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Stationary phase retention and preliminary application of a spiral disk assembly designed for high-speed counter-current chromatography
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Journal of Chromatography A, 1188 (2008) 164–170
Stationary phase retention and preliminary application of a spiral disk assembly designed for high-speed counter-current chromatography Xueli Cao a , Guanghui Hu a , Liangsheng Huo b , Xiaoping Zhu b , Ting Li a , Jimmie Powell c , Yoichiro Ito d,∗ a
Beijing Technology and Business University, Beijing Key Lab of Plant Resource Research, Beijing 100037, China b Beijing Technology and Business University, Mechanical Automation College, Beijing 100037, China c Machine Instrumentation Design and Fabrication, National Institutes of Health, Bethesda, MD 20892, USA d Bioseparation Technology, Biochemistry and Biophysics Center, Bldg. 10, Room 8N230, National Heart, Lung, and Blood Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA Received 23 November 2007; received in revised form 2 February 2008; accepted 6 February 2008 Available online 4 March 2008
Abstract A spiral disk assembly composed of five single-channel units was designed for high-speed counter-current chromatography (HSCCC). The retention of different solvent systems ranging from moderately polar to polar organic–aqueous systems to aqueous two-phase systems (ATPs) was investigated under different elution modes. The results indicated that the spiral disk assembly can produce excellent retention of stationary phase for moderately polar organic–aqueous solvent systems, such as chloroform–methanol–water (4:3:2) and hexane–ethyl acetate–methanol–water (1:1:1:1) by pumping lower mobile phase from head (H) to tail (T), and upper mobile phase from tail (T) to head (H) even at a high flow-rate (8 mL/min, Sf > 70%), regardless of whether the inlet is at the inner or outer terminal of the channel. This makes it possible for fast analysis of some small molecular compounds. This has been proved in the separation of mixtures of three flavones, including isorhamnetin, kaempferol, and quercetin. The spiral disk assembly can also provide satisfactory retention for polar to ATPS such as 1-butanol–acetic acid–water (4:1:5) (<3 mL/min, Sf > 70%), 12.5% poly(ethylene glycol) (PEG) 1000–12.5% K2 HPO4 –75% water (≤1 mL/min, Sf > 70%) and 4% PEG 8000–5% Dextran T500–91% water (≤0.5 mL/min, Sf > 50%) by pumping lower mobile phase from inner terminal (I) to outer terminal (O), and upper mobile phase from outer terminal (O) to inner terminal (I) at a low flow-rate, while this is not possible with the multilayer coil column. Acceptable resolutions were achieved when it was used for the separation of peptides such as Leu-Tyr and Val-Tyr, and proteins including cytochrome c and myoglobin, lysozyme and myoglobin, and fresh chicken egg-white proteins. © 2008 Elsevier B.V. All rights reserved. Keywords: High-speed counter-current chromatography; Spiral disk assembly; Retention of stationary phase; Two-phase solvent system; Aqueous two-phase system; Protein separation
1. Introduction The commonly used high-speed counter-current chromatography (type-J HSCCC) instrument (also known as coil planet centrifuge, CPC) usually uses a multilayer coil as a separation column. The column is made simply by winding a single piece of polytetrafluoroethylene (PTFE) tubing in several layers on a spool [1]. The HSCCC centrifuge based on multilayer coil can produce a highly efficient separation with good retention
∗
Corresponding author. Tel.: +1 30149 61210; fax: +1 30140 23404. E-mail address: [email protected] (Y. Ito).
