Microscopic Identification of Microgram Quantities of Galacturonic Acid and GIucuronoIactone Direct Synthesis of Hydrazone Derivatives by the Solvent Diffusion Technique LAWRENCE M. WHITE and GERALDINE E. SECOR Western Utilization Research and Developmenf Division, Agricultural Research Service, Deparfmenf o f Agriculfure, Albany 7 0, Calif.
U. S.
b A previously described solvent diffusion technique for the identification of some hexoses and pentoses has been extended to the identification of galacturonic acid as the 2,5-dichlorophenylhydrazone and glucuronolactone as the 2,4-dinitrophenylhydrazone. As little as 2 to 3 y of the pure or 10 to 15 y of the chromatographically separated sugar acid gives the test. METHODS for the identification of galacturonic and glucuronic acids are valuable because of the increasing importance of these compounds in physiological chemistry Irrigation solvents (3, 7 ) and specific sprays (4) have been described that permit the separation and tentative identification of these compounds on paper chromatograms. However, isolation of the pure x i d or formation and characterization of a derivative is usually required for positive identification. The solvent diffusion technique described in earlier papers in this series was used to synthesize microgram quantities of some hexose and pentose hydrazine derivatives (6, 8, 9 ) . It can also be employed to prepare characteristic derivatives of galacturonic acid and glucuronolactone and, when used in conjunction with paper chromatographic separations, gives specific tests for these compounds. This paper describes the methods used for the formation and recognition uf microgram amounts of galacturonic acid 2,5-dichlorophenylhydrazone and glucuronolactone 2,4-dinitrophenylhydrazone. DDITIONAL
METHODS AND OBSERVATIONS
Using the reagents indicated belox 2nd the techniques previously described (6, S ) , make the chromatographic ieparations by descending chromatography, ether wash the chromatogram, elute the test area of the chromatogram with water, and deionize the h a t e Prepare and assemble the dif-
fusion cell, and observe the progress of the synthesis. Compare the appearance of any reaction product with that of products formed under the same conditions by authentic compounds and with blanks prepared from water eluates of nonsugar-containing areas of equal size from the same chromatogram and the hydrazine reagent. 2,5-Dichlorophenylhydrazine Test for Galacturonic Acid. K a s h Whatman X o . 1 “special for chromatography” sheets with distilled water by descending chromatography for 24 hours, remove the water fiom the tray and allow the paper t o air-dry in place. Chromatograph 10 to 15 y or more of‘the galacturonic acid on the washed paper with ethyl acetate-pyridine-water (8 to 2 to 1) solvent for 16 hours. Treat the eluate with 1.7 mg. of 35- to BO-mesh Bmberlite IR-120 H resin, transfer to the slide (8), and apply sufficient freshly 2,5-dichlorophenylhydrazine ground (Eastman KO.4095) to the air-dried acid area on the slide to assure that some reagent crystals will remain undissolved for 1 to 2 hours. Add 1 MI. of 2-methoxyethanol (Eastman KO. P 2381)-acetic acid reagent (9 to 1 v./v.) to the well of the culture slide and immediately assemble the cell.
As little as 2 y of pure galacturonic acid will give a recognizable product within 10 minutes and 10 t o 15 y (amount applied to the paper) of the chromatographically separated acid will give a product within 1 hour. Larger amounts of the acid react more rapidly. Small and moderate amounts of the acid give a distinctive gel in the form of small, translucent, white patches with granular appearing surfaces, becoming semicrystalline on aging. Identification of large amounts of the acid is made immediately after the d 8 u sion cell is assembled and while the gel is forming because a dense blanket is finally formed that is not characteristic in appearance. The product synthesized is galacturonic acid 2,s-dichlorop henylhydrazone. 2,4-Dinitrophenylhydrazine Test for Glucuronolactone. Chromatograph 10 to 15 y or more of glucu-
ronolactone on the n-ater-washed Whatman S o . 1 ..special for chromatography” paper with ethyl acetatepyridine-water (8 to 2 to 1) solvent for about 5 hours or with n-butyl alcoholethyl alcohol-nater (10 to 1 to 2) solvent for up to 40 hours. Use a wellsaturated tank and start the irrigation of the paper as soon as possible after spotting the lactone solution. Treat the eluate with 5 mg. of a 35- to 60mesh mixture of 1 part of Amberlite IR-120 H and 2 parts (w./r.) of Duolite A-4, and transfer to the slide (8). Make two applications of a saturated solution of 2.4-dinitrophenylhydrazine (Eastman So. 1866) in ethyl acetate to the air-dried lactone area on the slide with the tip of a 1-mm. glass rod. Add 1 pl. of dilute acetic acid (1 to 200) to the Fell of the culture slide and immediately assemble thc cell. Pure lactone reacts to give a crystalline product within to 2 hours, but the chromatographically separated lactone requires to 16 hours. The time required depends on the amount of lactone present and the irrigant used in the separation. The test responds to 3 to 5 y of the pure or 10 to 15 y (amount applied to the paper) of the chromatographically separated lactone. The product is light yellow, short to long slender needles in brushes, sheaves, or hemispherulitic clusters. It grows slowly, almost always in a solvent pool around the edge of the dried lactone area. Ordinarily the needles continue to groiv for several hours. Maximum development of the test is not usually obtained before 16 hours. The product synthesized is glucuronolactone 2,4-dinitrophenylhydrazoneS DISCUSSION
Many sugars react with 2,5-dichlorophenylhydrazine (6). The nonspecificity of the reagent requires that galacturonic acid be separated from other sugars before the solvent diffusion test is made. The separation is easily accomplished by paper chromatography using ethyl acetate-pyridine-water solvent in which galacturonic and glucuVOL. 31, NO. 7, JULY 1959
