A CHROMATOGRAPRIC SEPARATION OF SOME
DYESTUFFS
JORN SHOW, III .ad RAXOLD 91. EEINE Bucknell University, Lawisburg, Pennsylvania
TAE
chromatop;mphic adsorption technique first The preparation of the column for adsorption is as described by Tswett in 1906 is seldom presented in the follon~s. A plug of g l m wool (D) ia tamped firmly introductory organic laboratory. A survey of the into the con~trictedportion of the adsorption tube and literature revealed that few of the separations effected rovelwl with a lnycr of sand (0 not more than 5 mm. can be adapted for student use. For this reason experi- thick. After the sand has been leveled off by gentle ments were d e v i . 4 to acquaint the student with the tapping of the tube with n ruler or dowel, the admrbent principles of chromatography. mixture (R), prepared by shnking together in a wideDyes of unrelated structure were selected for the mouthed bottle 24 grams of activated alumina (Alumistudy because of the rme of separation. P r e ~ i o l l ~num Company of America) and 2 grnms of Hyflo investigators' have fihown that dyes ran be qualitativrly Supercel (JohneAIanville), is added slowly and with r e x h d on adsorption columns and recently Rieman' continued t n ~ d n et:o the tube. Half the nuantities of pre.sented a method of alumina a n d kl&-aid reeommen(lwl nhovb also gave chromatographic analexcellent rrsult-3. The tapping prows9 insnm an ysis for eqtimnting small evenly paekrd column. quantities (> 1.0 mg.) of A piece of filter paper of the anme diameter aa the dyestuffs. For the prescolumn is cut and gently placed on the adsorbent so that ent work it wnsdesired to the surface of the aluminn will not be disturhed by the wparnte larger quantities elution process. of compounds of this The column is now washed with 95 per cent ethanol type. Activated aluminn and suction is gently applid. Eluant is continually \vns employed as the ndaclded so that the top of the column never runs dry. sorbent, and 95 per cent The water aspirator is adjusted so thnt the rate of flow ethanol and n-ater were is about 15 drops per minute. When the first drop of used for developing the eluate falls into the rerriver, the column is ready for chromntograms. T h e U.W. The eluate is allowed to come within 1mm. of the dye mixtures investitop of the alumina a t which time the dye is added all a t gnted werc: (1) methylonce. When the dye solution is 1 mm. nhove the enc blue and water-solrrnrlsorbent, the top of the tube is r i n d with 2 cc. of hIefluomcein,(2) methyl et hnnol. orange and methylme blue, (3) Victoria Hlue I3 and methyl ornogc. Water-Soluble Fluorescein and Methylene B l u . This Excellent results werc separation oRem no difficulty. Five mg. of waterobtnined in ench syxtem soluhle fluorescein (Coleman nnd Bell Co.) and 5 mg. of i n v e s t i g a t e c l . Two methylene blue (J. T. Rnker Chemical Co.) are placed widely .separated bands in n binrh @st tube and dissolved in 3 t o 4 cc. of 95 developed within a very per cent ethanol with vigorous shnking t o insure solushort time nnd solutions t,ion of the dyes. Ethanol is used as the developer. of individual dye comThe water-soluble fluoresrein is held tenaciously by the oonents were isolnted bv alumina while the hand of methylene blue begins t o rontinual elution of thk ( separate almcat immedintely. The separntion is concolumn. The total time w t l o n n-hu tinued nntil the eluate dropping into the receiver is required t o prepare the colorless. The receiver is emptied and water is now column, effect the separation, and collect the eluate is user1 as the elunnt. The wnter-soluble fluorescein three hours. immedintely migrates down the column. The apparatus umd for the separation is shown in the .llrlhyl Orangc and dfelhykne Blue. This separation illustration. The glass column ( A ) was prepared can hc nrhievrrl hy derrloping the column with ethanol. previously by sealing together two lengths of pyrex One mg. of methyl orange nnrl 5 mg. of methylene blue tuhing 8 and 14 mm. in diameter. are disqolved in 2.2 cc. of ethanol. The dye mixture is shakcn v e w thoroughly before pouring on the column. ' RUOOLI.P., AND 1'. JENSEN,HeIV. Chim. Ada.. 18.624(1935). ' I ~ I E M A N ,WM.,J. C n a ~ Eooc., . 18. 131 (1911). The methyl ornngr is nrlnortwl more strongly than the
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methylene blue. It can he collerted by eluting the column with water alter the methylene hlue ha. p a s 4 into the receiver. Mdhyl Orange and Vicforia Blue R. One mg. of methyl orange and 5 mg. of Victorin Blue I3 are die-
JOURNAL
OF CHEMICAL EDUCATION
solved in 2.2 cc. of 95 per cent ethanol. The rhromatogram is developed with ethanol. Methyl orange ngnin passes% the stronger nrlwrption affinity and i~ l o r n k l nenr the top of the colwnn. It mny he elufnl with water.
