Chromatographic Separation and Determination of Dicarboxylic Acids


1952. 491 mixture, the color reactions would indicate malonic acid to the exclusion of the other two acids. In practice the technique should be used a...
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V O L U M E 24, NO. 3, M A R C H 1 9 5 2 mixture, the color reactions would indicate malonic acid exclusion of the other two acids. In practice the technique should be used as follows:

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Develop a chromatogram of the unknoffn material and spray with bromophenol blue. Calculate the RF values for each spot. From these RF values, select acids for standards of comparison which have corresponding RF values. Prepare new chromatograms in quadruplicate, so that each standard acid is run adjacent to an unknown mixture. After drying the paper sheets, spray each chromatogram with the appropriate color-producing reagent and treat and observe as described under “Materials and Reagents.” ACKNOWLEDGMENT

The authors sincerely thank Eric Baer, University of Toronto, for furnishing the sample of glyceric acid, J. W. E. Chattfield, University of Chicago, for furnishing the samples of a, 0-, and a,-pdihydroxybutyric acids, and various members of the laboratory staff for supplying other acids. LlTERATURE CITED

(1) “Allen’s Commercial Organic Analysis,” 5th ed., Vol. I, p. 103, Philadelphia, P. Blakiston’s Son & Co., 1923.

Ibid., p. 769.

Allport, K. L., “Colorimetric Inalysis,” p. 410, London, Chapman and Hall, 1947. Boggs, L., Cuendet, L. S., Ehrenthal, I., Koch, R., and Smith, F., Nature, 166, 520 (1950). Brauer, Kurt, Chem.-Ztg., 44,494 (1920). Casares-Lopez, R., Biochem. Z., 284, 365 (1936). Ciusa, R., and Piergallini, A.. Gazz. chzm. ital., 45 (I),59 (1915). Feigl, F., and Frehden, O., Mikrochemie, 18, 272 (1935). Fenton, H. J. H., J . Chem. Soc., 6 5 , 899 (1894). Jaffe, E., Chimica ( M i l a n ) , 4,307 (1949). Lugg, J. W.H., and Overell, B. T., Australian J . S c i . Research P h y s . S c i . , 1, 98 (1948). Partridge, S. hI., N a t u r e , 158, 270 (1946). Partridge, S. M., and TTestall, R. G., Biochem. J . , 42, 238 (1948). Porter, W.L., Buch, M. L., and Willits, C. O., Food Research, 16, 338 (1951). Shriner, R. L., and Fuson, R. C., “Systematic Identification of Organic Compounds,” 3rd ed., p. 96, New York, John Wiley & Sons, 1948. Sumner, J. B., and Sisler, E. B., A r c h . Biochem., 4, 333 (1944). Rillits, C. O., and Porter, W. L., U. S. Dept. Agr., Bur. Agr. Ind. Chem. (Eastern Regional Research Laboratory), AIC268 (April 1950). RECEIVED for review August 15, 1951.

Accepted Sovember 19, 1951.

Chromatographic Separation and Determination of Dicarboxylic Acids, C to C 4

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TAKERU HIGUCHI, School of Pharmacy, University of Wisconsin, Madison, Wis.,NORIMAN C. HILL, T h e C. P . Hall Co., Akron, Ohio, AND GERALDINE B. CORCORAN, T h e C. P. Hall Co. of Illinois, Chicago, 111. Chemical oxidation of fatty acids yields mixtures of straight-chain dicarboxylic acids, but no analytical method was available for determination of the component acids. A n analytical method was developed suitable for routine determination of sebacic, azelaic, suberic, pimelic, adipic, and lower straight-chain dicarboxylic acids in mixtures containing any or all of these acids. The method is based on chromatographic separation of the acidic components on a partition column having pH 5.40 citrate buffer as the internal phase for separation of acids above adipic and water for separation of acids below adipic.

