Anal. Chem. 2000, 72, 1490-1494
Recycling Foam Countercurrent Chromatography Hisao Oka,*,† Masato Iwaya,‡ Ken-ichi Harada,‡ Makoto Suzuki,‡ and Yoichiro Ito§
Aichi Prefectural Institute of Public Health, Tsuji-machi, Kita-ku, Nagoya 462-8576, Japan, Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468-8503, Japan, and Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
A new sample injection method for foam countercurrent chromatography (CCC), named the “recycle injection system”, has been developed. In this system, the effluent from the liquid outlet is directly returned into the column through the sample feed line so that the sample solution is continuously recycled for repetitive foam fractionation. The utility of this system was demonstrated in the separations of microcystin extract and bacitracin complex from large volumes of sample solution. Microcystins were separated and enriched in decreasing order of hydrophobicity. Bacitracin A, a hydrophobic major component, in the bacitracin complex was highly enriched in the foam fraction and almost completely isolated from other components. This recycling foam CCC method may be effectively applied for separation and enrichment of various foam-active components from crude natural products. Recently, an innovation of the foam separation technology has been achieved by the development of foam countercurrent chromatography (CCC), which uses a long coiled separation column in a centrifugal force field.1 Introduction of a sample mixture into the coiled column, either batchwise or continuously, results in the separation of the sample components: molecules with a foam-producing capacity or foam affinity quickly move with the foaming stream and are collected through the foam outlet, whereas the remaining molecules are carried with the liquid stream in the opposite direction and eluted through the liquid outlet. The utility of foam CCC has been demonstrated for the fractionation of commercial bacitracin, a peptide antibiotic.2,3 Hydrophobic components were enriched with foam and collected in decreasing order of polarity. The results indicate that foamactive components can be effectively separated up to a sample size of 0.5 mL with batch sample injection using nitrogen and distilled water without a surfactant or other additives.2 Although the sample size can be further increased by continuous sample injection, the efficiency of separation is limited to a length of the separation column. * Corresponding address: (phone) 81-52-911-3111; (fax) 81-52-913-3641; (e-mail) hisaooka@alles. or.jp. † Aichi Prefectural Institute of Public Health. ‡ Meijo University. § National Institutes of Health. (1) Oka, H. In High-Speed Countercurrent Chromatography; Ito, Y., Conway, W. D., Eds.; Wiley, New York, 1996; Chapter 5, pp 107-120. (2) Oka, H.; Harada, K.-I.; Suzuki, M.; Nakazawa, H.; Ito, Y. J. Chromatogr. 1989, 482, 197. (3) Oka, H.; Harada, K.-I.; Suzuki, M.; Nakazawa, H.; Ito, Y. J. Chromatogr. 1991, 538, 213.
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The present method provides an excellent solution to the above problem by recycling the effluent through the separation column, which becomes equivalent to increasing the effective column length for foam separation. Consequently, this recycling injection method radically improves the efficiency of foam separation by manyfold. EXPERIMENTAL SECTION Reagents. Bacitracin was purchased from P-L Biochemicals, Inc. (Milwaukee, WI). Preparation of Extract of Microcystins. Water bloom samples were collected in Lake Suwa, Japan, in 1991 and designated as 917S. Lyophilized cells (1.0 g) were extracted three times with 40 mL of distilled water for 30 min while stirring, and the extracts were combined. HPLC Conditions. Bacitracin. HPLC column; Capcell Pak C18 (5 µm, 150 × 4.6 mm, i.d., Shiseido, Tokyo, Japan); mobile phase, methanol-0.04 M disodium hydrogen phosphate solution (62:38); flow rate, 1 mL/min; detection, 234 nm. Microcystins. HPLC column, Cosmosil 5C18-AR (5 µm, 150 × 4.6 mm, i.d., Nacarai, Osaka, Japan); mobile phase, methanol0.05 M phosphate buffer (58:42, pH 3.0); flow rate, 1 mL/min; detection, 238 nm. Recycling Foam CCC Apparatus. The apparatus used for the present study is a multilayer coil planet centrifuge that was designed and fabricated at the National Institutes of Health, Bethesda, MD. Since the design of this prototype was reported in detail elsewhere,2 only a brief description is given here: The apparatus holds a pair of column holders symmetrically on the rotary frame at a distance of 20 cm from the central axis of the centrifuge. The coiled column was mounted on the gear-side holder which undergoes a planetary motion in such a way that the holder rotates about its own axis and simultaneously revolves around the central axis of the centrifuge at the same angular velocity in the same direction. This synchronous planetary motion of the holder produces countercurrent movement of two phases through the coiled column, the heavier phase (liquid) moving toward one end of the coil called the head and the lighter phase (foam) moving toward the other end called the tail. The above planetary motion also prevents twisting of the flow tubes and permits the continuous passage of gas and liquid through the rotating column without the use of rotary seals. The separation column consists of 10 m long, 2.6 mm i.d. poly(tetrafluoroethylene) (PTFE) tubing (Zeus Industrial Products, Raritan, NJ) coaxially wound around the holder hub making a total capacity of 50 mL. As schematically illustrated in Figure 1, 10.1021/ac990974d CCC: $19.00
© 2000 American Chemical Society Published on Web 02/26/2000
Figure 1. Foam CCC system with recycle sample injection.
