V O L U M E 2 4 , NO. 7, J U L Y 1 9 5 2 quired reduction equivalent is read off the graph. The volume of ferricyanide required for a given ionic strength is given by:
MI. of ferricyanide
=
(
6
’)(g)
where ~.ris the ionic strength, T’T, the initial titration volume, and M, the molarity of the ferricyanide reagent. The reduction equivalent was set a t a value of 6.0 by this method. For routine control work this procedure may be useful. Readjustments of conditions are required as the ferricyanide solution ages. The linear dependence of the reduction equivalent on ionic strength is limited by the time factor involved in oxidizing increasing quantities of glucose, and the dilution factor with the larger titration volumes. It was possible to vary the ferricyanide concentration from 5 to 20 ml. of the 0.05 111‘ stock. For best results a fixed value of ferricyanide should be maintained. I n the microgram titrations no attempt was made to vary the ferricyanide concentration. The linear plot of Figure 4 holds only for a fixed glucose concentration (0.01 M). Variations in glucose concentration change the reduction equivalent (at a fixed ferricyanide concentration) and also because of a dilution effect. In the microgram region the glucose could be varied from 0.0012 to 0.00008 AI‘ without changing the reduction equivalent. A lyider limit should be possible in the milligram-scale titrations, n-here a larger titration volume is involved. However, the recommended procedure is to run a parallel standardization titration if the glucose Concentration differs appreciably from the standard glucose used in establishing the reduction equivalent. A n alternative procedure is to run a
1203 trial titration, calculate the glucose molarity, and accordingly dilute the titrant until its titer more nearly matches the standard. Using the derivative polarographic titration, small quantities of glucose were titrated using a total titration volume of 5 ml. Results indicate that about 20 micrograms of glucose can be titrated with an accuracy of about +5%. A summary of some determinations is given in Table I. I n Table I1 are results of several determinations run by the parallel standardization procedure discussed above. These titrations were run with a Leeds and Xorthmp p H meter. LITERATURE CITED
(1) Assoc. Offic. Agr. Chem.. J . Assoc. Ofic.Agr. Chem., 33, 816
(1950). (2) Assoc. Offic. Agr. Chem., “Methods of Analysis,” 7th ed., 1950. (3) Britton, H. T. S.,,and Phillips, L., Analyst, 65, 149 (1940). (4) Hassid, W. Z., ISD.ENG.CHEM.,AXAL.ED.,8, 136 (1936); 9, 228 (1937). (5) Heck, K., Brown, W.H., and Kirk, P. L., Mikrochemie, 22, 306 (1937). (6) Kirk, P. L., “Quantitative Ultramicroanalysis,” p. 202, Sew York, John Wiley & Sons, 1950. (7) Miller, B. F., and Van Slyke, D. D., J . Biol. Chem., 114, 583 (1936). (8) Reilley, C. K.,Cooke, W. D., and Furman, N. H., A N ~ L . CHEM.,23, 1223 (1951). (9) Stern, H., and Kirk, P. L., J . Biol. Chem., 177,37 (1949). (10) Van Slyke, D. D., and Hawkins, IT., Ibid., 79, 739 (1928). (11) Whitmoyer, R. B., IND.EXG.CHEII.,ANAL.ED.,6 , 268 (1934). (12) Kood, W.B., J . Bid. Chem., 110, 219 (1935). RECEIVED for review March 6, 1952. Accepted April 14, 1952.
