Bengt Lindblad, Goran Lindstedt,' and Hans-Garan Tiselius University
of
Lund
220 07 Lund, Sweden
I I
Counter-Current Distribution as a Student Laboratory Experiment
O n l y few laboratory exercises have been described in order to illustrate the principles of Craig's counter-current distribution. Arrequin, et a1.,2 andLeonard3haveused asystemof n-butanol and 0.05 M aqueous sodium bicarbonate to separate pairs of acidbase indicators. A serious drawback with this solvent system is that the solubility of n-butannl in water decreases when the temperature rises slightly above room temperature. Thus, if the temperature in the cuvette chamber in the photometer is not kept constant, emulsions are sometimes obtained before the absorbances are measured. Cooling the samples before reading eliminates this source of error, but then water vapor may condense on the cuvettes. Furthermore, centrifugation of the samples may be necessary when the phases separate poorly. These experimental difficulties may be instructive when teaching laboratory assistants, but beginners in chemistry, such as our medical students, find them frustrating. We have therefore used another solvent system which separates readily into two phases; and we have used two colored compounds which have distribution coefficients not too far from unity in this solvent system. The experiment has been used to demonstrate the principles of extraction and counter-current distribution as well as of spectrophotometric analysis. First, the absorption spectra are determined with solutions of each of the two compounds and the absorption maximum of each compound is noted (Fig. 1). The validity of the Lambert-Beer law is tested a t these two wavelengths for both compounds (Figs. 2 and 3), To whom reprint requests should be addressed. l A ~ B.. ~PADILIA, ~ . J.. ~. AND~ H E~R R ~.~NJ.... J.. CHEM. Eouc., 39, 536 (1962). LI:ON.\RD,C. B., Jn., J. C ~ MEDUC., . 44, 363 (1967)
WAVELENGTH nrn Figure 1. Absorption spectra for p-ominoozobenzene (open circler) and I-methylomino.4-N-toluidinoonthr~quiione (closed circler).
"
0
1
2 10-2 gll
4
3
Figure 2. Plot of mbsorbance a t 3 8 0 and 645 nrn versus concentration of I -methylomino-4-N-toluidino(1nthr(1quinone.
0
.--
1
2 10-2 g l l
4
3
Figvre 3. Plot of absorbance ot 3 8 0 and 645 nm versus concentration of paminoambenzene.
and the molar absorbancy indices are calculated. It is then possible to calculate the concentration of each one in a solution of both compounds by absorbance measurements a t these two wavelengths (dichromatic readings). It may be noted that the absorbance of p-aminoazobensene is negligible a t the wavelength for maximal absorbance of 1-methylamino-4-N-toluidinoanthraquinoue which facilitates the calculations. A mixture of the two dyes is then distributed between the solvent phases in five separatory funnels. The distribution of each compound is calculated from the results from the dichromatic readings (Fig. 4). The partial separation of the two compounds may be followed visually. After completion of the experiment the blue color from 1-methylamino-4-N-toluidinoanthraquinone dominates in the upper phase of the first funnels, with a maximum in the second funnel, whereas the yellow color from p-aminoazobenzene is chiefly localized in the lower phase of the last two funnels. I n some intermediate phases a green color shows the presence of a mixture of the two dyes. The distribution coefficients in the present two-phase solvent system are then determined, and these values are used to calculate the theoretical distribution of the compounds (Fig. 5). Volume 46, Number 8, Augusf 1969
/
525
I
I
1
2
3 4 FUNNEL NUMBER
5
Figure 4. Distribution in five separotory funnels of I-methylomino-4-Ntoluidinoenthraquinone lvpper port of the figure) and of p-aminoorobenzene llower port of the figure) between equal volumes of the two solvent pharer obtained by mixing water, methanol, acetone, and petroleum ether ( I : 1 : 3: 5).