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OMMERCIAL “azelaic acid” and a number of other straight-chain dicarboxylic acids are now being prepared commercially by oxidation of fatty acids. T h e crude reaction products from such reactions are mixtures of a number of dicarboxylic acids of varying chain lengths. A search of the literature disclosed, however, only a few references (2, S) to possible methods of determining the acidic components. Recently Marvel and Rands (1) showed that it was possible to separate certain straight-chain dicarboxylic acids by partition chromatography with water as the stationary phase. T h e method as presented, however, is not suitable for convenient and quantitative analysis of mixtures of widely differing composition, especially those containing large proportions of higher dicarboxylic acids. Because the higher acids are relatively insoluble in water, t o obtain separations for example, betn-een sebacic and suberic acids, it was necessary to employ mobile solvents-e.g., chloroform-with very little affinity for these acids. Because of the limited solubility in these solvents, the samples were dissolved by boiling and transferred in the form of supersaturated solutions onto the columns. It has been the authors’ experience that acids such as suberic and sebacic often crystallize under such conditions, being very sparingly soluble in chloroform a t room temperature; this results generally in poor recoveries or limits the proportion of the less soluble components t h a t can be tolerated in such samples.

The method presented here has been employed with minor variations for nearly two years as a control method. The procedure is based on the use of a buffered, stationary aqueous phase and is not dependent on use of highly supersaturated solutions. By the method described it is possible, furthermore, t o separate sebacic and all lower dicarboxylic acids individually from each other and from higher dicarboxylic acids. With the water column suberic acid is the highest straight-chain dicarboxylic that can be isolated as a single component from mixtures of these acids. EXPERhWENTAL

Apparatus. A 20-mm. borosilicate glass chromatographic column 45 cm. long is used. A close-fitting glass plunger is employed in packing the column. Packing the Column. Twenty-five grams of silicic acid (Mallinckrodt, chromatographic grade) and 25 ml. of the aqueous phase are thoroughly worked together in a 400-ml. beaker with a spatula. Roughly 200 ml. of 39, n-butyl alcohol-97% chloroform mixture are added and a homogeneous slurry is formed by vigorous stirring. This is packed incrementwise into the column, care being taken to prevent formation of air pockets or other forms of heterogeneity. Each increment is packed down uniformly and firmly with the plunger. Finally a circle of filter paper is placed on the top of the packing. Preparation of Sample Solutions. A 1% solution of the sample in 57, tert-amyl alcohol-95% reagent chloroform solution is prepared by dissolving a weighed amount of the material in the

ANALYTICAL CHEMISTRY

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correct proportion of warm tert-amyl alcohol and making to proper volume with chloroform. The same concentration in 10% tert-amyl alcohol-90% chloroform is prepared in a similar manner. Sample Addition and Development of Column. For determination of acids above adipic, a 5-ml. sample of the solution containing 5% tert-amyl alcohol is pipetted into a chromatographic column, buffered with 1 M (pH 5.40)citrate buffer. The usual chromatographic precautions are taken in adding the sample and in washing down following its addition. The eluting solutions generally had the following compositions

0-40 40-100 100-160 160-220 220-400

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2

10

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OB

*4W n

n-Butyl Alcohol, % 3

111.

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P

os

W I-

;0.4

5

A

10

a

20 35

all percentages being on a volume basis, the remainder being made up with chloroform. Eluate fractions of 10 ml. are collected.

0.2

0.0 0

0

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LE

16

20

E L U A T E FRACTIONS

a

24

rioML.

12

16

40

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Partition Chromatogram of Same Mixture

Figure 3.

Obtained from column charged with 2 M (pH 5.40) citrate buffer

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Figure 1. Partition Chromatogram of Mixture of Several Acids

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3 M L SAMPLE

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Column charged with 1 M (pH 5.40) citrate buffer

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For determination of adipic and lower acids a 5-mL sample of the solution containing 10% tert-amyl alcohol is pipetted into a column having water as the stationary phase. The following eluant compositions are used: 311.

n-Butyl Alcohol, %

0-60 60-120 120-240

10 20 35

2 0.40 2 0.3-

i

4

1

0.2

The rest of the procedure is the same as that of the preceding section.