Paper Chromatography of Nucleic Acid Derivatives D. C. CARPENTER New York State Experiment Station, Geneva, T. Y. HIS paper describes the separation by paper chromatography the common bases, nucleosides, and nucleotides employing several single-phase mixed solvents in which 1-propanol, isoamyl, and tetrahydrafurfuryl alcohols, and an aqueous solution of a buffer salt are used to control the pH a t desired levels during the operation. Vischer and Chargaff (6) seem to have been the first to apply paper chromatography to the separation of nucleic acid derivatives; they located the purin and pyrimidine bases on the paper by forming insoluble mercury salts on guide strips and converting these salts to black sulfides. Reguera and Asimov ( 4 ) pointed out that purin bases may be located by forming insoluble silver salts on strips arid converting them to brown chromates. Nucleosides or nucleotides of these bases do not give the mercury or silver tests. Hotchkiss (2) employed 1-butanol and 2.5% ammonium hydroxide (1 to 1) as solvent for developingthe chromatogram and the stepwise elution of the separated components for identification by absorption spectrum. Carter ( 1 ) pointed out that ribonucleotides do not move in the solvent systems previously reported. He noted that resolution of nucleotides could be achieved by a two-phase solvent system composed of isoamyl alcohol and an aqueous solution of buffer salt such as citrate or phosphate (butanol-aqueous urea was also used). The spot Tyas located by the quenching of ultraviolet fluorescence, as noted by Markham and Smith ( 3 ) . I t is obvious that two-phase solvents cannot be employed in column chromatography and the authors have been interested in developing a buffered single-phase solvent that could be employed to handle much more material than on paper sheets, when new substances must be separated and characterized. Isoamyl solvents travel a great deal faster on the chromatograph, giving a run in about 24 hours, while butanol solvents require around 40
hours. 1-Propanol has been employed as well as isoamyl alcohol and tetrahydrofurfuryl alcohol has been added to the propanol (or isoamyl)-aqueous solvent mixture in the ratios propanoltetrahydrofurfuryl alcohol-buffer (2 :1: I ), and amy1 alcoholbuffer (1: 1: 1). 7I hese mixtures are about saturated with respect to 0.08 M citrate, and maintain the pH at 3.02, 5.66, and 7.92, respectively. A series lyith 0.1 11 acetate buffer a t pH 3.03 is also included. PREPARATION OF MATERIAL
The buffer salts were from reagent grade material and the organic solvents employed were redistilled before use. The nucleic acid derivatives were commercial samples which had been recrystallized and which showed the presence of a single component on the chromatogram. It was difficult to convert the commercial samples of the barium salts of adenosinediphosphate and triphosphate to their sodium salts by dissolving the barium salt in 0.2 iV nitric acid ( 5 )and removing barium as sulfate by adding the calculated amount of sodium sulfate. Such samples are alwavs less homogeneous than those prepared by base exchange with“ the calculated amount of sodium sulfate alone. Even brief contact with nitric acid causes hydrolysis. EXPERIMENTAL
Solutions of the various nucleic acid derivatives were made by weighing out 5-mg. samples of each, dissolving in 1 ml. of 0.08 -11 citrate buffer, and diluting with water to 5 ml. in a volumetric flask. All solutions were protected by toluene. Spots were made in duplicate of a number of compounds simultaneously on large sheets of Khatman KO.1 filter paper, using ultraniicropipets and employing 0.010- and 0.025-mg. samples of nucleic acid derivative for each spot. The large prepared sheets were hung in pairs from the stainless steel trough in the chromatograph box, and after the air in the box had been saturated with the solvent, the experiment \vas started by adding solvent to the trough. I n the e.qeriments reported the direction of motion of the solvent was descending.
ANALYTICAL CHEMISTRY
1204
_ _ _ ~
___
~
Table I.
Summary of Rf Values
1-Propanol-Tetrahydrofurfuryl Alcohol plus Buffer Salt (2 : 1: 1) 0.10 * y a m momum acetate, 0.08 .lf Potassium Citrate p H 3.02 pH 3.02 p H 5.66 p H 7.92
Purins Adenine Ad en o 3in e Adenylic-3 Adenylic-5 Adenosine diphusb3hate Adenosine triphosphate Guanine Guanosine Guanylic acid Xanthine Xanthosine Hypoxanthine Inosine Pyrimidines Cytosine Cytidine Cytidylic acid Thymine Uracil Uridine Uridylic acid
Isoamyl .Ucohol-Tetrahydrofurfuryl Alcohol plus Buffer Salt (1: 1 :1) 0.08 'iT. Potassium Citrate p H 3.02 p H 5.66 p H 7.92
0.53 0.36 0.07 0.12 0.37 0 45 0.37
0.44 0.36 0.49 0.62 0.04 0.40 0.49 0.37 0.07 0.39 0.38 0.45 0.39
0.45 0.38 0.20 0 44 0.05 0.05 0.50 0.35 0.07 0.54 0.27 0 45 0.37
0.52 0 46 0.06 0.08 0.05 0.05 0.54 0.38 0.05 0.41 0.18 0.48 0.41
0 58 0.57 0.35 0.28 0.07 '0.08 0.63 0,53 0.67 0.59 0.56 0.59 0,56
0.65 0.57 0.31 0.27 0.08 0.08 0.56 0.52 0.43 0.60 0.40 0.56 0.52
0.55 0.54 0.20 0.20 0.07 0.70 0.63 0.49 0.18 0.58 0.32 0.53 0.49
0.42 0.38 0.10 0.70 0.56 0.53 0.16
0 62 0.42 0.09 0.71 0.62 0.59 0.15
0.60 0.43 0.08 0.70 0.61 0.59 0.17
0.64 0.47 0.07 0.72 0.64 0.61 0.10
0.68 0.50 0.26 0.77 0.68 0.66 0.43
0.65 0.51 0.26 0.73 0.66 0.63 0.39
