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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 wide-
Dyes of unrelated structure were selected for the mouthed bottle 24 grams of activated alumina (Alumi-
study 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 anal- excellent 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 pres- column 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 nd- aclded 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 investi- top of the alumina a t which time the dye is added all a t
gnted werc: (1) methyl- once. When the dye solution is 1 mm. nhove the
enc blue and water-solrr- nrlsorbent, 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 water-
obtnined 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 solu-
short time nnd solutions t,ion of the dyes. Ethanol is used as the developer.
of individual dye com- The water-soluble fluoresrein is held tenaciously by the
oonents were isolnted bv
rontinual elution of thk (
column. The total time
w t l o n n-hu
alumina while the hand of methylene blue begins t o
separate almcat immedintely. The separntion is con-
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
' RUOOLI.P., AND 1'. JENSEN,HeIV. Chim. Ada.. 18.624(1935). shakcn v e w thoroughly before pouring on the column.
' I ~ I E M A N ,WM.,J. C n a ~ Eooc.,
. 18. 131 (1911). The methyl ornngr is nrlnortwl more strongly than the

methylene blue. It can he collerted by eluting the solved in 2.2 cc. of 95 per cent ethanol. The rhromato-
column with water alter the methylene hlue ha. p a s 4 gram is developed with ethanol. Methyl orange
into the receiver. ngnin passes% the stronger nrlwrption affinity and i~
Mdhyl Orange and Vicforia Blue R. One mg. of l o r n k l nenr the top of the colwnn. It mny he elufnl
methyl orange and 5 mg. of Victorin Blue I3 are die- with water.