0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.02.073
of stationary phase for a variety of two-phase solvent systems, especially for those organic–aqueous systems widely used for the separation of small molecular compounds in natural products [2,3]. However, it often fails to retain a satisfactory amount of the stationary phase for highly viscous, low interfacial tension aqueous two-phase systems (ATPSs), that are useful for the separation of biological macromolecules [4]. The cross-axis CCC has been investigated and applied for separation of macromolecules using ATPS [2,4], but due to its complex design, lower stationary phase retention and lower peak resolution, the cross-axis CCC was not commercialized until now. In the conventional type J high-speed CCC centrifuge, the retention of stationary phase almost entirely depends upon the Archimedean
X. Cao et al. / J. Chromatogr. A 1188 (2008) 164–170
screw force produced by the planetary motion of coil, which can be improved by forming a spiral tube configuration to increase the radially acting centrifugal force gradient. However, in the traditional spiral column design, the spiral pitch is limited by the outer diameter of the PTFE tubing. During recent years, a spiral disk column design was devised to increase the pitch of the spiral [5–7]. In this paper, using a multiple spiral disk assembly designed and manufactured in our laboratory, we have investigated the retention of a series of solvent systems including both typical organic–aqueous systems and ATPSs. In addition, its preliminary application in the separation of small molecular flavones, peptides, and macromolecular proteins was demonstrated. 2. Experimental 2.1. Design and development of the apparatus A type-J coil planet centrifuge with a 9.7 cm revolution radius was designed as seen in Fig. 1A, the dimension is 66 cm long, 50 cm wide, and 58 cm high. A spiral disk assembly was mounted on one side and a counterweight on the other side to balance the planet centrifuge system as shown in Fig. 1B, which is a top view of the inside without the upper cover. The β values
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range from 0.24 to 0.78. The column consists of five single-spiral disks, each of them sandwiched by a pair of PTFE septum, they are held between a pair of stainless-steel flanges using a multiple set of screws around the inner and outer edges. The top flange is equipped with a plastic gear, which engages to an identical stationary gear mounted on the central shaft of the CPC machine. Each disk made of polymonochlorotrifluoroethylene (Kel-F) has a single-spiral groove (2.5 mm wide, 2.0 mm deep, 4.0 mm pitch), which starts at the inner terminal (I) and ends at the outer terminal (O) (see Fig. 1C). It forms a spiral channel when sealed with a PTFE septum equipped with a transfer hole (see Fig. 1D). When a liquid is introduced from a flying lead and a hole made on the upper flange, it flows straight through the hole on a septum to reach the inner terminal (I) of the spiral channel on the first disk, then to the outer terminal (O), and through a hole inside the outer terminal (O) to reach the other side of the disk, and then it follows through a radial narrow groove (1 mm wide and 1 mm deep) to reach the point just behind the inner terminal (I) and then through the transfer hole on the septum to reach the inner terminal (I) of the next disk. In this way, the spiral channels in different disks are connected in series. Finally, the liquid exits the disk column through the hole made on the lower flange and flows out of the machine through another flying lead. The liquid
Fig. 1. Photos of the prototype J coil planet centrifuge equipped with a spiral disk column. Dimension: 66 cm × 50 cm × 58 cm. (A) The front appearance of the machine. (B) Top view of the inside without upper cover. (C) The spiral disks with a single-spiral channel. I, inner terminal; O, outer terminal. (D) The septums equipped with a transfer hole.
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X. Cao et al. / J. Chromatogr. A 1188 (2008) 164–170
can also be introduced in a reversed way. The total capacity of the above-mentioned column is 74 mL. 2.2. Reagents Hexane, ethyl acetate, methanol, chloroform, 1-butanol, acetic acid, potassium phosphates (K2 HPO4 and KH2 PO4 ) were purchased from Beijing Chemical Reagent Co. (Beijing, China) in analytical grade. Poly(ethylene glycol)s (PEGs) 1000 and 8000, Dextran T500, and test samples including leucyl-tyrosine (Leu-Tyr), valyl-tyrosine (Val-Tyr), myoglobin, cytochrome c, lysozyme, conalbumin, and ovalbumin were all obtained from Sigma–Aldrich (St. Louis, MO, USA). Isorhamnetin, kaempferol, and quercetin were prepared in our laboratory. Reagents used for electrophoresis include acrylamide, N,N methylene-bisacryl amide, sodium dodecyl sulfate (SDS), ammonium persulfate, bromophenol blue, -mercaptoethanol (Sigma–Aldrich). 