The Quantitative Separation of Some Dyestuffs' An Application of the Chromatographic Method
WILLIAM RIEMAN I11 Rutgers University, New Brunswick, New Jersey
1HE purpose of this paper is t a present a method of chromatographic analysis suitable for performance by college juniors. The method is designed to be fairly rapid and inexpensive, rather than to achieve the greatest accuracy possible. Water was chosen as the solvent to decrease the expense and the fire hazard. The solutes to be separated and determined are three commercial dyestuffs, uiz., victoria blue B, crystal violet, and auramine. Ruggli and Jeusen2 separated these and other dyes qualitatively with specially activated alumina as the adsorbent. In our method, satisfactory results are obtained with Merck's Reagent ignited aluminum oxide just as it comes from the bottle, without any time-consuming activation process. The apparatus is illustrated in Figure 1. The tube T,15 cm. long, is cut fr0m.a combustion tube with an internal diameter of 1.5 cm. Above it is fitted a 50ml. dropping funnel F. The lower end of the tuhe is connected through a stopcock S to a 60-ml. side-arm pyrex test tuhe R, preferably graduated. Suction is applied to R through the safety trap P. A n opentube mercury manometer (not shown) is connected to P. The tuhe T should be dry a t the beginning of a determination. The dropping funnel is removed, and a wad of absorbent cotton (C in Figure 1) is put in the bottom of the tuhe. The cotton is firmly tamped down with a half-inch wooden dowel. The cotton should 0.1 g. of Johusextend one cm. up the tube. One g. Manville's Hflo-Supercel and 14 + 1 g. of Merck's Reagent ignited aluminum oxide &e mixed by shaking together for two minutes in a dry cork-Stoppered widemouth bottle. This mixture (A in Figure 1) is then transferred to the tube with the aid of a wide-stem funnel. The tuhe is tapped continually with the dowel durinp this addition. Then a disc of filter vauer, 1.5 cm. in diameter, is placed on top of the adsorbent. The stopcock is c'osed, and a vacuum of 20 * Then the stopcock is cm. of is with the dowel a few 'penedp and the tube is more times. The height of the column, including the cotton, is 10.6 to 12.3 cm. The Upper Part the tube is now with a tion 0.002 M with respect to both primary and secondary sodium phosphates. This solution, hereafter called the buffer,has a pH of 7.04. The liquid runs down the
'1[
tube slowly, displacingmost of the interstitial air. More buffer is added to the tuhe as necessary to keep the column always covered with liquid. As soon as the huffer starts to drop into the receiver, the column of adsorbent is ready for use.
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Presented before the Division of Chemical Education at the 100th meeting of the A. C. S., Detroit, Michigan, September 12, 1940. RUGGLI AND JENSEN, "Die chromatographische Adsorptionsanalyse in Anwendung auf wbserige Ldsungen kfinstlicher organischer Farbstoffe."Hdu. Chim. Ada, 18,62443 (1935).
Then the stopcock is closed'without disturbing the vacuum in the trap. The level of huffer is adjusted to he 2 cm. above the top of the column. Then 0.100 ml. of a solution of the unknown dye mixture in 95 per cent ethanol is added to the The unknowns should he prepared to contain in 0.100 ml. a maximum of 1.00 mg. of total dye a minimum of 15 7 of each constituent, After addition of the dye, the stopcock is opened. whenthe meniscus is ahout 5 mm. above the top of the column, the upper part of the tube is rinsed do& with Some buffer from a wash bottle. When the meniscus settles again to about 5 mm. from the adsorbent, the funnel aled with buffer is put in place, and its stopcock is opened. Care must be taken not to let
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the top of the column be exposed to air. If the vacuum is maintained at 20 cm., the liquid will flow a t the rate of 0.7 to 1.1 ml. per minute. As the dyes descend through the column, the separation occurs. Victoria blue is most tcnaciously adsorbed and remains in the upper part of the column. Auramine is least adsorbed and separates as a wide yellow band in the bottom of the adsorbent. Crystal violet, with intermediate adsorbability, forms a distinct band in the center. men the auramine reaches the the separation is complete. At this point, about 20 to 30 ml. of turbid but colorless filtrate will have been collected. The lower edge of the blue band is 2 to 6 nun. from the top of the column, depending on the composition of the mixture. A white band of thickness varying from a fraction of a millimeter to 10 mm. appears between the blue and purple bands. The lower edge of the purple band is 20 to 74 mm. from the top. No white band separates the purple from the yellow. However, the lower part of the purple zone and the upper part of the yellow zone are relatively poor in the respective dyes. Thus a good separation can be obtained mechanically. As soon as the auramine reaches the cotton, the stopcock of the dropping funnel is closed, and the funnel is removed. The liquid remaining above the column is allowed to flow into it, and air is then sucked through the column for five minutes to remove the excess liquid. Then the suction is shut off,and the tube is removed. If drops of liquid remain above the column, they are removed by wiping with a cotton wad on a wooden splint. The small quantity of victoria blue lost in this process is neglected. With the aid of a microspatula, the filter paper and the upper (blue) layer of adsorbent are removed and transferred to a 100-ml. beaker. Care must be taken not to contaminate the blue adsorbent with the violet layer. Then the upper part of thq tube is wiped again with the cotton wad, lest some remaining victoria blue contaminate the crystal violet. TheSviolet layer is then removed and transferred to another 100-ml. beaker. It is impossible to remove all the violet adsorbent without some slight contamination with the auramine. This contamination, however, is not sufficient to interfere with the colorimetric determination of either dye. The upper part of the tube is wiped again. The cotton and yellow zone are then removed through the lower end of the tube and put into a third 100-ml. beaker. Into each beaker is put about 25 ml. of 95 per cent ethanol to desorb the dyes. Each beaker is stirred, and the contents filtered through paper. A paper 9 cm. in diameter sufficesfor the victoria blue, but 12.5cm. papers are required for the other two dyes. The filtrates and ethanolic washings are caught in graduates. The washing is continued until 50 ml. have been collected from the adsorbent with victoria blue, but 100 ml. must be collected in the other two cases to get reasonably complete recovery of the dyes. Each filtrate is
then agitated and compared with a standard solution of the respective dye in a Duboscq colorimeter. The standard victoria blue should contain 100 y in 50 ml. The other standards should contain 100 y in 100ml. The results are shown in Table 1. TABLE I UWCOPPBCTBD RESULTS
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"". knosn
victorin BIX~ crynot violat .r Avrominc N ~ m wTake" F o u " ~ Enar Tokm Found Error Takan Found Error 1 45 64 +I9 140 99 41 220 215 5 2 180 200 +20 220 18s 35 500 37s -122
5
50
52
8
440 50
454 38
9*
+2 +14 -12
+
,
;;! it: is
11
--
-
;:
4 650
: 120 99 - 21 55 460 450 10 190 ,,, ,,, -- , ,
-
;!
