Purification of 4, 5, 6, 7-Tetrachlorofluorescein by pH-Zone-Refining

Chapter 17

Downloaded by PENNSYLVANIA STATE UNIV on June 28, 2012 | http://pubs.acs.org Publication Date: May 15, 1995 | doi: 10.1021/bk-1995-0593.ch017

Purification of 4,5,6,7-Tetrachlorofluorescein by pH-Zone-Refining Countercurrent Chromatography Effects of Sample Size, Concentration of Eluent Base, and Choice of Retainer Acid 1,3

2,4

1

Kazufusa Shinomiya , Adrian Weisz , and Yoichiro Ito 1

Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 7N322, Bethesda, MD 20892 Office of Cosmetics and Colors, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Washington, DC 20204 2

Various factors involved in pH-zone-refining countercurrent chromatography were investigated in the purification of 4,5,6,7tetrachlorofluorescein, using a two-phase solvent system composed of diethyl ether-acetonitrile-10 mM ammonium acetate (4:1:5). Trifluoroacetic acid (TFA) was added to the sample solution as a retainer acid. The results indicated that 1) increasing the amount of dye improved the yield of pure compounds; 2) increasing the concentration of ammonia in the solvent system increased the concentration of analytes in the mobile phase and shortened the retention time of the major peak; and 3) ammonium acetate in the solvent system could be eliminated and TFA in the sample solution could be replaced by acetic acid without significantly affecting the purification. Countercurrent chromatography (CCC) is a form of liquid-liquid partition chromatography that does not use a solid support (7). The technique avoids complications caused by chromatographic supports, such as adsorptive sample loss and denaturation, tailing of solute peaks, contamination, etc. CCC has been widely used for many years to separate components of natural and synthetic products (2). Recently, a new preparative CCC technique called pH-zone-refining CCC was developed (5) as a consequence of an incidental observation that, during the purification of a bromoacetylated thyroxine analog, the product formed an unusually sharp elution peak (4). Further studies revealed that bromoacetic acid present in the 3

Current address: College of Pharmacy, Nihon University, 7-7-1, Narashinodai, Funabashi-shi, Chiba 274, Japan Guest researcher at Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892

4

This chapter not subject to U.S. copyright Published 1995 American Chemical Society In Modern Countercurrent Chromatography; Conway, W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


17. SHINOMIYA ET AL.

Purification of TCF by pH-Zone-Refining CCC

219

sample solution as a reaction product formed a pH gradient in the mobile phase that concentrated the acidic solute at the steep rear border of the gradient zone. The introduction of various organic acids in the sample solution produced similar peaksharpening effects for many other acidic compounds including the hydroxyxanthene dyes used in the present study. When pH-zone-refining CCC was applied to the purification of a larger quantity of the hydroxyxanthene dye, the sharp elution peak underwent a drastic change (5): The major component in the sample mixture formed a flat broad peak with constant pH behind the pH-gradient zone in the aqueous phase because the mobile phase had become saturated with sample. Within this pH plateau, minor impurities were efficiently excluded according to their partition coefficients, moving toward either end of the plateau and forming sharp peaks. The method was found capable of purifying large quantities of compounds, even if they were only partially dissolved. One of the advantages of this method over other purification techniques is that the efficiency in terms of both yield and purity is improved as the amount of sample is increased. In the chapter by Weisz et al., a quantitative aspect of the separation of components of commercial 4,5,6,7-tetrachlorofluorescein (TCF) is described in terms of recovery, yield, and purity as well as identification of contaminants from commercial TCF samples. The present chapter focuses on the effects of important experimental parameters such as amount of sample, concentration of eluent base in the mobile phase, and choice of retainer acid on purification of TCF. A series of experiments has been performed with a high-speed CCC centrifuge and a two-phase solvent system composed of diethyl ether-acetonitrile-10 mM ammonium acetate (4:1:5). Experimental Reagents. TCF was obtained from Aldrich (Milwaukee, WI). Diethyl ether, acetonitrile, methanol, and trifluoroacetic acid (TFA) were all glass-distilled chromatographic grade and purchased from Burdick & Jackson Laboratories (Muskegon, MI). Other chemicals including ammonium acetate, ammonium hydroxide (aqueous ammonia), and acetic acid were reagent grade and obtained from J.T. Baker Chemical Co. (Phillipsburg, NJ). Apparatus. The present studies were performed using a commercial high-speed CCC centrifuge (P.C. Inc., Potomac, MD). The design of the apparatus is described in detail elsewhere (5). The column used for the present experiments was a semipreparative multilayer coil consisting of 16 coiled layers with a total capacity of about 325 mL. The entire column was made from a single piece of polytetrafluoroethylene (PTFE) tubing, 170 m x 1.6 mm i.d. (Zeus Industrial Products, Raritan, NJ). The β value (the ratio of the revolution radius to the distance from the holder axis to the coil), which is an important parameter determining the hydrodynamic distribution of the two solvent phases in the rotating coil, ranged from 0.5 at the internal terminal to 0.85 at the external terminal. The speed of the apparatus was regulated up to 1000 rpm by a speed control unit (Bodine Electric Co., Chicago, IL).