2.3. Preparation of solvent systems and sample solution The following two-phase solvent systems were prepared: chloroform–methanol–water (4:3:2, v/v), hexane–ethyl acetate–methanol–water (1:1:1:1, v/v), 1-butanol–acetic acid–water (4:1:5, v/v), 12.5% PEG 1000–12.5% K2 HPO4 –75% water (w/w), 4% PEG 8000–5% Dextran T500–91% water (w/w). Each solvent system was prepared by thoroughly equilibrating it in a separatory funnel at room temperature and two-phases separated shortly before use. The sample solutions were prepared by dissolving the sample mixture in the lower or upper mobile phase used for the separation. The sample solution for chicken egg-white was prepared according to the method described by Awade et al. [8]. One volume of fresh chicken egg-white was mixed with two volumes of 0.05 M Tris–HCl (pH 9.0) containing 0.4 M NaCl and 10 mM -mercaptoethanol, and the mixture was gently stirred overnight. The solvent system composed of 16% PEG 1000–12.5% (K2 HPO4 + KH2 PO4 )–71.5% water, pH 8.0 was employed for the separation of chicken egg-white protein according to Shibusawa et al. [9]. The sample solution for CCC was then prepared by adding 0.8 g of PEG 1000 and 0.6 g of potassium phosphate (mixture of monobasic and dibasic potassium phosphates) to 3.6 g of the egg-white solution, to fit with the composition of the solvent system used for the separation. 2.4. Studied on the retention of different solvent systems A series of experiments was performed to evaluate the retention of different solvent systems in different rotation directions, different mobile phases, different flow rates and different flow ways. The experiments were carried out as follows: the column was firstly filled entirely with the stationary phase, and then the apparatus was rotated at 800 rpm in a certain direction while the mobile phase was pumped into the column at a given flow-rate and flow direction. A graduated cylinder was used to collect the stationary phase eluted out from the column. After the
dynamic equilibrium was established without carryover of the stationary phase, the volume of stationary phase pushed out from the column was measured to determine the percentage retention of stationary phase or Sf . Then the flow-rate was increased to another value, more stationary phase was pushed out, and the Sf was calculated again. In this way, the retention of stationary phase at different flow rates was measured. The experiments were performed in two rotation directions: clockwise and counter clockwise viewing from the tube flying lead side which was opposite to the channel grooved side of the disk. In other words, viewing Fig. 1C from the reversed side of the drawing. When the column is rotated clockwise, the outer channel terminal (O) in each disk was the head (H), and inner terminal (I) was the tail (T), and vice versa. The following eight different flow modes were examined. In this notation, the phase pumped (the mobile phase) is indicated by the first letter (L or U for lower or upper phase). The second letter indicates the terminal the mobile phase enters (I or O for the inner or outer terminal of the channel). The third letter indicates the head (H) or tail (T) nature of the inlet terminal. Clockwise rotation was used for modes 1–4 and counter clockwise rotation for modes 5–8. (1) L–I–T: lower mobile phase pumped from inner tail to outer head; (2) L–O–H: lower mobile phase pumped from outer head to inner tail; (3) U–I–T: upper mobile phase pumped from inner tail to outer head; (4) U–O–H: upper mobile phase pumped from outer head to inner tail; (5) L–I–H: lower mobile phase pumped from inner head to outer tail; (6) L–O–T: lower mobile phase pumped from outer tail to inner head; (7) U–I–H: upper mobile phase pumped from inner head to outer tail; (8) U–O–T: upper mobile phase pumped from outer tail to inner head. In the present study, the above eight elution modes were examined for each solvent system, except for the PEG–Dextran system in which four elution modes were tested. 2.5. Preliminary separation procedures Based on the above retention experiments, preliminary application of the spiral disk assembly was performed on the separation of both small molecular compounds and macromolecules, including typical flavones (isorhamnetin, kaempferol, and quercetin), dipeptides (Leu-Tyr, Val-Tyr), and proteins (cytochrome c, myoglobin, etc.). After a certain elution mode was chosen, each separation was carried out as follows: the column was first filled with the stationary phase, and then the mobile phase was pumped through the column while the machine was rotated at 800 rpm. Once the equilibrium was established, the sample solution was loaded through an injector valve.
X. Cao et al. / J. Chromatogr. A 1188 (2008) 164–170
The effluent was continuously monitored at 280 nm using a UV detector and fractions collected.
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3. Results and discussion 3.1. Study on the retention of stationary phase
2.6. SDS-PAGE analysis Chicken egg-white proteins were analyzed by SDS-PAGE (polyacrylamide gel electrophoresis) which was carried out with 15% separation and 4% stacking gels. The gels were stained with 0.1% (w/v) Coomassie Brilliant Blue R 250 in 24% (v/v) ethanol containing 8% glacial acetic acid, and destained with 25% ethanol containing 8% glacial acetic acid.
Although the design and development of the above spiral column was intended to improve the retention of the highly viscous, low interfacial ATPS, and finally to be used for separation of bio-macromolecules such as proteins, a series of additional solvent systems including moderately polar and, polar organic–aqueous solvent systems were also investigated to evaluate its versatility for future applications. The retention
Fig. 2. Stationary phase retention of different solvent systems under different elution modes. *(A) Chloroform–methanol–water (4:3:2, v/v); (B) ethyl acetate–nhexane–methanol–water (1:1:1:1, v/v); (C) n-butanol–acetic acid–water (4:1:5, v/v); (D) PEG 1000–K2 HPO4 –water (12.5:12.5:75, w/w); (E) PEG 8000–Dextran T500–water (4:5:91, w/w). The triangle line present the results obtained by Dr. Ito [5].