the coiled column is equipped with five flow tubes, the liquid outlet and the N2 inlet at the head end and the foam outlet and liquid inlet at the tail end, while the sample inlet opens at the middle portion of the coil. A needle valve is placed on the liquid outlet line to regulate the liquid flow through the head of the coil. The general procedure for foam CCC using recycle sample injection is as follows: (1) Clamp the liquid feed inlet (no liquid feed is employed), (2) rotate the column at 500 rpm, (3) fully open the needle valve 1, (4) introduce nitrogen gas at 80 psi through the gas feed line, (5) introduce the sample at a flow rate of 9.0 mL/min through the sample feed line for 20 min, (6) stop the pumping, (7) close the needle valve 1, (8) resume the pumping at a flow rate of 1.0 mL/min, (9a) when foam emerges, fractionate effluents from foam outlet at 2.5 min intervals, (10) increase the flow rate to 1.5 mL/min, (9b) when there is failure to elute at step 8, increase the flow rate to 1.5 mL/min or until the foam is eluted, and (11) analyze the fractionated eluate by HPLC. RESULTS AND DISCUSSION In foam CCC, the sample solution is introduced from the sample inlet at the middle portion of the coiled column where it is immediately mixed with N2 and the generated foam moves with the N2 stream toward the foam outlet located at the tail (Figure 1). Since the coiled column consists of a 10 m long tube, the foam is forced to travel through a 5 m long narrow coiled path before it reaches the foam outlet. As the foam travels through the column being exposed to a gradually decreasing pressure gradient, every bubble is expanded while the excess fluid is removed by centrifugal force. Therefore, it is reasonable to assume that the foam is subjected to a repetitive process of coalescence, eruption, and regeneration before reaching the foam outlet at the tail. Consequently, successful foam CCC requires a strong foamproducing capability and foam stability of analytes. A lack of either capacity would result in its failure unless the column length is much reduced. As mentioned earlier, foam-active components in the sample solution introduced into the coiled column generate foam and quickly move with N2 toward the tail, while the rest of the components are carried with the liquid stream in the opposite direction and accumulated at the head end of the column. Since the column capacity is limited, the volume of the sample solution thus retained at the head of the column affects the traveling distance of the foam. Thus, reducing the retained sample volume increases efficient column space, which permits the elution of strongly foam-active components with high foam stability. Less foam-active components may be eluted if the effective column length is reduced by increasing the retained sample volume.
In the foam CCC system, there are two major factors to determine the foam retention volume: One is the opening of the needle value located at the liquid collection line, which regulates the liquid output, and the other, a flow rate of the sample solution through the sample feed line. Thus, it is assumed that by manipulating these two variables foam-active components may be efficiently separated according to their foam stability. Our preliminary studies, however, have shown that manipulating the needle value failed to effectively regulate the foam retention volume for separation of natural products. Therefore, we have considered a different approach to improve the separation by recycling the effluent from the liquid outlet back into the sample feed line. In the present study, this recycling foam CCC technique is applied to the separation of microcystin extract and bacitracin components, both possessing sufficient foam activities. Separation of Microcystins. Microcystins are hepatotoxic cyclic peptides produced by cyanobacteria with foaming properties.4 We extracted microcystins from the 917S with distilled water and obtained an extract. A 100 mL aliquot of the extract was subjected to recycling foam countercurrent chromatography by varying the flow rates of the sample solution. Figure 2A shows a typical HPLC chromatogram of an extract from cyanobacteria bloom sample 917S containing microcystins. We chose peaks 1 (microcystin RR), 2 (microcystin YR), 3 (microcystin LR), and 4 (microcystin LR-s) to evaluate the separation efficiency of the present foam CCC. The extract yielded foam fractions at flow rates between 3.0 and 5.0 mL/min (Figure 3), and we obtained 16 fractions from the foam collection line. Distribution of microcystins in the foam fraction is shown in Figure 4. In the first fraction, peak 5 containing the most hydrophobic component is enriched. Then, in a decreasing order of hydrophobicity, enrichment of peaks 4, 3, 2, and 1 is followed with their maximum values in fractions 2, 4, 6, and 14, respectively. The HPLC analyses of the original sample and foam fractions indicated by arrows in Figure 3 are shown in Figure 2. In the first fraction, peaks 4 and 5 show enrichment by 192 and 230 times that in the original sample solution, respectively. Peaks 2-5 were enriched 224-282 times in the fourth fraction. Although peaks 2 and 3 were enriched 232 and 151 times, respectively, in the sixth fraction, enrichment of peaks 4 and 5 was reduced to 51-52 times. In the 11th, 14th, and 15th fractions, peak 5 disappeared, whereas peaks 1-4 continued to elute from the foam collection line. These results indicate that the recycling foam CCC method can effectively separate and enrich microcystin components according to their hydrophobicity from a large volume of the sample solution. Bacitracins. Bacitracin (BC) is a basic cyclic peptide antibiotic which consists of more than 15 components including two major components, BC-A and BC-F. Recently, the structures of other components have been elucidated.5 Figure 5A shows an HPLC chromatogram of the bacitracin complex. In the present study, we paid special attention to six peaks, i.e., peaks 4, 6, and 8-11 to evaluate foam separation and enrichment in recycling foam CCC. According to the procedure described in the Experimental Section, recycling foam CCC of bacitracin was performed using a (4) Harada, K.-I. In Toxic Microsystis; Watanabe, M. F., Harada, K.-I., Carmaichael, W. W., Fujiki, Eds.; CRC Press: New York, 1995; Chapter 6, pp 103148. (5) Ikai, Y.; Oka, H.; Hayakawa, J.; Matsumoto, M.; Saito, M.; Harada, K.-I.; Mayumi, T.; Suzuki, M. J. Antibiot. 1995, 48, 233.
