Anion-Exchange Chromatography of Some Aromatic Organic Acids in Ferric Chloride-Organic Solvent Medium Kil Sang Lee, Dai Woon Lee, and Euy Kyung Yu Lee Department of Chemistry, Yonsei University, Seoul, Korea The elutions of some aromatic organic acids from a column of Dowex 1-X8, chloride form, have been studied by using ferric chloride in organic solvent as the eluant. Several separations of organic acids were performed using eluting solution suggested by the distribution data. The concentrations of organic acids in the eluates were determined by spectrophotometry.
ANIONEXCHANGE CHROMATOGRAPHY has been widely used for the separations of organic acids. Aliphatic and aromatic organic acids (94 organic acids, including tartaric, citric, benzoic, and salicylic acid) have been eluted with formic acid (0 to 25M) from a column of Dowex 1 in the formate form by C. Davies, R. D. Hartley, and G . J. Lawson ( I ) . N. E. Skelly and W. B. Crummett (2) have used an acetic acid-methanol solution for the separation of the isomers of hydroxybenzoic acid with Dowex 2-X8, acetate form. On the other hand, anion-exchange chromatography in metal solution media which can react with organic acids adsorbed on the resin to form strong non-adsorbable complexes has been used in the separation of organic acids. Zinc acetate solution has been employed as a useful eluant (3) in the separation of a number of organic acids, such as galactonic, mannonic, and gluconic acid. K. S. Lee and 0. Samuelson (4) examined magnesium acetate as an eluant in the separation of various organic acids, including oxalic, malic, galactaric, and maleic acid, with Dowex I-XS, acetate form, and found this method was valuable. 0. Samuelson and B. Johnard have used a copper acetate eluant which forms a colored complex band when the lactic and glycolic acids are separated. In this paper, however, the analysis of the eluant has been carried out by an oxidation method (5). In earlier papers (1-5)it has been shown that aromatic acids, such as 2-hydroxybenzoic acid, are adsorbed strongly by the anion exchange resin and thus a greater concentration and volume of eluant solution and a longer time are required to elute such acids. In addition, the procedure for the analysis of the eluate is not simple. In the present work, a ferric chloride-organic solvent solution which reacts with some aromatic organic acids such as salicylic and aromatic hydroxamic acids to form colored stable non-adsorbable complexes was tried as the eluant for the separation of organic acids. The elutions were carried out with the chloride form of Dowex 1-X8, 200-400 mesh. The elution behavior of organic acids adsorbed on the resin could be observed visually as organic acids reacted with the ferric ion in the eluant to form colored zones of Fe(II1)-organic acid
(1) C. Davies, R. D. Hartley, and G. J. Lawson, J. Chromatogr., 18, 47 (1965). ( 2 ) N. E. Skelly and W. B. Crummett, ANAL.CHEM.,35, 1680 (1963). (3) T. Isaksson, U.-B. Larsson, and 0. Samuelson, Acta Chem. S c a d . , 20, 1965 (1966). (4) K. S . Lee and 0. Samuelson, Anal. Chim. Acta., 37,359 (1967). (5) 0. Samuelson and B. Johnard, Su.Kem. Tidskr., 73,586 (1961). 554
ANALYTICAL CHEMISTRY, VOL. 42, NO. 6, M A Y 1970
