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 200 and 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, it 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,
~
V O L U M E 24, NO. 7, J U L Y 1 9 5 2
1205 _______
Table I.
Fluorene and analogs
Compound Fluorene 9,9'-BiAuorene Biphenyl p-Terphenyl !?,lO-Dihydroacridi?e Anthrone Dibenzofuran Dibenaothiophene Dihenzoselenophene
Color Blue-green Blue-green Colorless Colorless Light yellow Red Colorless Colorless Colorless
The fact, that 2-acetylfluoreiionc gives a violet color in alkaline alcohol is not surprising. The molecule has two electron-attracting groups in the m- position and also has an activated methylene group. Thus, t,he niolecule can react with itself in alkaline solution t o form the violet complex. 2-Nitrofluorenone, like nz-dinitrobenzene ( 6 ) , rea& with ketones containing an act,ive methylene group (Table V). The sensitivity of the reaction is shown by the fact' that cg-clopentanone gives a discernible color reaction at a dilution of 1 to 10,000 n-it,h an alkaline sat,ur:ited alcoholic solution of 2-nitrofluorenone.
Table 11. Fluorene Derivatives Compound E:lectron-attractiIig 2-Chlorofluorone 2-Nitrofluorene 2-Nitro-7-chlorofluorene 2-Benzoylfluorene 2-acetyl fluorene
2-Benzalaminofluore ne 2-Cinnamalaminofluorene 2-(4'-Dimethylaminohenzalamino)fluorene 2,5-Dinitrofluorene 2,7-Dinitrofluorene 2-Kitro-7-benzoylfluorene 2,7-Diacetylfluorene 2,2'-Difluorenyl ketone Electron-repelling 2-Fluorenediazonium chloride 2-.4minofluorene 2-Acetoxyfluorene 2-Dimethylaminofluorene 2-Acetylaminofluorene 2-Benzoylaminofluorene 2- (o-Carhoxyhenzoylamino) fluorene 2-p-Tosylaminofluorene 2,7-Diaminofluorene Phenyl-2-fluorenylcarbinol Phenyl-2-fluorenyl-S-phenylthiocarbinol 2-(4'-Dimethylaminophenylazo)fluorene 2- (4'-Nitrobeneenesulfonylamino) fluorene
Color
T E S T P R O C E D U R E F O R T A B L E S I , 11, .%ND 111
Hliie-green Blue-green Blue-green Blue-green Green Green Green Green Blue-green Blue-green Blue-green Blue-green Blue-green
To a n-arm solution or suspension of 0.2 gram of the ('ompound in 2 ml. of acetone was added 0.5 gram of powdered eodiuni hydroxide. The mixture was refluxed gently over a steam bath for 5 minutes. h n y change in color Tvas noted.
Brownish Yellow Yellow Light pink Light pink Yellow Pink White ppt. Brownish Pinkish Yellowish Brownish Red-brown
T E S T PROCEDURE FOR TABLE V
Table 111. Influence of Substituents with Active EI?-drogen Compound 2-(o-CarboxybenzoyI)fluorene Fluorene-2-sulfonic acid 2-(4'-Hydroxybenzalamino) fluorene 2-(2',4'-Dihydrosyheni:alaiiii1io)fluorene
Color White ppt. Pink Ye1low pp t , Pellow ppt.
like the ni-dinitrobenzenes, give a negativc reaction in alkaline alcohol. The striking fact that 2,4,5,i-tet~ranitrofluorcnone forms a violet, color in alkaline alcohol is not surprising, for so does 1,3,5-trinit~robcnzenc. This can he attributed t o the fact that both molecules have the requisite order of activity ioi, rea(:tion with the et.hoxide ion to form complexes of this tJ-pi, ( s w a ) :
?-
T E S T P R O C E D U R E F O R T A B L E IV
To a solution or suspension of 0.1 gram of t,he compound in 2 ml. of acetone or alcohol was added 0.5 gram of powdered sodium h>-droxide. The color IWS read in 2 minutes.
T o a solutioii or suspension of 0.1 gram of the ketone in 2 nil. of a saturated alcoholic solution of 2-nitrofluorenone was added 0.5 gram of poTT-dered sodium hydroxide. The color was read within Peveral minutes.
Table IV. Compounds with Two Meta Electron-Attracting Groups Comk>ound 2-h-itrofluorenone 2,5-Dinitrofluorenone 2,7-Dinitrofluorenone 2,4,7-Trinitrofluorenone 2.4.5.7-tetranitro fluorenone 2-Acetylfluorenone 2-(2'-Piperidinoethyl) Riiorenonrcarhoxvlatr
Color with Acetone Violet Violet Violet Violet Violet Purple (on warnling) Violet-bromna
.. . .
Brown-red" Light violet Red violet
2-Nitroanthraqninonr 2,4-Dinitrophenylhydsazine Purple m-Dinitrobenzene Blue-riolet l i Presmce of acidic hydrorren interferes.
.,...
..
cOi&ie,-. ~~
-
Table \-. Ketones Conilioiind Acetone Ifethyl rth>-l ketone Methyl isobutyl ketone I l e t h v l n-anirl krtone Acetophenone Cyclohexanone Cyclopentanone 2-Acetylnaphthalcnr
0-
I
I .
2-acetyl fluorene
2,7-Diacetylflnorene
Canbacak ( 5 , 6) has postulated that it is possible for the mdinitrobenzcnes to react with a series of anions, such as CH,COCH2-, CHJO-, OH-, iYH2-, CK-, etc., to give colors of decreasing intensity dependent on the stability of the quinonoid complex. Thc similarity of the formulas, and of the color reactions with alkaline acetone and alkaline alcohol, of the 2-nitrofluorenones and the nz-dinitrobenzenes, is definitc evidence that the violet color is due in all cases to one general over-all reaction. This is thc reaction in alkaline solution between a compound containing an aromatic ring M ith two or three electron-attracting gioups in nz-positions and a suitably activated anion t o give a violet t o purple complex of uncertain structure. In this respect, compounds containing a methylene group, activated by an electron-attracting group, form suitably activated anions in alkaline solution.
Color 1f-it h rllcohol Ye11o~ Light green Light green Green-brown Dark violet Violet
Color Violet Violet Violet Violet Violet Violet Violet Violet Violet Violet
ACKYOW LEDGRIEZlT
The author is indebted to F. E. Ray for his cooperation and his expert help in asscm1,ling the paper. LlTERATURE CITED
(1) Canback, Teodor, F a m i . I?ei,!/, 46, 802 (1947). (2) I b i d . , 48, 153 (1949). (3) Ihid., p. 217. (4) Canback, Teodor, J . P h a r m . Pharmacoi.. 1, 201 (1949). (5) Ibid., personal comniunication. ( 6 ) Canback. Teodor, Scensk F a r m . Tid., 54, 1 (1950).
(7) France, H., Maitland, P.. arid Tucker, 9. H.. J . Chevz. Soc.. 1937, 1739.
Hartough. H. D., AXAL.? H E M . , 23, 1128 (1951). (9) Maitland, P., a n d Tucker, S. H., J . Cheni. SOC.,1929, 2559 (8)
RECEIVED f o r review Fehrriary 1 1 , 1052. Arcepted April 2 1 , 14.52.