In Modern Countercurrent Chromatography; Conway, W., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

220

MODERN COUNTERCURRENT CHROMATOGRAPHY

The solvent was pumped with a reciprocating pump (Accu-Flo pump, Beckman Instruments, Inc., Palo Alto, CA, or Milton Roy Minipump, LDC/Milton Roy Co., Riviera Beach, FL), the effluent was monitored with a UV detector (Uvicord S, LKB Instruments, Inc., Bromma/Stockholm, Sweden), and fractions were collected (Ultrorac, LKB Instruments, Inc.).


Preparation of Two-Phase Solvent System and Sample Solutions. The solvent system was prepared by mixing diethyl ether, acetonitrile, and 10 mM aqueous ammonium acetate at a volume ratio of 4:1:5. The solvent mixture was thoroughly equilibrated in a separatory funnel at room temperature. After the two phases were separated, aqueous ammonia (ca. 28%) was added to the lower mobile phase to adjust the pH to the desired level. The sample solution was prepared by dissolving 350 mg of TCF in 10 mL of the solvent system consisting of equal volumes of each phase and then acidifying the solution by adding 200 uL of TFA, unless otherwise specified. The addition of TFA to the sample solution converted TCF to the hydrophobic lactoid form, which partitioned mostly into the upper nonaqueous phase. Because of reduced solubility at the lower pH, the sample formed a precipitate in the upper phase. To disperse the precipitate, the mixture was sonicated, and the resulting homogeneous suspension was injected into the CCC column without filtration. As described earlier, the present method permits loading an undissolved sample into the column (5). CCC Separation Procedure. The purification was performed using a standard procedure previously described (5). The multilayer coil was first entirely filled with the stationary phase (upper nonaqueous phase), and the sample solution was injected through the sample port. Then the mobile phase (lower aqueous phase) was pumped into the column at a flow rate of 3 mL/min while the apparatus was rotated at 800 rpm. The absorbance of the effluent was continuously monitored at 206 nm with a Uvicord S detector, and 3-mL fractions were collected in test tubes. After the purification was complete, the apparatus was stopped and, by connecting the inlet of the column to a pressured N line, the column contents were emptied into a graduated cylinder to measure the volume of the stationary phase retained. 2

Analysis of CCC Fractions. An aliquot of each fraction was diluted with a known volume of methanol, and the absorbance was manually determined at 254 nm with a Zeiss PM6 spectrophotometer. Fractions were also analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC). The HPLC system consisted of a Model LC-6A pump (Shimadzu Scientific Co., Columbia, MD), a manual injector (Rheodyne, Berkeley, CA), a Model SPD-6A detector and a Model C-R5A recording data processor (both Shimadzu Scientific Co.), and a Capcell Pak C18 column, 13 cm x 0.46 cm i.d. (Shiseido, Tokyo, Japan). The flow rate of the mobile phase, composed of methanol-0.1 M ammonium acetate (1:1), was 1.0 mL/min, and the effluent was monitored at 254 nm.


