Oswaldo 8. S. Godinho Universidade Estaduol de Campinos 13100 Campinos, C.P. 1170, Brosil and Gilberto 1. Braga Faculdade de Farrnoc~a e Odontolog~ode Rlbe1r6o Preto Ribeircio Preto, Brasil
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An Experiment Illustiating Countercurrent Chromatography with Simple Apparatus
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When the partition coefficients of two solutes between two immiscible solvents differ greatly, the solutes can be separated by a single extraction in a separatory funnel. However if the difference between the partition coefficients is not sufficiently great a macroscale multistep process called countercurrent distribution or a microscale process called partition chromatography can be utilized. Countercurrent chromatography (1-7) differs from conventional partition chromatography in that it is performed in the absence of solid support. For this reason it is also called liquid-liquid partition chromatography without solid support. It also differs from the countercurrent distribution method in that it is a continuous nonequilibri.~m process. Interesting experiments involving the separation of indicators which illustrate the countercurrent distribution method have been described (8, 9) To illustrate countercurrent chromatography we propose an apparatus and an experiment involving the separation of the indicators Phenol Red (PR) and Bromcresol Purple (BCP). The use of indicators enables the separation to he followed visually. A 0.05 M N a ~ C 0 3aqueous solution and n-butanol were used as the mobile and stationary phases, respectiveIv . The behavior of the system using the arrangement described in the experimental section is similar to that ohtained with the system described by Tanimura et al. (5) to develop droplet countercurrent chromatography. The possible utilization of an arrangement such as ours is suggested by the paper of Ito and Bowman (3) where they discuss the various forms of countercurrent chromatography.
The experiments presented in this paper are only illustrative of the use of our apparatus. Experiments with the aim of calculating the efficiency of the system or of comparing experimental and calculated values of retention volume of a given solute have not been made. However, we
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Figure 1. Design of apparatus. (A) Cross section of woad support; (8) Longitudinal section of apparatus. L, latex tubing. F, places where fhe Polyefhylenetube is flaltened, G, glass tubing: (C)Distribution of mobile and stationary phases in the helical tube.
static pressure of the liquid column in the buret it is maintained full of the mobile phase, with the aid of a syphon (Figure 1.). However the flow rate must be checked from time to time, and regulated if necessary. Fractions of 0.6 ml are received, 2.4 ml of 0.05 M NazC03 aqueous solution saturated with butanol is added to each, and the absorbance of the solution is measured at 560 and 590 nm. The solution is kept cold before the absorbance measurements are made, to prevent the appearance of some turbidity 19). Discussion
The elution curves obtained by using 32-, 50-, and 70turn coils and a flow rate of one drop evely 5 s are shown in Figures 2A, ZB, and 2C, respectively. The elution curves A, B, and C of Figure 2 show that the separation of the two solutes is improved by increasing the number of turns in the coils. In spite of the use in these experiments of different amounts of solutes it can be concluded that a n increase in the number of turns in a coil increases the efficiency of the system. The efficiency of the system is also increased by decreasing the tuhe diameter and by decreasing the flow rate. However there is a minimum to the diameter of tube which it is possible t o use for a given solvent pair. For example with the solvent pair used in this experiment (n-hutanol-0.05 M Na2C03 aqueous solution) the minimum diameter tubing is 2 mm. In respect to the flow rate it was observed that it cannot he increased to above one drop/s because all the hutanol then tends to he displaced from the tuhe. T h e efficiency in countercurrent chromatography is calculated by using the formula used in gas chromatography Fraction n u d r F i g u r e 2. Elution curves obtained. (A) s a m p l e r c o n t a i n i n g 25 g g of e a c h PR and B C P : number of furns of t h e c o i l . 32. IB) s a m p l e s c o n t a i n i n g 12 p g of PR and 18 p g of B C P : n u m b e r of t u r n s of t h e coil. 50, (CI s a m p l e s c o n t a i n i n g 19 pg of P R and 38 p g of BCP. n u m b e r of t u r n s of the c o i l ,
70.