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of stationary phase of different solvent systems under different operating conditions was summarized in Fig. 2, in which the retention of stationary phase was expressed as Sf (%). The results were compared with those reported by Ito et al. [5]. For moderately polar solvent systems such as chloroform– methanol–water (4:3:2) and hexane–ethyl acetate–methanol– water (1:1:1:1), better retention was obtained in the following four elution modes: U–I–T; L–O–H; U–O–T; L–I–H. For these moderately polar organic–aqueous systems, better retention of stationary phase can be achieved only by pumping lower mobile phase in the head (H) to tail (T) direction, or upper mobile phase in the tail (T) to head (H) direction, regardless of whether the inlet is located at the inner (I) or outer (O) terminals. This was consistent with that of the classical multilayer column on the Jtype CPC [2]. In addition, relatively high retention of stationary phase can be maintained under these four conditions even at a higher flow-rate of mobile phase, especially when lower phase was used as mobile phase where, retention of the stationary phase of over 70% can be attained in the column as the flow-rate increases even to 8 mL/min. More than 50% of stationary phase can still be retained under a higher flow-rate of 16 mL/min. This may due to the big cross-section area of the rectangular channel. This result suggests that the above spiral disk column could be used for fast analysis or preparative separation of some small molecular compounds, and this has been proved by our later experiments. For polar solvent systems such as 1-butanol–acetic acid– water (4:1:5, v/v/v) with lower interfacial tension, the retention value of stationary phase was lower compared to the above less polar systems. The best retention was obtained in the following modes: L–I–T; U–O–H; L–I–H. It seems that for the polar solvent system, higher retention of stationary phase can be achieved by pumping lower mobile phase from inner terminal (I) to outer terminal (O), and upper mobile phase from outer terminal (O) to inner terminal (I), no matter that when the terminal is the head or the tail. This result agrees with the results reported by Ito et al. using a similar spiral disk assembly [5] with an exception that for when the upper mobile phase was pumped from the outer terminal (O) in the tail to head (U–O–T) elution mode, steady retention of stationary phase could not be achieved, although this phenomenon cannot be explained at this moment. A similar phenomenon was also observed for the PEG 1000–12.5% K2 HPO4 –75% water system as discussed below. Unlike the less polar solvent system, increased flow-rate of mobile phase reduces the retention of stationary phase and Sf can be reduced to less than 20%. However, at a lower flow-rate (<3 mL/min), Sf can still maintain a high value (>70%) in L–I–T and U–O–H elution modes. The retention trends for 12.5% PEG 1000–12.5% K2 HPO4 –75% water were similar to that of 1-butanol–acetic acid–water (4:1:5). In this solvent system the increase of flow-rate results in more serious loss of the stationary phase from the column, reaching the retention lower than 10% at a flow-rate of 5–7 mL/min. However, a high retention level of about 70% can still be obtained at a low flow-rate of less than 1 mL/min, or even higher retention of over 80% can be attained
in the L–I–T mode. For the 4% PEG 8000–5% Dextran T500–91% water (w/w) system, the following four conditions were tested: L–I–T, U–O–H, L–I–H, and U–O–T. Among them, the upper mobile phase can produce relatively high retention of stationary phase under a lower flow-rate (<2.0 mL/min), where nearly 50% of lower stationary phase can be retained at a flow-rate of 0.5 mL/min. The retention value this dextran solvent system was much lower than that of above-mentioned systems due to its high viscosity and small difference in density between the two phases. As compared in Fig. 2, the retention values for these solvent systems including 1-butanol–acetic acid–water (4:1:5, v/v/v),
Fig. 3. Separation of a mixture of isorhamnetin, kaempferol, and quercetin using chloroform–methanol–water (4:3:2) system in L–O–H mode. Rotation speed: 800 rpm; sample: 2 mg mixture in 5 mL mixture of stationary and mobile phase; flow-rate: 2 mL/min (A), 4 mL/min (B), 8 mL/min (C); detection: 280 nm.