17. SHINOMIYA ET AL.

Purification of TCF by pH-Zone-Refining CCC

221


Results and Discussion Typical Elution Profile and HPLC Analyses of CCC Fractions. Figure 1A shows a typical chromatogram from CCC of TCF. The purification was performed with a solvent system of diethyl ether-acetonitrile-10 mM ammonium acetate (4:1:5) in which the pH of the lower mobile phase was adjusted to 9 by the addition of ammonium hydroxide. The other experimental conditions were as follows: Column, semipreparative multilayer coil with 16 coiled layers and 325-mL total capacity; revolution, 800 rpm; mobile phase, lower aqueous phase; flow rate, 3 mL/min; fractionation, 3 mL or 1 mL/tube. In this chromatogram, the absorbance is shown by two solid lines: The thick line indicates the direct detection at 206 nm, and the thin line indicates the manual measurement (after dilution of an aliquot) of the absorbance at 254 nm with the PM6 Zeiss spectrophotometer. Although the two absorbance curves show similar trends, the direct tracing at 206 nm offers significant advantages as follows (Y. Ito et al., pH-Zone-Refining Countercurrent Chromatography: A New Technique for Preparative Separation, in this monograph): Direct monitoring of absorbance produces a smooth plateau, demonstrating a constant concentration of solute. In the manual measurement, dilution of concentrated solutions becomes a main source of error, resulting in an irregular plateau level. In addition, the sharp peaks at the plateau boundaries are well preserved in the direct tracing. In the manual measurement, these peaks become much broader because of the large volume of each fraction, 3 mL/tube. Because of these advantages, the chromatograms in Figures 2-4 were obtained by direct detection at 206 nm. (Direct detection at 254 nm produced an off-scale chromatogram at the plateau region.) One disadvantage of direct detection at 206 nm is that the absorbance, although responsive to changes in concentration, is not linear with concentration and no scale is given. The absorbance scale for manual measurement at 254 nm is adjusted for dilution of the fractions. The dotted line in the chromatogram indicates the pH of the fraction as measured manually with a pH meter (Accumet 1001, Fisher Scientific Co., Fair Lawn, PA). The pH of the effluent decreases sharply at the.mobile phase front (SF), and then decreases gradually until it rises sharply at 69 min, when an impurity (peak b) elutes. This first pH drop corresponds to elution of acetic acid from the ammonium acetate in the mobile phase. After the sharp rise from 5.6 to 6.5 (b), the pH again starts to fall (c). This second pH drop is produced mostly by the elution of TFA. The pH increases sharply at 95 min, when another impurity (peak d) elutes. Then the first pH plateau forms at pH 6.2 and 98 to 104 min, which corresponds to elution of a relatively large amount of a colorless impurity. At the end of this plateau, the pH rises to 8.1, forming a long second plateau that corresponds to the elution of the major component, TCF. At the begimiing of this main pH plateau (f), impurities form a sharp peak. Finally, the pH curve returns to the original level after 170 min, which coincides with the elution of the last impurities (peak h). HPLC analyses of aliquots of several of the fractions were performed as indicated by arrows in the chromatogram (b-h). As shown in Figure ΙΒ-a, the original sample consisted of one major component, TCF (ca. 86% of the total peak area) and various impurities with a broad range of retention times. The HPLC


222

MODERN COUNTERCURRENT CHROMATOGRAPHY

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Purification of 4, 5, 6, 7-Tetrachlorofluorescein by pH-Zone-Refining

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