complexes on the resin. It was not necessary, therefore, that all of the column effluents be collected and analyzed because the colored eluate-containing complex could be separated from the other eluate fractions. The concentration of organic acids could be determined colorimetrically without any treatment of the effluents. Compared with the methods mentioned above, therefore, this present process is much less tedious, far simpler in procedure, and less time-consuming for the elution and analysis. EXPERIMENTAL
Resin and Reagents. A strongly basic anion-exchange resin, Dowex 1-X8 (200 to 400 mesh), was washed with methanol, then converted to the chloride form by passing a large excess of aqueous 5M KC1 through a column of the resin. After rinsing with demineralized water and then with methanol, the resin was air dried. Stock solutions of organic compounds (0.02M) were prepared by dissolving organic compounds (salicylic, 5-nitrosalicylic, 5-sulfosalicylic, salicylohydroxamic, benzhydroxamic acid, acetylacetone, and oxine of analytical reagent quality) in a water-methanol solution. Ferric chloride solutions for column elution were prepared by adding measured volumes of water and organic solvent to weighed amounts of FeCl3.6HZO. These eluants must be freshly prepared when needed in order to prevent the hydrolysis of the ferric chloride. Salicylohydroxamic acid was prepared by the following procedure. HONH3Cl(72 g) is added to 250 ml of methanol, refluxed, mixed with 120 g of KOH in 200 ml of methanol, the KC1 filtered off, the filtrate treated with 100 ml of methylsalicylate, and the mixture left overnight. Then CU(OAC)~ solution is added, the dark-greenish precipitate dispersed in methanol, decomposed with HzS, and the mixture filtered and evaporated to give salicylohydroxamic acid, m.p. 165 ==I 2 "C. Benzhydroxamic acid (130 f 2 "C) was prepared by same method. These hydroxamic acids were verified by UV and IR spectrophotometry. Procedure. The chromatographic column was a glass column (52.5 cm X 0.6 cm diam.) filled with Dowex 1-X8 resin. The column was washed with water and then with a water-organic solvent identical with the eluant solvent. The surplus water-organic solvent was drained from the column until the liquid level was about 1 mm above the top of the resin. Two tenths ml of the organic acid solution to be separated was then forced into the column using nitrogen gas: The liquid was again drained to within 1 mm of the resin. Then the inside wall of the column was rinsed with about 0.5 ml of organic solvent and the liquid again drained almost to the resin. This rinsing and draining was repeated twice more. A supply of eluant was then connected to the top of the chromatographic column by a technique employing a Beckman Accu-Flo pump. A flow rate of 0.2 to 1.0 ml per minute was maintained throughout the elution. After the organic acids were completely eluted, the column was washed first with dilute hydrochloric acid (about 0.02M) and then with water in order to remove any ferric chloride remaining in the column. If the column is washed with water only, hydrolysis of the ferric chloride may occur.
Table I. Volume Distribution Coefficients of Some Organic Acids and Compounds in Various Concentrations of Methanol, 0.05M FeC13, with Dowex 1-X8, C1- Form, 200- to 400-Mesh Resin. Flow Rate: 1.0 ml/min Methanol, Acid and compound HnO 20 30 50 65 80 90 Salicylic acid 0.82 0.28 0.28 O.Sb 3.994 1.29” 0.28~ 5-Sulfosalicylic acid 7.83 4.33 3.65 3.59 4.53 5-Nitrosalicylic acid 1.83 1.03 Salicylohydroxamic acid 2.04 1.03 0.35 0.22 Benzhydroxamic acid 0.15 0.05 0.08 0. 25b 0.02 Oxine 0.15 0.08 Acetylacetone 0.08 0.02 0.02 0.05b Eluant: 0.01M FeC13. Flow rate: 0.25 mlimin.