21 1
2
-
3 1 FUNNEL NUMBER
I 5
Figure 5. Theoretical distribution in five seporotory funnels of i-methylamino-4-N-toluidinoanthraquinone (upper port of the figure) and of paminoozobenrene llower port of the figure) between the solvent phases referred to in Figure 4. Dirtribvtion coefficients which hove been used ore 2.37 and 0.29, respectirely.
Procedure
The phases are prepared by mixing water, methanol, 4-N-toluidinoanthraquinone and 10 ml from the solution acetone, and petroleum ether (boiling range 40-60°C) of p-aminoazobenzene, and the volume is made up to 50 in the proportions 1 : 1:3: 5. Two liters are made for ml with the upper phase. To the four other funnels are each experiment. After separation into two phases added 50 ml of the same phase; this makes out the the upper phase consists of almost only petroleum ether, stationary phase. Fifty ml of the mobile or lower phase whereas the lower phase is a mixture of water, methanol, are added to the first funnel which is then shaken. and acetone. The two phases are separated and stored The students are told to open the funnel a t intervals in closed bottles. The dyes used are l-methylamino-4during this procedure. When the phases have sepaN-toluidinoanthraquinone (Farbenfabriken Bayer A G, rated the lower phase is transferred to the second funnel Leverkusen, West Germany), obtained as "oil-blue and a new 50-ml volume of the lower phase is added to ASL" from ICeho AB, Stockholm, Sweden, and pthe first funnel. The procedure is then repeated until aminoazobenzene, purchased from BDH, Ltd., Poole, all separatory funnels contain two phases which have Great Britain. Stock solutions of the dyes are made been brought into equilibrium with each other. The immediately before the experiments and are prepared separation of the two dyes is then obvious from the apby dissolving about 40 mg (exact weight noted) of each pearance of the funnels. The absorbance of each fracdye in 100 ml of the upper phase. The solutions may tion is determined a t 380 nm and 645 nm and the conhe stored only in closed bottles as petroleum ether centration of each compound is calculated by using the evaporates readily. Suitable concentrations for meavalues for the molar absorbancy indices. It will be surements of absorption spectra are about S mg/l of apparent that sensitivity of the human eye is much p-aminoazobenzene and about 20 mg/l of l-methyllower a t 350 nm than a t 645 nm as compared with the amino-4-N-toluidinoanthraquinone. The stock soluphototubes. tions are diluted with the upper phase, and measureThe distribution coefficients for the two dyes in this ments are made in stopped cuvettes. Absorption spectra phase system are determined by adding an aliquot (1-2 are determined between 350 and 750 nm. Absorption ml) of the respective stock solution to a separatory maximum for 1-methylamino-4-N-toluidinoanthriaqui- funnel with the two phases. The funnels are shaken, noncis around645 nm and forp-aminoazobenzenearound and when the phases have separated, the concentrations 3S0 nm (Fig. 1). The validity of the Lamhert-Beer of the compounds in each phase are determined spectrolaw is tested with accurately. diluted stock solutions. photometrically. I n a typical student experiment, the Figures 2 and 3 show the results obtained by reading the distribution coefficients obtained were 2.37 for 1absorbances of solutions of the two dyes a t these wavemethylamino-4-N-toluidinoanthraquinone and 0.29 for lengths. The concentrations in Figures 2 and 3 are p-aminoazohenzene. These values are used to calcugiven in gram per liter and not in mole per liter belate the theoretical distribution (Fig. 5). cause the purity of commercial l-methylamino-4-NNote in addition: The choice of a blue and a yeUow toluidinoanthraquinone, which varies between different dye, which are the Swedish national colors, was deterhatches was not specified by the mauufacturer. Howmined by need for dye-stuffs with suitable distribution ever, from didactical reasons, students are requested coefficients, and was not an expression of the nationto calculate the molar ahsorbancy indices as if the comalistic sentiments of the world today. mercial products were pure compounds. Acknowledgment The counter-current distribution is performed in five 2.50-ml separatory funnels. To the first funnel are The technical assistance of Mrs. Lsa Ndrgird is added 10 ml from the stock solution of l-methylaminogratefully acknowledged. 526
/
Journal of Chemical Education