0.64 0.50 0.13 0.71 0.64 0.63 0.21
0.55
0.47 0.48 0.46
When the solvent front had progressed to within 40 to 50 nim. of the bottom of the sheet, the chromatograph box was opened and the sheets were removed and suspended by a pair of clips in a current of air until dried. The locat,ion of the spot was found by examination in ultraviolet light and the Ri value was calculated as usual. The temperaturevaried between 200and 250 C, during runs, but closer temperat,ure control was not required. DISCU SSIOK
The R f values obtained are given in Table I . I n general, when t.etrahydrofurfuryl alcohol is introduced into the solvent the free base t,ravels fastest, the nucleosides next, and t8henucleotides slowest' a t a given pH. This is t'he opposit,e of the
order recorded by Carter for isoamyl alcohol and buffer salt. Increasing the p H generally lowers the rate of travel of the nucleotides, making separations from their hydrolysis products someeasier' Choice O f the primary ployed regulates the general rate more than pH. By selection of the most, favorable p H conditions, the bases (except, cytosine and uracil) may be separated, although two runs may be required n.ith different solvent,^; also possible is the separation of the nucleosides by use of different solvents. Separation of the bases from their reppective nucleosides and nucleotides is comparatively easy, except for uracil and ita nucleoside. Separation of the nucleotides from one another is somen-hat bett,er at, pH 3 than 5.6 or 7.9. The presence of isoamyl alcohol increases the rate of t,ravel over that found when I-propanol is part of the solvent. Addit,ion of tetrahydrofurfury1 alcohol to the solvent results in don-er travel except for guanine and xanthine. LITERATURE CITED
(l) c. J . am.Chem. 729 1466 (lg50). ( 2 ) Hotchkiss, R.D., J . 1759 315 (1948)* 13) Markham, R., and Smith, J. D., .Vuture, 163, 250 (1949). (4) Reguera, R. M., and dsimov, I., J . -Am. Chem. Soc., 72, 5781
(1950). (5) T'mbreit, W. W., Burris, R. H., and Stauffer, J. F., "Manometric
Techniques arid Tissue Metabolism," p. 204, Minneapolis, Burgess Publishing Co., 1949. (6) l-ischer, E., and Chargaff, E., J . Bid. C'hem., 168, 781 (1947); 176, 703 (1948).
,,,,
liEce.,.t~o f o r Decel,,her 1y51, .4ccepted February 2 1 , , g , J B , .journal paper so.881 of the xe,,.y o r k state~ ~ station.~
Differentiating Color Test for Fluorene Derivatives EUGENE SAWICKI, Cancer Research Laboratory, University of Florida, Gainesville, Fla. S 1929 Maitland and Tucker (9) reacted fluorene with acetone in the presence of potassium hydroxide to obtain a compound a hich analyzed m ClsHloO and melted at 76" to 78" C. Eight years later, France, Maitland, and Tucker ( 7 ) proved thc structure of this compound to be methyl p-9-fluorenyl- p-methyl-npropyl ketone. The authors reported that a dark blue-green color was formed in this reaction. Fluorene was also condensed with mesityl oxide in the presence of potassium hydroxide t o give the same compound in poorer yield. Again a green color was obtained. This color could have been due to reaction on the fluorene molecule or t o impurities. I t was found in this laboraton that the formation of color was due t o interaction on the fluorene molecule for many fluorene cornpounds reacted in a similar manner. The mechanism for this color reaction has not been thoroughly investigated as yet but owing to the striking findings, i t a-as thought desirable to report the results to date in view of thc analogous work on thiophenes by Hartough (8). It is probable that the 9- position in fluorene is the position attacked, for electron-attracting groups in the 2- position activate the 9- position and do not hinder the color test, while electron-repelling groups in the 2- position which deactivate the 9- position do not give the blue-green color. The reaction of fluorene compounds n i t h acetone, in the presence of alkali, arranges fluorene derivatives into three distinct groups. I n the first group are included fluorene itself, and fluorene derivatives with an electron-attracting group in the 2-
position. These compounds give a blue-green color in the testI n the second group are fluorene derivatives with an electronrepelling group in the 2- position. These compounds do not give any vivid color. I n Table I it is shown that the blue-green color is specific for the fluorene molecule, while in Table I1 the two groups of fluorene derivatives are compared'. Acidic hydrogen interferes with the test for compounds in the first group, as s h o w in Table 111. In the third group, shovm in Table IV, are the fluorenone derivatives and other compounds with electron-attracting groups in t,he ni- positions. Thcsc compounds give a violet color with acetone in the presence of alkali. Canhack (1-6) has made a vigorous st,udy of the react,ion in alkaline solution between m-dinitrobenzenes and conipounds containing activated methylene groups. He postulates that in the rcact,ion between m-dinitrobenzene and acetone in alkaline solution a violet quinonoidlike compound is formed,
ro-
s
0 2
1-
where "the bond between the benzene nucleus and the acetone ion must be regarded as a bond between a dipole and an ion with many resonance forms" ( 2 ) . The mono- and dinitrofluorenones,
~
