Table IV.
Sample 1 2
Analysis of Lanthanum for Nitrogen
Vacuum Fusion, Wt. 0 104 0 105 0 093 0 0114 0 0119 0.0119
Kjeldahl, wt. 70 0 1002 0,0998 ..
.. ..
..
(Table IT). Only three comparisons were made because of the greater interest in the more difficult oxygen determination. I n the operating procedure the samples are dropped into the bath at 1300" C. and the bath is then heated to 1900" C. As the nitrogen appeared to be liberated completely a t
1600" C., most of the nitrogen should be pumped out of the furnace well below 1900" C. and the temperatures a t which volatilization of nickel becomes troublesome. Since precision and accuracy were obtained at 1600" C., t h e procedure as developed for evolving the oxygen should be satisfactory for nitrogen. No standard samples containing knoii n amounts of hydrogen n ere analyzed. Hydrogen seemed to be rapidly and completely evolred from either an iron or a nickel bath a t 1600" C. As the hydrogen is easily eyolved and should not be adsorbed to a greater extent than carbon monoxide, the standard oxygen procedure should give accurate results for hydrogen. ACKNOWLEDGMENT
The authors would like to thank F. H.
Spedding of the Ames Laboratory for supplying the lanthanum metal used in the study. They would like also to acknowledge the work of H. J. Svec and J. E. Capellen in developing the isotope ratio method of gas analysis and for performing the analyses. LITERATURE CITED
(1) Daane,
H., "T'apor Pressure of 1,anthanum and Praseodymium," U S. -4tornic Energv Commission,
AECD-3209 ( A u ~ 1, . 1991). (2) Guldner, IT. G., Beach, A . L., ,4SAL. CHEM.22, 366 (1950). ( 3 ) Kroll, IT. J., Schlecton, A . IT., Truns. Electrochem. SOC.93, 247-58 (1948). (4) Kalter, D. I., ANAL CHEM.22, 297303 (1950).
RECEIVED for reviex May 10, 1956. Accepted Kovember 19, 1956. Contribution N o . 488. JTork performed in the rimes Laboratory of the U. S. Atomic Energy Commission.
Paper Chromatographic Detection of Galacturonic and Glucuronic Acids MILDRED
GEE
and R. M. McCREADY
Western Utilization Research Branch, Agriculfural Research Service, United States Department of Agriculfure, Albany 1 0, Calif.
,A procedure for the tentative identification of galacturonic acid on paper chromatograms in the presence of glucuronic acid is described. The mixture containing glucuronic acid is partially lactonized and chromatographed. Characteristic migration rates, the hydroxamic acid-ferric ion test for lactones, and the specific lead acetate test for galacturonic acid served to identify these substances,
9 with sodium hydroxide, and then it produces a single spot. The present report describes a procedure whereby a combination of the basic lead acetate test (3, 4) and the reaction of hydroxamic acid with ferric ion used for lactones and esters ( I ) served to identify galacturonic and glucuronic acids by paper chromatography. EXPERIMENTAL
P
CHROhlATOGRAPHY is used widely for the tentative identification of the sugars produced b y the hydrolysis of polysaccharides. Most sugar mixtures are resolved with the proper irrigating solvent, or if resolution is poor they are selectively detected b y a particular indicator. Mixtures of glucuronic and galacturonic acids are difficult to separate b y irrigating with the usual chromatographic solvents, although a mixture of pyridine, ethyl acetate, acetic acid, and water (6) can be used to separate these substances. Solutions of glucuronic acid usually contain glucuronolactone and produce double spots on a chromatogram (6). Glucuronolactone can be hydrolyzed to sodium glucuronate b y titration to p H APER
Paper Chromatography. Aqueous solutions of t h e uronic acids were applied t o W h a t m a n No. 1 filter paper sheets with micropipets and t h e sheets were then dried in air at room temperature. T h e papers were irrigated in t h e ascending direction (2) with a solvent of ethyl acetate, pyridine, mater, and acetic acid in a ratio of 5:5:3:1 (6) and dried in air without heating until they were free from acetic acid. Appropriate color reagents were then applied as described below.. Fivemicroliter amounts of solution to be tested produced spots of 10-mm. diameter upon application on Whatman KO. 1 paper. After irrigation, the spots had diffused t o a diameter of 15 mm. Uronic Acids. Five-microliter amounts of lY0 uronic acid solutions were applied t o paper sheets and t h e sheets were dried, irrigated, and tested with t h e following results.