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ronic acids are nearly immobile while pentoses, hexoses, and some disaccharides move rapidly and are completely removed from the origin in the irrigation period recommended. Longer irrigation may be required t o remove some of the slower moving di- and trisaccharides. Glucuronic acid, which does not separate from galacturonic acid in this solvent, does not give a visible reaction product at or below the 100-7 level and is inhibitory only if present in a large excess over the galacturonic acid. Any interference by glucuronic acid in the galacturonic acid test may be eliminated by lactonizing the glucuronic acid to the ?-lactone (1, 2) before chromatography. The 2-methoxyethanol-acetic acid reagent may be replaced as the diffusing solvent by an equal amount of dioxane, methyl alcohol containing up to 5% water, or 2-ethoxyethanol. However, the test is not as sensitive with these solvents as it is with the 2methoxyethanol, although in some cases a mo’re crystalline product is obtained. Kater-elutable materials in some lots of chromatographic paper caused the formation of large amounts of long, regular, colorless prisms or bars that obscured the presence of the gel-like product in the galacturonic acid test. These materials and certain inorganic ions cause inhibition in this test and also in the 2,4dinitrophenylhydrazine test for glucuronolactone. To obtain a high sensitivity with these tests, the paper must be washed with distilled water by descending chromatography
for a t least 24 hours, the chromatogram must be ether-washed immediately after it is removed from the tank, and the water eluate must be treated with the ion exchange resin or resins as specified. Even with these precautions small amounts of inhibitory materials are not removed. This slight inhibition in addition to losses due to incomplete elution and some occlusions by the ion exchange resin or resins (8) account for the difference in the sensitivity of the test for pure and chromatographed acid or lactone. Glucuronic acid is identified as glucurolactone 2,4-dinitrophenylhydrazone after chromatographic separation as the lactone. Previously, 2,4-dinitrophenylhydrazine was used with organic solvents as a reagent for identifying microgram amounts of a number of hexoses and pentoses (6, 8). However, when water or water containing a trace of acetic acid is used as the diffusing solvent, very few sugars give a visible reaction product at or belon. the 100-7 level, probably because of the low solubility of the 2,4-dinitrophenylhydrazine. The lactone is separated readily from all common sugars except ribose, fucose, and rhamnose in the n-butyl alcohol-ethyl alcohol-water solvent and from all common sugars in the ethyl acetate-pyridine-water solvent in the times recommended. Of the common sugars migrating near glucuronolactone on the chromatogram, only fucose a t high levels gives a product under the conditions of the test. Although the
appearance of the 2,4--dinitrophenyihydrazone of fucose differs from that of the lactone, the certainty of the test is increased if fucose is not present. If this sugar is present in the test solution, or if a rapid test is required, the chromatographic separation should be made with ethyl acetate-pyridine-water as solvent instead of n-butyl alcoholethyl alcohol-water. However, tests performed after chromatography in the latter solvent are slightly more sensitive; the product is more typical in appearance and it appears slightly sooner at low levels than when the ethyl acetate-pyridine-water solvent is used. LITERATURE CITED
(1) CharalampouB, F. C., Lyras, C., J . Biol. Chem. 228, 1 (1957).
(2) Eisenberg, F., Jr., Field, J. B., Ibid., 222, 293 (1956).
(3) Fischer, F. G., Dorfel, H., 2.physiol. Chem., Hoppe-Se ler’s 301, 224 (1955). (4) Gee, M., Mc&eady, R. M., ANAL.
CHEM.29, 257 (1957). (5) Mandl, I., Neuberg, C., Arch. Biochem. Biophys. 35, 326 (1952). (6) Secor. G. E.. White. L. M.. ANAL. CHEM.‘27, 1998 (1955)’. (7) Smith, F., Spriestersbach, D., h’atuie 174, 466 (1954).
.,
(8) White, L. M., Secor, G. E., ANAL.
CHEM.27, 1016 (1955). (9) Ibid., 28, 1052 (1956).
RECEIVED for review November 10, 1958. Accepted February 24, 1959. The mention of commercial products does not imply that they are endorsed or recommended by the Department of Agriculture over others of a similar nature not mentioned.
Carbon and Hydrogen Microdetermination by Automatic Combustion Control ERVIN STEHR Texaco Research Center, The Texas
Co., Beacon, N. Y.
b An automatic method for the microdetermination of carbon and hydrogen is described in which the rate of combustion is accurately controlled by the changes in pressure which develop in the combustion tube from the vaporization and burning of the sample. By use of a diaphragm-type pressure switch, movement of the sample heater is stopped when the pressure rises above an equilibrium pressure established by a choking plug; when the pressure again drops to the equilibrium point, motion of the heater is resumed, Thus, a uniform cycle of combustion is automatically effected for samples ranging from solids to 1274
ANALYTICAL CHEMISTRY
volatile liquids. Improvements in the apparatus include a new pressure regulator and flowmeter for the oxygen gas, a precision mortar, and a grooved track for adjusting the position of the separate units in the train.
T
accuracy of microdeterminations of carbon and hydrogen largely depends upon the care exercised in burning the sample. Too rapid or erratic burning causes low results due to incomplete combustion. If this phase is unduly extended, time is wasted and fewer samples can be run per day. I n manual operations, the control of HE
the combustion is entirely a matter of judgment as to the selection of proper conditions for burning a variety of samples. Samples may vary from volatile liquids to refractory solids, requiring entirely different cycles for combustion. Mechanical devices to control the movement of the movable heater have been used to effect more uniform burning and many such units have been described. Some devices (4, 6, 7) were designed for uniform travel of the movable furnace, while others (1, 3, 6) employed variable speeds to extend the time required for passage of the movable heater over the sample area. Although the use of such units has helped to bring