-
RESULTS AND DISCUSSION
Nature of the Fe(Il1)-Organic Acid Complexes. Salicylic 5-nitrosalicylic (7), and 5-sulfosalicylic acid (8) react with acidic ferric chloride solution to produce colored ferric complexes. Oxine (8-hydroxyquinoline) reacts with the ferric ion, in acidic organic solvent-water solution, to give the dark green ferric oxinate complex (9). The application of the ferric acetylacetonate complex (10) in organic solvent to the determination of iron(II1) has been studied. It has been reported that in solutions with pH values less than 2.0-2.5, Fe(II1) and acid (salicylic, 5-nitrosalicylic, 5-sulfosalicylic, salicylohydroxamic, and benzhydroxamic) react in a 1 :1 ratio to form a stable complex. It can be seen from the results that the formation of a colored non-adsorbable complex between the ferric ion and the organic acid leads to consequent reduced sorption on the anion resin (lower Dc). The authors suggest that the stabilities of the complexes in the eluant solution, the ionization constants of the acids and other factors may have considerable influence on the elution pattern. But there seems to be no clearly defined general relationship between the factors ( L e . , stability and ionization constants) and the elution behavior. Elution of Organic Acids and Compounds. The peak elution volumes (D) were determined from a great number of runs in a 0.05M ferric chloride solution containing organic solvents with single acids or with mixtures of acids. From these values the volume distribution coefficients (Do)were obtained from the equation (11): (6),
e/X’
=
DU + e
where X ’ is the corrected column volume and e the void fraction of the column. In this calculation, the authors used the value ( E 0.390) for the void fraction of the column as measured by Materova (12) for Dowex 1-X8 resin, 200-400 mesh. The eluant concentration was kept at 0.05M and the percentage of methanol was varied between 0 and 90%. The (6) A. Agren, Acta Chem. Scand., 9,49 (1955). (7) 2. L. Ernst and J. Menashi, Trans. Faraday SOC.,59, 2838 (1963). (8) A. Agren, Acta Chem. Scand., 8,266 (1954). (9) A. Albert, Biochem. J., 54,646 (1953). (10) T. Shigematsu and M. Tabushi, Japan Analyst, 8, 710 (1959). (11) 0. Samuelson, “Ion Exchange Separations in Analytical Chemistry,3’Almquist and Wiksell, Stockholm; Wiley, New York, 1963, p 126. (12) E. A. Materova, Z . L. Vert, and G. P. Grinberg, J . Gen. Chem., USSR,24,957 (1954).
0.5
0.4 0 U )
5
2c
a3
v)
2
0.2
0.1
0.0
0
10
20
30
50
40
Eluate Volume,
60
70
ml.
Figure 1. Influence of various organic solvents on curve of 5-nitrosalicylic acid 1. O.05M FeC13-50Z propanol 3. 0.05M FeCl&OZ methanol
2. 0.05M FeC13-50x acetone 4. 0.05M FeC13-50x ethanol
results obtained with five organic acids and two organic compounds are shown in Table I. The elution of these organic acids in aqueous eluant is extremely tedious and the elution curves are very broad. When a water-miscible organic solvent is added into the eluant, the curve becomes narrower and sharper. This fact is demonstrated in the elution of salicylic acid where elution with 0.01M FeC13aqueous solution gave 15 ml to 112 ml of eluant (Table I); with 0.01M FeCl3-3Oz methanol, between 10 ml and 42 ml; and with 0.01M FeC13-50% methanol, between 6.0 ml and 20 ml. It has generally been observed that the Du value decreases with increasing methanol percentage (Table I). However, this is not always the case. In the case of 5-sulfosalicylic acid, the Du value for 80% methanol is higher than that for 65%. The small difference in Du values between the methanol solutions and the aqueous eluant in the cases of benzhydroxamic acid, oxine, and acetylacetone shows that these are eluted so quickly that the concentration of the organic solvent has negligible effect on the elution. The effect of the nature of the organic solvent on the elution is seen in Table I1 and Figure 1. In the present work, the ANALYTICAL CHEMISTRY, VOL. 42, NO. 6, MAY 1970
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I
I
I
I
I
I
I
I
I
10
20
BO
A0
50
60
70
80
90
