the presence of the charged chelates was taken into account. Distribution Coefficients and Stability Constants. To calculate the for-
mation constants for the acetylacetonates, the distribution equation may be rearranged to give: K i . . .,, =
+
+
D(H+)" Ki(HR)wKi(H+)"-l K i . . . (-1) (HR),n-lKi*-' (H+)
....
+
( K D ,- D)(HR)w" Kin
Table
II.
Approximate Formation Constants for Acetylacetonates
K D~
Ion PHin .4hminum( 111)~ 1.75 0.67 Beryllium(I1)a 1.10 Copper(I1) Gallium(111) 1.20 Hafnium(IV) 1.75 Iron(II1) 0.07 5.65 Lead(I1) 1.66 Uran 1 zinc&) 5.30 Zirconium(IV) 1.50 From Steinbach (10). * Assumed value.
1% K l 19.9 14.5 14.5 21.0 27.0 21.0 6 13.9 5 28
9.5 41 6.5 28.6 6.6
100Ob
4.9 65.5 1.8 2.5
Comparison 22.3 (9) 14.63 (8) 15.16 (8) 23.6 (9) 26.2 (9) 14.15 (8) 8.81 (10)
0
Table 111. Formation Constants for Acetylacetonates (25"C.) Ion Log Ki Log Kz Log K J Log Kd Log K f 21.51 Aluminum(111). 8.25 7.35 5.91 22.3 (9) Comparison (30" 7.9 5.8 8.6 14.54 6.37 Beryllium(11)a 8.17 14.63 (8) Comparison (20' 7.88 6.75 15.80 Co per(I1) 8.96 6.84 15.16 (8) 8omparison (200 8.31 6.85 21.56 6.29 7.79 Gallium(111). 7.48 23.6 (9) Comparison (30 5.7 9.5 8.4 Hafnium(IV) 6.35 28.10 6.38 6.64 8.73 23.8i 8.71 9.27 Iron( 1II)b 5.89 26.2 (9) Comparison (30" 7.4 9.8 9.0 7.00 2.49 4.51 Lead(1) 14.13 Uranvl 8.87 5.26 14.15 (8) Cokparison (30" C.) 7.66 6.49 Zinc(I1) 5.06 3.66 8.72 Comparison (30" C.) 4.98 3.83 8.81 (10) Zirconium(1V) 8.38 7.58 7.26 6.86 30.08 Calculated from experimental data from Steinbach (20). * Based on assumed value of KD, = 1000.
(3)
At 50% extraction, the distribution ratio, D,equals unity. If as an approximation, all terms except (H+)" are neglected in the numerator, an approximate value for the over-all formation constant for the acetylacetonates may be calculated from the experimental value of pH,,, (the pH for 50% extraction) and the maximum extraction reached, KD,. The values for the formation constants calculated in this manner are shown in Table 11. Although the conditions of temperature, metal ion concentration, and ionic strength are not identical, some agreement is apparent between the values of the formation constants obtained from the extraction and potentiometric (8-10) methods. I n the case of iron(II1) acetylacetonate, the exact value of K D ,cannot be determined as the extraction approximates loo%, and some very sensitive technique of analysis, like that of radiometric estimation, would have to be utilized for determining the ratio of the metal in the organic and the aqueous phases. It was through the use of this technique only that Rydberg (16) was able to calculate the K D , for thorium acetylacetonate in a water-benzene system. In water-acetylacetone system, similar limitations affect the determination of K D ,for cerium(III), indium(III), and thorium(1V) acetylacetonates (11,WO).
The value of KD,, in cases where it is low, would be determined with sufficient precision by the extraction technique to permit calculation of the formation constants. The effect of the presence of the charged chelates would reasonably be more important where KD, is low. However, even in the case of thorium acetylacetonate, where the KD, is high [315 30 in water-benzene system (IS)], the presence of the intermediate charged chelates cannot be neglected (19) and the approximate equation does not give correct values for the formation constants. Similar observations have been made in other systems (3-6). Where the value of K D ,has been determined, the stepwise formation constants can be calculated if the complete equation is applied. There would be n number of unknowns for a metal ion with a charge n, due to the successive K,. formation constants, K 1 , Kz, To get those values, n equations would be required. These can be obtained from the extraction curve by finding the distribution ratio, D, at different pH values. Mathematically, it is possible to call) culate even K D , by utilizing (n equations. However, the precision of the experimental data does not justify the calculation of this quantity. Thus in the case of iron(II1) acetylacetonates, varying values of K D , were obtained
+
... .
