ANALYTICAL CHEMISTRY
1366 the polarographic measurements are those used for the data of Table 11. The solutions should contain lithium chloride and thallium chloride, and the pH should be 5 to 6. However, the pH of the solution (unless highly buffered) is no measure of the p H at the electrode interface during reduction of the inosow. EXPERIMENTAL SECTION
The inosose used in the work was prepared by the method of Posternak ( 3 ) using nitric acid oxidat,ion and was purified 1)). .ix recrystallizations from water. I t had a melting point of 200" (', (decomposed). Composition of Solutions for Polarographic Analysis. Saml)lw of inosose were prepared by diluting aliquot portions of 0.01 -21 solutions. Solutions were buffered with 0.1 Jf potassium phosphate buffers. Thallous chloride was used as a refercncc. foi. half-wave reduction potentials. the concentration being 0.0005 31 in all cases. Lithium chloride, 0.1 M, ivas used as the supporting electrolyte in t,he solution employed for the determination of inosose. Polarographic Procedure. The determinations wttrc niatlt. x i t h a Heyrovsk; hlodel 10 niicropolarograph (Sargent ) quipped Lvith a photographic recorder and an .iyrton shunt. .ill polarograms were made at a sensitivity of 1 to 40. The galvanometer deflrction was calibrated a t various ratios of galvanomtJter currant to cell current by means of a standard resistance. A 1-mm. deflection at a sensitivity of 1 to 40 was equal to 2.91 X lo-: ampere of cell current. The capillary const,ants were: T I I =
0.00278 gram of meicury per second and t = 2.79 average seconds per drop. .4ll polarograms were obtained using a voltage increment of 0.5 volt per minute. Calculation of Polarogram Data. Hdf-wave potentials reported are all referied to the normal calomel electrode. They n ere calculated froin the known constant value €or thallous ion of -0.48 volt and the value of 0.1815 volt pei centimeter experimentally determined for the particular instrument by determining the distance between half-wave points for thallium and barium ( E l i 2= 1.94 volts). The Heyrovsk? method of tangents was used for the measurement of half-nave potentials and wave hrights on the polarograms. The accuracy wm mithin f 0 . 5 nim. = fO.O1 volt or f l 5 X 10-8 ampcie at a sensitivity of 1 to 40. ACKNOWLEDGMENT
The> :Idvice and encouragement of S. AI. Cantor during the p r u p r c w of this work are gratefull? acknowledged as well as the m i i r t ( y of -1, I,. E:ltic,r :ind Henry COYin permitting its puh1ic:ttion. LITERATURE CITED
11) Cantor. S.AI., and Peniston, Q. P.. J . Am. Chem. Soc., 62, 2113 (1940.) ( 2 ) Fletcher, H. G., A d m n c e s i n Carbohydiate Chem., 3, 62-5 (1948). (3) Posternak, T., H e h . C h i m Acta., 24, 1045 (1941). HEcKIl-r:o for review February 6, 1952. Accepted .ipril 23, 1952.
Separation of Vanillic and Protocatechuic Acids by Chromatography IRWIN A. PEARL
4ND
DONALD L. BEYER
The Institute of Paper Chemistry, Appleton, Wis.
-ISILIJC
and prot,ocatechuic acids have been found to-
\'gether in acid fractions obtained from the alkaline oxidat,ion
at high temperatures of lignin materials and from the caustic fusion below demethylation teniperat,ures of partially methylated phloliotannins and phlobophenes. In the past it has been practicall!, impossible t o separate these acids and to estimate them separately. Instead, the usual practice has been to determine the mixed acids and estimate the vanillic acid from the methoxyl value of the mixture. The lead acetate procedure for the separation of vanillic acid from ot,her acidic products of oxidat,ion of lignin materials by means of silver or mercuric oxides ( 3 )was 1111successful in the separation of prot,ocatechuic acid from vanillic acid. The present paper rep0rt.s a simple chromatographic procedure for the quantitative separation of these two acids. The procedure is the same as that employed earlier for the separation of vanillin and s!-ringaldehyde ( 4 ) , except that t,he developer used in this case is 20 to 1 benzene-ethanol. The acidswere separated by this developer on a column of acid-n-ashed Magneeol and located by R ferric chloride streak. I n a study of the R, values of several naturally occurring phenolic materials, Bate-Smith and Westall ( 1 ) showed that vanillic and protocatechuic acids could h c qualitatively separated by paper chromatography. EXPERlMENTAL
Separation of Vanillic and Protocatechuic Acids. A solution of i 4 . 0 mg. of vanillic acid and 71.5 mg. of protocatechuic acid in 15 nil. of 20 t o 1 benzene-ethanol was adsorbed on a column (35
mm. in diameter and 200 mm. in length) of acid-wmhed Magnesol (3.) prewet with benzene, and the chromatogram was developed w t h 180 ml. of 20 to 1 benzene-ethanol. I n this instance, freshly prepared acid-washed blagnesol (4)was unsuitable for the separation of protocatechuic acid and vanillic acid, but after standing overnight in open pans, t,he absorbent propcrties were modified to
the ppint where the separation noted 1%-asobtained. The column was extruded and streaked with ferric chloride solution, using a capillary-tipped pipet ( 4 ) . The ferric chloride streak produced a hlue-green color with the protocatechuic acid band and a reddishIironn color with the vanillic acid band. Protocatechuic acid occupied a well-defined zone between 37 and 116 mm. from the top of the column, and vanillic acid another well defined zone at 132 to 160 mm. The column was cut a t 124 mm. from the top and t3ach zone was eluted with an excess of acetone and the acetone solutione wrre evaporated to dryness. Protocatechuic acid melting a t 193" t o 195" C. and vanillic
K h m dcveloped with 20 t,o 1 ticnzcxwethanol, vanillic acid gives a reddish-brown color with ferric chloride reagent but, when developed with developers less rich in ethanol, no color is developed. \l*hereas vanillic acid in aqueous solution will give a reddish-brown color with ferric chloride when lukewarm, but not cold (2). in a l ~ ~ o h the o l color ip devclopcd even at room t,eniperaturc.. LITERATURE CITED
(1) Bate-Smith, E. C., and Westall, R. G., Biochin,. et Biophys. A c t a . , 4,427 (1950). ( 2 ) Fischer. E., and Freudenberg, K . . .4nn., 372, 48 (1910). (3) Pearl, I. A , , J . i l m . Chem. Soc., 71, 2196 (1949). (4) Pearl, I. A , , and Dickey, E. E . . I h i d . , 73, 863 (1951). (5) Ibid.,74, 614 (1952). (6) Tiemann, F., and Haarmann, K , , Be,.., 7, 618 (1874). RhCE1l.F-D
for reriew ;\larch 3, 1952. Accepted ;\fag 12. 1%2.
VI11 in t h e series, "Studies of Lignin and Related Products. see ( 5 ) .
"
This is paper For paper VI1