discuss the possible use of our apparatus for the study of such important aspects of countercurrent chromatography. Experimental
Apparatus A polyethylene tube with an internal diameter of 2 mm is wound helically on a wood support, whose form and dimensions are shown in Figure 1. The tube must be properly flattened at the small plane surfaces, S, at the top and the bottom of the wood support. In this way the passage of the liquid from one half-turn to another is partially obstructed. The polyethylene tubing is immened in a beaker containing hot water before use in order to avoid breakage at the points where it is flattened. The first and the last turns of the t"bing must he sealed at the wood support with adhesive tape. The polyethylene tubing is connected to a 25-ml buret bv means of a ~ i e c eof latex tubinc which is inserted in the system near the first turn. A glass tube ii also connected to the extremity of the polyethylene tube by means of another piece oflatex tubing Procedure
With the aid of the buret the helical tube is filled with hutanol which has been previously shaken with 0.05 M Na2C03 aqueous solution. Then the same carbonate solution is introduced into the tubing by means of the buret with s flow rate of one drop every 5 s. With the glass tube used at the extremity of our apparatus, 100 drops of aqueous solution corresponds to 0.6 ml. At the end of this operation the aqueous solution has replaced the butanol in approximately one half of each turn of the coil and the aqueous solution starts to arrive at the end of the tube. In this stage one observes droplets of water passing through the hutanol phase. If butanol is used in place of water as the mobile phase, the butanol passes through the water and arrives at the end of the tubing. In this ease however there is no formation of droplets. A 0.025 ml volume of 0.05 M NazCOS aqueous solution containing phenol red and bromcresol purple is injected into the latex tubing near the first turn by means of a hypodermic syringe. The buret eontaining the aqueous solution is opened so that the flow rate is regulated to the desired value. Since the flow rate depends on the hydro-
where N is the number of theoretical plates, R is the retention volume, and W is the peak width volume ( I , 2, 5). However, in our particular case the volume of aqueous solution left in the tuhe, which is not involved in the partitioning process, must be subtracted from the retention volume 15). For this reason. if it is desired to calculate the efficiency o f the system, it is preferable to use the apparatus with the coil vertical. In this confieuration all aaumus solution entering the tube is in t h e form of dropletsLpassing through the hutanol phase. In this case R is the proper retention volume of a given solute. Another question is how to calculate the retention volume of a given solute from its partition coefficient. The relation between the retention volume, Vr,, and the partition coefficient, K, of a given solute is given by the equation
where VM is the volume occupied by the mobile phase and Vs is the volume occupied by the stationary phase in the tuhe. The partition coefficient K is given by the equation: K = Cs/Cn where CS is the molar concentration of solute in the stationarv nhase and CM is the molar concentration of solute in theWm.ohilephase. w e have checked the experimental d a t a obtained bv Tanimura e t al. 15) and ohserved that the relation by eqn. (2) is obeyed. As they worked with a system similar t o that described in this paper this relation should he valid in our apparatus. However experiments with the aim of determining the agreement between experimental and calculated values of V Rhave still t o he made. Literature Cited Ill Ito.Y..a n d h w m a n , R.I... Scknee. 167,281 (19701. 121 Im. Y.. and Bowman, R . L . J Chromnfn~r.S c i . 8.31il19701. 121 ito, Y.. and Bowman. R.L.,Anol. C h r m . 43,69A 11971). I41 1to.Y.andBowman. R.L.,.Science. 17:s. 420119711 IS) Tanimura.T.. Pirano. .l..l.. lro. Y..and mowman. R.~ . . s c i k n r e169.64 11970). Harada. R., Kimura. E.. and Nunopaki. K.. .va. 16) Ito. Y.. Weinstein. M.. Aoki, I.. Lure. 212.986 119661. l 11. I71 [to. Y., Aoki. I..Kimura, E., Nunneaki. K.. and Nunopaki. Y.. ~ n o chem., 1679I1969l. I81 Arrewin. B..Padilhs.l., end Her.4n. J.. J. CHEM. EOUC.,39.539119621. 191 Leonard. JI., C. B.. J. CHEM. EDUC.. 41.363 11967).
Volume 52. Number 4, April 1975
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