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Figure 2. HPLC analysis of microcystins in foam fractions: (A) original sample, (B) fraction 1, (C) fraction 4, (D) fraction 6, (E) fraction 11, (F) fraction 14, and (G) fraction 15.
Figure 3. Elution curve of microcystins from foam line. Flow rate (mL/min): fractions 1-2, 3.0; fractions 3-4, 3.5; fractions 5-8, 4.0; fractions 9-13, 4.5; fractions 14-16, 5.0.
100 mL sample solution at two different concentrations of 300 and 50 ppm. At 300 ppm, foam eluted at flow rates between 1.0 and 4.0 mL/min (Figure 6) and we obtained 73 fractions from the foam collection line. Distribution of bacitracin components in these foam fractions is shown in Figure 7, and the HPLC analyses of the original sample and foam fractions (indicated by arrows in Figure 1492 Analytical Chemistry, Vol. 72, No. 7, April 1, 2000
Figure 4. Distribution of microcystins in foam fractions: Flow rate (mL/min): fractions 1-2, 3.0; fractions 3-4, 3.5; fractions 5-8, 4.0; fractions 9-13, 4.5; fractions 14-16, 5.0.
6) are shown in Figure 5. In the first fraction corresponding to peak 11 (BC-F), the most hydrophobic component is enriched 196 times that in the original sample solution (Figure 5). Then, peaks 8 (BC-A), 4, and 6 were eluted from the foam collection line in a decreasing order of hydrophobicity with their maximum values in fractions 22, 52, and 59, respectively (Figure 7). All components show substantial enrichment relative to those in the
Figure 5. HPLC analysis of bacitracin (300 ppm) in foam fractions: (A) original sample, (B) fraction 1, (C) fraction 22, (D) fraction 52, and (E) fraction 59.
Figure 6. Elution curve of bacitracins (300 ppm) from foam line. Flow rate mL/min): fractions 1-3, 1.0; fractions 4-29, 1.5; fractions 30-41, 2.0; fractions 42-51, 2.5; fractions 52-59, 3.0; fractions 6063, 3.5; fractions 64-73, 4.0.
original sample solution. As shown in Figure 5, peak 8 (BC-A) in fraction 22 was enriched 22 times and almost isolated from other components as observed in our previous study.2 After elution of peak 8 (BC-A), still more hydrophilic components were collected from the foam collection line. The overall results of the above studies show that foam CCC separates bacitracin components according to their hydrophobicity, yielding efficient enrichment of their components ranging from 10 to 196 times. The present method was applied to a sample solution of bacitracin complex at a concentration of 50 ppm, which failed to
Figure 7. Distribution of bacitracin components (300 ppm) in foam fraction. Flow rate (mL/min): fractions 1-3, 1.0; fractions 4-29, 1.5; fractions 30n 41, 2.0; fractions 42-51, 2.5; fractions 52-59, 3.0; fractions 60-63, 3.5; fractions 64-73, 4.0.
yield foam fractions in the standard foam CCC technique using a batch sample injection.6 The recycling foam CCC technique successfully eluted the foam at flow rates of 4.0 and 4.5 mL/min, and we obtained 12 fractions. As observed in the 300 ppm sample solution, each component was eluted in decreasing order of hydrophobicity. Although too little BC-A (peak 8) was isolated as compared with the 300 ppm sample, each component was separated and enriched 18-246 times, which substantially exceeds the results obtained from the 300 ppm sample. The above studies (6) Oka, H.; Iwaya, M.; Harada, K.-I.; Murata, H.; Suzuki, M.; Ikai,Y.; Hayakawa, J.; Ito, Y. J. Chromatogr., A 1997, 791, 53.
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demonstrate that the present recycling foam CCC technique can be applied to a dilute sample solution that is not separable by batch sample injection. CONCLUSION In this recycling foam CCC system, the effluent from the liquid outlet is directly returned into the column through the sample feed line so that the sample solution is continuously recycled. The utility of this method is demonstrated in the separation and
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enrichment of microcystin and bacitracin complexes from a large volume of sample solution. The method elutes the foam-active components in the order of their hydrophobicity. The present method may be effectively applied to separation and enrichment of various foam-active components from crude natural products. Received for review August 25, 1999. Accepted January 5, 2000. AC990974D