; SEBACIC

I

E L U A T E F R A C T I O N S ( 5 ML. 1

Figure 4. Partition Chromatogram of Sample of Singly Recrystallized Sebacic Acid Eimer end Amend C.P. sebacic acid obtained from column charged with 1 M (pH 5.40) citrate buffer. Smaller sample oize and smaller fraction size used to obtain greater resolution

Table I. Mixture 1

2

Figure 2.

Partition Chromatogram of Same Mixture as in Figure 1

Column charged with 1 M (pH 5.20) citrate buffer

Analysis of Fractions. Five milliliters of absolute alcohol are added to each fraction, which is then titrated with 0.04 to 0.05 N alcoholic sodium hydroxide. m-Cresol purple is used as the indicator. The alcohol is added to sharpen the end point and to permit single-phase titration of the chloroform solutions.

Recovery of Acids from Synthetic I)Iixtures .4cid Added, 1Ig. Benzoic 1 0 . 0 Sebacic 1 0 . 0 Azelaic 10.0 Suberic 1 0 . 0 Pimelic 10.0 Adipic 10.0 Benzoic 1 0 . 0 Azelaic 2 0 . 0 Suberic 10.0 Pimelic 1 0 . 0 Adipic 10.0

Acid Found, M g , pH 5.40 PH j.20 11.0 9.3

9.9 9.9 9.9

.. .. .. .... ..

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9.9 9 9 9 9

10.9 IS 4

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10 0

RESULTS A N D DISCUSSION

The degree of separation of a mixture of benzoic, sebacic, azelaic, suberic, pimelic, and adipic acids (10.0 mg. each) is shown in Figure 1. One molar (pH 5.40) citrate buffer was used on the column. In comparison, on the partition column charged

V O L U M E 24, N O . 3, M A R C H 1 9 5 2

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with only water, the first three peaks shown appear as a single peak even nith stritight chloroform as eluant. The effect of a slight change in the buffer pH is illustrated in Figure 2, where the same mixture was analyzed on a column buffered a t pH 5.20 (1 M citrate). The separation between sebacic and benzoic acids seems to be lost and the other components appear correspondingly earlier. Because of the dibasic nature of the acids concerned, the effective partition coefficient is very sensitive to pH. The effect of a higher buffer concentration is demonstrated in Figure 3, where 2 ,I4 (pH 5.40) buffer was used on the same mixture as above. The degree of separation between the first two peaks here is much less than in the first instance. This behavior can be attributed in part to the salting out of the un-ionized acidic components from the water phase, K i t h smaller sample size it is apparent that sharper peaks can be obtained. KOparticular advantage can be gained in most cases, as the separations seem to be complete, but in some instances the increased resolution may be of value. For example, a 3-ml. sample of Bingly recrystallized Einier and Aniend C.P. sebacic acid was chromatographed on 1 111 (pH 5.40) buffered column. The results of titration of 5-1d. fractions collected from the column are shown in Figure 4. The smaller sample volume permitted resolution of two impurity peaks ahead of the major component. The same, however, could have been accomplished with the Etandard size sample by raising the pH of the buffer very slightly. In Table I are listed some recovery data for known mixtures of acids. The values were calculated from the sums of the volumes of the standard base consumed by fractions represented under each peak minus the corresponding blank corrections. The data

presented seem to indicate that benzoic acid carries ~ i t it h on elution an appreciable proportion of the fraction following it. This behavior is not exhibited by any of the dicarboxylic acids, 99 to 100% recovery being usually obtained in the cases given here and in numerous other runs in these laboratories. The elution chromatogram of a mixture of adipic, glutaric, and succinic acids resembles that given for the higher acids. I n all cases 99 t o 100% recoveries were obtained. As the separation of the lower acids is adequately covered by Marvel and Rands, no discussion of this aspect is represented here. tert-Amyl alcohol was used in dissolving the saniples becauce of the appreciable tendencies of primary alcohols to form esteis with the organic acids, I n certain cases where n-butyl alcohol was used as the solvent, a significant amount of the sample acid v a s found t o have undergone esterification during the dissolution process. This source of error is greatly minimized by using the tertiary alcohol. The primary alcohol can be used for elution purposes, as the process does not involve warming, the acids are mainly in their salt form and in the aqueous phase, and the system is practically neutral, owing to the buffer present. LITERATURE CITED