ezs 52 164
1
- 25 1 5i -- 263 - 82
'The disc of fitter paper war put in the beaker with the vioiet adoorbent
The low results for auramine and crystal violet are probably due to incomplete desorption of these dyes by the ethanol and to decomposition of these dyes, catalyzed by the alumina. The positive errors for victoria blue require a different explanation. They are due to retention of some crystal violet by the disc of filter paper and subsequent desorption of this crystal violet along with the victoria blue. In most cases the quantity of crystal violet thus contaminating the victoria blue is too small to enable the eye to detect a change in the quality of the blue color. In numbers 1 and 4, however, where the ratio of crystal violet to victoria blue was large, a distinct violet tint was observed when the desorbed victoria blue was examined in the colorimeter. Table 1 reve@s a rough correlation between the.quantity of crystal violet in the unknown and the error for victoria blue. T o test the hypothesis that crystal violet is retained by the disc of filter paper, solutions 9 and 10 were prepared with a large ratio of crystal violet to victoria blue, and they were analyzed by the foregoing procedure except that the discs of filter pap& were put in the beakers with the violet bands. Table 1 reveals that this modification gives good results. This modification cannot be applied unless the filter paper has a violet color, i. e., unless the ratio of crystal violet to victoria blue is large. The foregoing discnssiok of the nature of the errors .suggests the possibility of computing approximate corrections for them. The following empirical corrections were selected. (1) I t was assumed that for every 100 y of crystal violet found (uncorrected), 7 y of crystal violet were mixed with the victoria blue. Therefore 7 per cent of the uncorrected crystal violet found was substracted from the victoria blue found. (2) The uncorrected quantity of crystal violet found was increased by 23 per cent-7 per cent for retention by the disc of iilter paper and 16 per cent for decomposition and incomplete desorption. Of course, in the modified procedure (unknowns 9 and 10) only 16 per cent was added. (3) The quantity of auramine found was increased by 16 per cent to account for incomplete
desorption and decomposition. The corrected results are given in Table 2. TABLE 2 Coaarrcra~R s s m r s
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vic1orio Blue T o k m Found E I I ~ 45 57 +I2 7 180 187 - 8 190 182 15 5 -10 1 50 51 420 451 +31 140 158 +lS 440 447 7 50 38 -12 2 10 12
+
+ + +
ina Error +2@ - 82 0 - 2 +75 10 3 5 0 -17
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+
It may be concluded that the recommended procedure yields results with a relative error of * 13 per cent or better for quantities of dyes over 90 y and a maximum absolute error of * 12 y for smaller quantities. It remains now to explain several steps in the procedure. "HyJ3o-Supercel" is added to the adsorbent to increase its porosity and thus to decrease the time required for the determination. The chromatogram is developed with 0.002 M pri-
mary sodium phosphate and 0.002 M secondary sodium phosphate rather than with water to improve the separation. The action of the phosphate solution is not entirely a matter of buffering, for other buffers of the same pH do not have the same effect. Adsorption of the phosphate undoubtedly plays some role. The unknowns are prepared and added in ethanolic, rather than aqueous, solution to increase their stability. Auramine is particularly unstable in water. The solutions in ethanol were stored in the dark but subjected frequently to ordinary daylight for a period of five months without decomposition. The use of ethanol as a solvent requires a rather concentrated solution of unknown lest sufficient ethanol be introduced with the unknown dye mixture to affect the chromatogram. After the apparatus is assembled and the standard solutions prepared, the student can easily run a determination in two three-hour laboratory periods. During the first period, the chromatogram must be developed, the dyes separated, and the ethanol added to each fraction. Otherwise, if the dyes are left in contact with alumina in aqueous medium, excessive decomposition may occur. The second period is then used for the filtrations and colorimetric determinations.