Table 11. Effect of Various Organic Solvents on Volume Distribution Coefficient, Dv 0.05M FeC18-50% organic solvent, with Dowex 1-XS, C1- form 200- to 400-mesh resin. Flow rate: 1.0 ml/min Acid and compound Methanol Ethanol Acetone Propanol Salicylic acid 0.55 0.62 0.69 0.35 5-Sulfosalicylic acid 3.56 4.06 4.19 3.18 5-Nitrosalicylic acid 1.83 2.24 1.63 1.03 0.05M FeCI3-20%organic solvent, with Dowex 1-XS, C1- form 200- to 400-mesh resin. Flow rate: 1.0 ml/min
Salicylohydroxamic acid Benzhydroxamic acid Oxine Acetylacetone
1.03 0.02
0.15 0.02
1.16 0.05 0.15 0.02
0.82 0.08 0.15
0.69 0.02 0.12
0.02
0.02
0
Eluate Volume, %I. Figure 3. Separation of benzhydroxamic(B), 5-nitrosalicylic (Nz)and 5-sulfosalicylic acid@) by elution with 0.05M FeCls8 0 x methanol. Flow rate: 0.75 ml/min. Separation of salicylohydroxamic acid(SH) and 5-nitrosalicylic acid(NJ by elution with 0.05M F e C l d O Z ethanol, flow rate: 0.6 ml/min 0.7
0 . 0 5 ~ PCl,-
4
80% Methanol ( 0 - 5 )
7
0
0
10
20
30
40
50
60
70
80
90
Eluate Volume, ml. Figure 2. Separations of salicylic (SI)and 5-sulfosalicylic acid(Su), acetylacetone(A) and salicylic acid(&) by elution with 0.05M FeC1,-methanol S1---Su: 50% Methanol, flow rate: 1.20 ml/min A- - - -SZ: 90% Methanol, flow rate: 0.2-0.3 ml/min
elution order of organic acids is as follows: propanolmethanol-ethanol. No relationship was found with acetone. As shown in Tables I and 11, the Du values cf benzhydroxamic acid, oxine, and acetylacetone are very small. Therefore, to examine the degree of adsorption of these compounds on the resin and to determine whether these organic compounds can be eluted by an eluant containing no ferric ions, the elutions were performed using a 20% ethanol-water solvent. These investigations revealed that among these three compounds only acetylacetone can be eluted within the 10 to 18-ml volume range of the ethanol-water solvent. It can, therefore, be concluded that acetylacetone is adsorbed weakly on the resin so that the formation of ferric acetylacetonate does not affect the Du value to a great extent. Separations. From the results presented in Tables I and I1 the separation of mixtures of organic acids and organic compounds have been considered. In the separation of a mixture of salicylic and 5-sdfosalicylic acid (0.2 ml of the mixed solution containing 2.76 and 10.17 mg of the respective acids) salicylic acid was separated quantitatively from 5-sulfosalicylic acid by elution with a 0.05M FeCIr-50% methanol solution (flow rate: 1.2 556
ANALYTICAL CHEMISTRY, VOL. 42, NO. 6, MAY 1970
0
(0
20
30
40
50
60
70
80
90
Eluate Volume, mi. Figure 4. Separation of oxine(0) and 5-sulfosalicylic acid (SI, SZ)by stepwise elution with FeCl,-organic solvent ml/min). This elution curve is shown in Figure 2. The data show that with the same conditions, including flow rate, there is a slight overlapping of the elution curves corresponding to acetylacetone or benzhydroxamic acid and salicylic acid. This overlapping can be eliminated by increasing the methanol content (90%) and decreasing the flow rate (0.2 to 0.3 ml per min) and concentration of the mixture. As shown in Figure 2, acetylacetone and benzhydroxamic acid (not shown on the elution curve to avoid complication) can thus be separated from salicylic acid. The separation of 3.06 mg of salicylohydroxamic acid or 4.35 mg of oxine (not shown in Figure) from 3.66 mg of 5-nitrosalicylic acid with 0.05M FeC13-50 % ethanol is presented in Figure 3. This separation is possible only when
~
~
~
_
_
_
_
~
Table 111. Absorption Maxima (mp) of Organic Acids and Organic Compounds in 0.05M FeC13-Organic Solvent
Methanol, Acid and compound Salicylic acid 5-Sulfosalicylic acid 5-Nitrosalicylic acid Salicylohydroxamic acid Benzhydroxamic acid Oxine Acetylacetone
Ethanol,
50
80
90
550 520 530 570
560 530 530
580
560 660 510
570
660