+
when data from different experimental points were used. Slight variation in the extraction value from say 99.6 to 99.9% alters the K D ,value from 249 to 999.
In the case of zinc acetylacetonate, the KD, is only 1.8, pH112is 5.3, and the value obtained from the extraction data is considerably lower than that obtained from the potentiometric measurements (Table 11). Steinbach (20) had considered this K D ,to be erroneous because of the possibility of hydrolysis of zinc. However, the acetylacetonate can be expected to be more stable than the hydroxide and even a t p H 7, the relative concentrations of the hydroxyl ions (10-7M) and acetylacetonate (10-1.7M) would favor the formation of the acetylacetonate. As such the value of 1.8 for K D , for zinc acetylacetonate cannot be considered erroneous and the major factor to be considered is the presence of the intermediate chelates. Taking two values of extraction a t different pH values (D = 1.00, pH = 5.30; D = 0.25, p H = 4.35)from the curve obtained experimentally, the formation constants for the chelate and the intermediate have been calculated. These calculations give a value of 8.7 for the logarithm of the over-all formation constant, R,, while log K I and log Kz are 5.1 and 3.7, respectively. These values may be compared with the values obtained potentiometrically-8.81, 4.98, and 3.83 (10). VOL. 31, NO. 5, MAY 1959
925
When metal ions with an oxidation number of 3 or 4 are considered, the solution of the various equations requires the application of determinants. These calculations have been made for a number of acetylacetonates (Table 111). On allowing for the intermediates, the maximum change in log K , (f3.78) took place in the case of zinc where the KD,is the lowest (1.8) and pHlla is the second highest (5.30) and the least changes, +0.04 and f0.23, took place in the case of beryllium and uranyl where the K D , values are the highest (41 and 65.7) and the pH1,a among the lowest (0.67 and 1.66). Thus the effect of the presence of charged intermediates is enhanced when KD,is low or the extractions take place at higher pH values. In addition to the presence of charged chelates, the theoretical calculations will be affected by hydrolysis and changes in the complexity of the molecular species. However in the system studied, the close agreement between the formation constants obtained from extraction and potentiometric studies (Table HI), suggests that interference from such causes is negligible. I n the case of zinc(I1)
acetylacetonate, hydrolysis can be ruled out as a factor of any consequence. The low concentrations of metal ions ( l O - a M ) used in this study, along with the fact that the extraction behavior of copper(I1) acetylacetonate has been correctly predicted (Figure 1) from the value of the formation constants obtained independently by potentiometric methods, indicate that increases in molecular complexity, if any, are not significant.