(1) Marvel, C. J., and Rands, R. D., J . Am. Chem. SOC.,72, 2T42 (1950). (2) Stafford, R. W., Francel, R. J., and Shay, J. F., ANAL.C m a r . , 21. 1454 (1949). (3) Swann, M. H., Ibid., 21, 1445 (1949). RECEIVED for review August 29, 1951. Accepted October 2, 1951. Presented before Section 2, Analytical Chemistry, a t t h e X I I t h International Congress of Pure and Applied Chemistry, New York, N. Y., Septemher 10 t o 13, 1951.

Determination of Thermal Conductivity of Copper and Deoxidized Copper-Iron Alloys Apparatus and Technique 3T. J. GOGLIA, Georgia Institute of Technology, Atlanta, Ga., AND G . A. HAD-KINS Purdue University, Lafayette, Ind.

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SURVEY of the literature reveals few data regarding the

effect of small amounts of iron on the thermal conductivity of copper. In the design of copper-bonded brake drums and special heat exchangers a knowledge of this effect is important. An investigation was undertaken, to design and construct apparatus suitable for thermal conductivity measurement and to study a system composed of copper containing small amounts of iron. This report describes the apparatus and presents data on the thermal conductivity of copper containing small amounts of iron at temperatures of 150°, 300",and 500" F. DESCRIPTION OF APPARATUS

An apparatus was designed so that cylindrical specimens 0.5 inch in diameter and 6 inches long could be used. Each specimen bar was silver-soldered into a bar 1 inch in diameter and 3.75 inches long. The bar, B , as shown in Fi ure 1, acted as a heat source for the specimen. A water jacket, softsoldered to the top of each bar, served as a heat sink. This jacket consisted of a copper tube 2.5 inches in outside diameter and 0.5 inch long with copper disks silver-soldered on each end to form an enclosed chamber. The water was introduced into the jacket through a copper tube, 0.5 inch in outside diameter silver-soldered into a hole in the top disk. A split 1.5-inch circular baffle ring was soldered concentrically in the jacket to create turbulence and

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J. E. DEVERALL,

increase the transfer of heat from the specimen to the water. The outlet tube was mounted through the top copper disk, extending to the bottom disk on the outside of the baffle ring. The specimen extended u p into the jacket 0.125 inch, so that the inlet n nter impinged on top of it. Thermocouple wells were niounted in the elbows which CTtended from the inlet and outlet connections. These wells were of copper tubing inch in outside diameter, flattened arid soldered on one end. They M ere soldered into the elbows in a vertical position, extendin about 1 inch into the tube, and as much as possible in line with t%e direction of flow. Oil was placed in the wells to get good thermal contact bet\\-een the 1mll of the thermocouple well and the thermocouple. Thermocouples were mounted on the specimen bar in the following manner. A reference point was scribed on each bar b> a surface gage, so that the point on each bar would be at the same height from the bottom of the bar B. This was done to orient properly the pairs of thermocouples on the specimen A , and specimen guard ring, F . Lines were scribed every inch above and below this reference point for the location of six thermocouples on each bar. Spacing was arranged so t h a t the extreme thermocoup. 1es were 0.25 inch from the exposed ends of the bar. A fine cut Tvas made a t each mark as shallow as was necessary to permit the 30-gage copper-constantan thermocouples to be placed lust flush with the surface of the bar. The thermocouples were then peened in by closing the groove with a small chisel. Evtreine care was taken to distort the surface as little as possible. The thermocouples were stri ped of insulation, cleaned with emery cloth, and wound t i g h y together hefore placing in the cut. h s