ethanol is used as the solvent of the eluant because in the cases of the other solvents the elution bands of the two acids overlap, as suggested from the elutions of 5-nitrosalicylic acid shown in Figure 1. The chromatogram shown in Figure 3 demonstrates the separation of three acids using a 0.05M FeC13-80 % methanol. Acetylacetone (not shown in Figure 3) which has almost the same elution band as benzhydroxamic acid is eluted first, then 5-nitrosalicylic acid, followed by 5-sulfosalicylic acid. The time required for this separation was about two hours. The data show that 5-sulfosalicylic acid is eluted much later than other acids such as oxine, acetylacetone, benzhydroxamic, and salicylohydroxamic acid and it is convenient therefore to speed up its elution by increasing the eluant concentration and changing the eluant solvent after the other acids have been eluted. An example of this stepwise elution is demonstrated in Figure 4. Spectrophotometric Determination of Organic Acids and Compounds. During the elution of an organic acid and compound through the column, the acid or compound forms a
530
50
Propanol, 90
50
580 530 520 570 540 670
550
530 510 540
490
colored ferric organic acid (or compound) complex on the resin and then this colored band is eluted. The colored fractions of the eluate are collected and analyzed by measuring the absorbance against an eluant blank at the wavelength of the Fe(III)-organic acid (and organic compound) complex. It was observed, as shown in Table 111, that the absorption maxima of the complexes varied slightly with change in ratio of organic solvent to water and with change of organic solvent. The concentration of organic acid and compound was calculated from calibration runs with standard solutions. Results obtained from the analysis of standard solutions containing 5 mg of organic acids and compounds gave an average recovery of 99.24 % of the acids and compounds with a standard deviation of 0.39. RECEIVED for review November 10,1969. Accepted February 2, 1970. This work was financially supported by the Ministry of Education of the Republic of Korea to which the authors express their gratitude.
Determination of Phosphides and White Phosphorus in Biological Materials by Neutron Activation Analysis S. S. Krishnan and R. C. Gupta' The Centre of Forensic Sciences, Province of Ontario, 8 Jarvis Street, Toronto 2, Ontario, Canada
A method for the determination of nanogram amounts of toxic phosphorus, ;.e., phosphides and white phosphorus, in biological materials has been developed. The technique consists of distillation of phosphorus as phosphine and collection of the phosphine as silver phosphide by reaction with silver nitrate. The silver phosphide is then oxidized to silver phosphate by chlorine and the amount of phosphorus is determined by thermal neutron activation analysis. A radiochemical separation procedure involving ionexchange and precipitation methods is used for the separation of phosphorus-32 activity induced during the neutron activation. The technique can be adapted to differentiate phosphine which is produced as a product of putrefaction in tissues, metal phosphides, and white phosphorus. The method is capable of uniquely identifying phosphorus and is sensitive down to 10 ng of phosphorus. It enables quantitative analysis of phosphorus at nanogram levels with an accuracy of +lo%. This constitutes an important advance for applications in the medico-legal field. Requests for reprints should be addressed to Dr. R. C. Gupta, Office of the Chief Medical Examiner, Los Angeles, Calif., U. S. A.
THEDETECTION of toxic phosphorus, i.e., phosphorus in the form of metallic phosphides such as zinc phosphide or white phosphorus, in liver or other biological materials is of toxicological importance especially in medico-legal cases. While several methods are available ( I ) for the determination of natural phosphorus existing in biological materials as phosphates, no quantitative procedure has yet been reported for the determination of toxic phosphorus when occurring in nanogram quantities. Curry et al. ( 2 ) have described a procedure for qualitative detection of toxic phosphorus in biological materials. In using their method, extreme caution and extensive experience is necessary in recognizing the positive color reaction if only nanogram amounts of phosphorus are present. (1) W. T. Mullins and G. W. Leddicotte, NAS-NS Report 3056, Dept. of Commerce, U. S. Government Printing Office, Washington D. C., March 1962. (2) A. S. Curry, E. R. Rutter, and L. Chin-Hua, J. Pharm. Pharmacol., 10, 635 (1958).
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