(8) Izatt, R. &I., Fernelius, W. C., Block, B. P., J . Phys. Chem. 59, 235 ( 19551. (91 I z a h , R. M., Fernelius, W. C., Haas, C. G., Block, B. P., Zbid., 59,170(1955). (10) Izatt, R. &I., Haas, C. G . , Block, B. P., Fernelius, W. C., Zbid., 58, 1133 (1954). (11) Krishen, Anoop, Ph.D. thesis, Uni-
ACKNOWLEDGMENT
vent Extraction in Analvtical Chemistry,,, Wiley, New York, "1957. (15) Nasanen, Reino, Lumme, Paavo, Mukula, A. L.. Acta Chem. Scand. 5,
The authors gratefully acknowledge the financial assistance of the V. S. Atomic Energy Commission. LITERATURE CITED
(1) Allsopp, K. E.,Arthur, T.E.,ASAL. CHEY.23, 1883 (1951). ( 2 ) Biltz, W., Clinch, J. A, 2. anorg. Chem. 40, 218 (1904). (3) Dvrssen. David, Svensk Kern. Tidskr. 68.212 f1'956). (4) Dyrssin, hfargareta, Rec. trav. chiin. 75, 748 (1956). (5)'Eck, C . L. van P. van, Zbid., 72, 529 (1953). (6) Hantzsh, A., Desch, C. H., Ann. Chem. Liebigs 308, 1 (1902) ( i )Hevesy, G. von, Logstromp, 11..Ber. deut. chem. Ges. 59, 1800 (1926). '
.,
versity of Pittsburgh, Pittsburgh, Pa.,
1957. (12) Krishen, h o o p , Freiser, Henry, ANAL.CHEX 29, 288 (1957). (13) McKaveney, J. P., Freiser, H., Zbid., 29, 290 (1957). (14) Morrison, G. H., Freiser, H., "Sol-
1199 (1951). (16) Rydberg, Jan, Zbid., 4, 150 (1950). (17) Rydberg, Jan, Arkiv K e m i 8, 113 (1955).
(iSj z& d., 9, 95 (1955).
(19) Rydberg, J a n , Svensk Kem. Tidskr. 67, 499 (1956). (20) Steinbach, J. F., Ph.D. thesis, University of Pittsburgh, Pittsburgh, (2
CHEhl. 25, 881 (1 (22) Ibid., 26, 375 (23) Van Uitert, L.,
vania State Colle,,, RECEIVEDfor review August 7, 1958. Accepted December 8, 1958.
Polychromatic Technique for the Identification of Amino Acids on Paper Chromatograms EDWARD D. MOFFAT' and RALPH 1. LMLE Division of Biochemistry, Naval Medical Research Unit No. 4, U. S. Naval Training Cenfer, Great lakes, 111.
A simplified polychromatic technique was developed to study the amino acid composition of protein hydrolyzates; the previous two-dimensional chromatographic systems were not entirely satisfactory, because of the time consumed and the inherent variables encountered in determining the reproducibility of Rl values. Twenty common, naturally occurring amino acids in amounts as low as 1.2 +y were completely differentiated after 6 hours of resolution when sprayed with ninhydrin-cupric nitrate (N-CN) and were readily identified by their characteristic color complexes. This method was equally satisfactory in the detection of single amino acids or mixtures on a unidimensional chromatogram in n-butyl alcohol-acetic acid-water (4: 1 :5 ratio) solvent system, although some possessed only slightly different R,'s-i.e., leucine-isoleucine. 926
ANALYTICAL CHEMISTRY
P
chromatographic techniques have contributed much toward simplifying the analysis of numerous multicomponent systems, such as mixtures of sugars, amino acids, and inorganic ions (4). Many methods for the identification of amino acids on a paper strip chromatogram are satisfactory. However, this report presents a simplified procedure that was developed in these laboratories. Investigations have recently been conducted to find a selective indicator to identify amino acids. Many indicators containing ninhydrin have been used, but with the exception of the yellow color of proline, they do not differentiate shades of red, violet, and purple. The excellent procedure of Hardy et al. was not applicable to the present problem because of its limited application (identification of 11 commonly occurring amino acids), such as the differentiation of leucine from isoAPER
leucine ( G ) . I n the quest for a polychromatic method for the identification of amino acids, released from a protein moiety, the following indicators were employed: 0.25% w./v. ninhydrin in acetone ( l 7 ) , 0.3% ninhydrin in 95% ethyl alcohol ( l a ) , 0.201, ninhydrin in water-saturated n-butyl alcohol ( 6 ) , 4% ninhydrin in pyridine (I@, and the method reported by Levy in 1953 ( 8 ) . Sone of these reagents proved to be as selective t o color formation as the reagent described, which is a modification of the ninhydrin-copper complex method cniployed by Levy and Chung (5). EXPERIMENTAL
TKO standard amino acid mixtures containing 10 amino acids in each were 1 Present address, Grove Laboratories, I X ~ C8877 . ~ 1,adue Rd., Clayton 24, Mo.