Glucuronic acid lactonized by heating a 1% solution a t p H 2 and 80" C. for 30 minutes gave double spots when chromatographed. Glucuronic acid titrated to p H 9 with sodium hydroxide gave a single spot. Galacturonic acid apparently does not lactonize under these conditions and gal-e but a single spot when chromatographed. Color Reagents. TKOgrams each of aniline and crvstalline trichloro-. acetic acid hydrate were dissolved in 100 ml. of ethyl acetate. T h e dried chromatograms t o be tested were dipped in this solution and permitted t o d r y in air for 15 minutes. T h e dried paper sheets were then heated for 5 minutes a t 95' C. T a n spots on a white background appeared in t h e presence of 10 y or more of reducing uronic acids. A saturated, aqueous solution of basic lead acetate was prepared and filtered. T h e dried paper chromatograms to be tested for galacturonic acid were dipped rapidly through the aqueous lead acetate solution, blotted to remove excess solution, and heated for about 1 minute in a cwrrent of live steam. -4 brick-red spot on a n-hite background was produced with 25 y or more of galacturonic acid. Hvdroxamic Acid-Ferric Ion Test for Lactones ( I ) . T h e solution of alkaline hydroxylamine should be prepared fresh before use. Seven grams VOL. 29, NO. 2 FEBRUARY 1957
257
of hydroxylamine hydrochloride were dissolved in 100 ml. of methanol. Potassium hydroxide, 7 . 2 grams (85% pure), was dissolved in 100 ml. of methanol. Equal volumes of these solutions were mixed and the precipitated potassium chloride was filtered off. Ferric chloride (1 gram, lumps) was dissolved in 100 ml. of 1% hydrochloric acid. After irrigation and drying, the chromatogram to be tested for lactones was sprayed with the freshly prepared alcoholic hydroxylamine solution, allowed to stand for 15 minutes, and sprayed with the acidic ferric chloride solution. Mauve to red spots on a vellOM background indicated the presknce of 10 y or more of uronic acid lactones. Procedure for Tentative Identification of Uronides in an Unknown Solution. A solution suspected of containing galacturonic and glucuronic acids was adjusted to p H 2, heated for 30 minutes a t 80" C., and spotted in replicate on a paper sheet with appropriate standards of galacturonic a n d glucuronic acids and glucuronolactone. T h e spots were applied in such a manner that the whole sheet was irrigated and dried and then cut into three strips, each containing t!ie series of knowns and unknowns. One dried strip was tested as described with the aniline-trichloroacetic acid reducing indicator. Spots appeared a t RGA(distance of migration compared n-ith galacturonic acid a t 100) of 100, 116, and 339, corresponding to galacturonic and glucuronic acids and glucuronolactone. The solvent front advanced 30 cm. and galacturonic acid had moved 6 cm. The lactones mere revealed b y applying the hydroxylamine and ferric chloride sprays as described. The colored spot resulting from the reaction of hydroxamic acid with ferric ion occurred a t RGA339 for glucuronolactone.
Table 1.
Migration Rates and Color Reactions of Various Uronic Acids"
Color Reaction Zironic Basic lead Hvdroxamic Bcid RGA acetate acid Galacturonic 100 Dark brick-red None Digalacturonic 28 Medium yellow-orange None Trigalacturonic 8 Medium yellow None G1ucu ronic 116 Light tan None Mannuronic 145 Faint tan None Glucuronolactone 339 Light tan Mauve Mannuronolactone 298 None Mauve 50 y of various uronic acids applied as 5-bl. spots on Whatman No. 1 filter paper and irrigated with ethyl acetate-water-pyridine-acetic acid solvent.
4 third paper strip was tested with the basic lead acetate dip. A brick-red spot at RGA100 indicated the presence of galacturonic acid. RESULTS
The migration of various uronic acids on paper sheets with the pyridineethyl acetate-water-acetic acid solvent is shown in Table I. Glucuronic, galacturonic, and mannuronic acids and their lactones n-ere separated by this solvent. The color reactions of uronic acids and lactones tested with the basic lead acetate reagent and the test with hydroxamic acid and ferric ion are shown in Table I. Galacturonic acid gives a dark brick-red color with the basic lead acetate. Di- and trigalacturonic acids, produced from pectic acid by enzymic hydrolysis and likely to occur occasionally with galacturonic acid, reacted with basic lead acetate to give orange and yellow colors, respectively, and they ~ and 8. Glucuronic migrated a t R G 28
and mannuronic acids as well as glucuronolactone reacted, but gave such faint yellow colors that they would not be likely to interfere with the specific lead acetate test for galacturonic acid. Both lactones reacted strongly in the hydrosamic acid test \vith ferric ion but the free acids did not. LITERATURE CITED
(1) Abdel-Akher, N., Smith, F., J . Ani. Chem. SOC.73, 5859-60 (1951).
(2) Block, R. J., LeStrange, R., Zweig, G., "Paper Chromatography," Academic Press, New York, 1952. (3) Ehrlich, F., Ber. 65, 352-58 (1932). (4) Ehrlich, F., Guttmann, R., Zbid., 6 7 , 073-89 11934). ( 5 ) Fischer, F. G.; Dorfel, H., H o p p e Seyler's 2. Physiol. Chem. 301 (4-6), 224-34 (1955). ( 6 ) Partridge, S. M., Biochem. J . 42, 23848 (1948).
RECEIVED for revie17 August 5, 1956. Accepted Sovember 17, 1956. Mention of specific products does not imply endorsement by the Department of Agriculture.
New Indicator for Complexometric Titration of Calcium in Presence of Magnesium GARY P. HILDEBRAND and CHARLES N. REILLEY Department of Chemistry, University of North Carolina, Chapel Hill, N . C.
,A new indicator for calcium (C1202, Calcon) allows an accurate complexometric titration of calcium in the presence of magnesium a t pH 12.3 in diethylamine buffer. The end point is easily detected as a sharp change from pink to pure blue. The indicator forms a 1 to 1 complex with calcium, as shown b y a study of continuous variation. The stability constants for the calcium, magnesium, and zinc complexes were determined to b e 105J, 1,'.'O and 1 012J, respectively. Strontium forms a very weak complex, 258
ANALYTICAL CHEMISTRY
whereas the barium complex is barely detectable. This is logical, in view of the increasing size of these metal ions and the steric requirements of Calcon.
C
is fast becoming a standard method for the estimation of calcium and magnesium. When both are present, the titration conditions are adjusted so that, during the second titration, either magnesium or calcium is precipitated and/or removed, and the other is titrated. OhfPLEXOMETRIC TITRIMETRY
For the titration of the sum of calcium plus magnesium, a n ammoniacal solution (pH 10) is usually employed and Eriochrome Black T (Color Index 203) is used as the indicator. This titration usually presents little trouble. The details of this procedure and methods for eliminating interferences of certain metal ions often encountered in hard water were studied in the classic paper of Diehl, Goetz, and Hach (4). For the second titration, magnesium is often precipitated as magnesium hydroxide by addition of potassium hy-