Fig. 4. Separation of two peptide leucyl-tyrosine (Leu-Tyr), valyl-tyrosine (ValTyr) using 1-butanol–acetic acid–water (4:1:5, v/v) system in U–O–H mode. Rotation speed: 800 rpm; sample: Leu-Tyr (7 mg) + Val-Tyr (5 mg) in 1 mL mobile phase; flow-rate: 2 mL/min (A), 4 mL/min (B); detection: 280 nm.
X. Cao et al. / J. Chromatogr. A 1188 (2008) 164–170
12.5% PEG 1000–12.5% K2 HPO4 –75% water (w/w), and 4% PEG 8000–5% Dextran T500–91% water (w/w) were similar to those reported by Ito et al. [5]. The overall results suggested that the spiral disk column can produce excellent retention of stationary phase for moderately polar organic–aqueous solvent systems even at high flow-rate, and this makes it possible for fast separation of some small molecular compounds. Meanwhile, the spiral disk column can also provide satisfactory retention for polar to aqueous twophase systems at lower flow-rate, and this makes it possible to be used for separation of some polar to water soluble small molecular compounds and macromolecules such as proteins.
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phase at different flow rates in the L–O–H mode. As the flowrate was increased from 2 to 8 mL/min, over 70% of stationary phase was retained, and almost the same resolution was achieved while the separation time was reduced over four times from 90 to about 22 min. 3.2.2. Separation of peptides Fig. 4 shows the separation of two peptides including leucyl-tyrosine (Leu-Tyr), valyl-tyrosine (Val-Tyr) using 1butanol–acetic acid–water (4:1:5, v/v) system by pumping the upper mobile phase in the U–O–H mode. The two peptides were separated with the peak resolution of Rs ≈ 1.0 at flow rates of both 2.0 and 4.0 mL/min.
3.2. Preliminary separation applications Based on the above studies, the performance of the spiral disk assembly was examined for the separation of the following compounds, including typical flavones, peptides, and proteins. 3.2.1. Separation of flavones Fig. 3 shows the separation of three flavones, isorhamnetin, kaempferol, and quercetin, using the chloroform– methanol–water (4:3:2) system by pumping the lower mobile
Fig. 5. Separation of protein mixtures using 12.5% PEG 1000–12.5% K2 HPO4 –75% water system. Rotation speed: 800 rpm; flow-rate: 1 mL/min; detection: 280 nm. (A) Sample: 0.3 mg cytochrome c + 0.3 mg myoglobin in 1 mL mobile phase; elution mode: L–I–T. (B) Sample: 0.2 mg lysozyme + 0.2 mg myoglobin in 3 mL mobile phase; elution mode: U–O–H.
3.2.3. Separation of proteins The above spiral disk assembly was applied to the separation of several protein mixtures using the 12.5% PEG 1000–12.5% K2 HPO4 –75% water system (see Fig. 5). Fig. 5A shows the separation of cytochrome c and myoglobin in the L–I–T mode. The two proteins were well separated. Fig. 5B displays the separation of lysozyme and myoglobin in the U–O–H mode. Fig. 6 presents the separation of chicken egg-white using the 16% PEG 1000 and 12.5% potassium phosphates polymer phase system at pH 8 in the L–I–T mode (see Fig. 6A).
Fig. 6. Separation of chicken egg-white using 16% PEG 1000 and 12.5% potassium phosphates aqueous solution at pH 8 in L–I–T mode and followed by U–O–H mode. (A) HSCCC chromatograms. Rotation speed: 800 rpm; sample: 5 g of chicken egg-white solution prepared in above-mentioned method; flow-rate: 1.5 mL/min; detection: 280 nm. (B) SDS-PAGE analysis of chicken egg-white fractions obtained from (A). Lanes: 1, peak 1; 2, peak 2; 3, peak 2 tail; 4, lysozyme; 5, ovalbumin; 6, conalbumin; 7, chicken egg-white.
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SDS-PAGE analysis in Fig. 6B reveals that conalbumin and ovalbumin can be well separated, while lysozyme was not detected in either the CCC chromatogram or the SDS-PAGE analysis due to its low concentration.
Acknowledgements The authors thank the National Natural Science Foundation of China and the Beijing Municipal Natural Science Foundation for the financial supports.
4. Conclusions References Our retention studies indicated that the spiral disk assembly can produce higher retention of stationary phase for moderately polar solvent systems even at a high flow-rate of the mobile phase, and it can also provide satisfactory retention for polar to aqueous two-phase systems at a lower flow-rate, which is not possible with the conventional multilayer coil column. The preliminary separation of test samples proved that the spiral disk column can produce efficient separation for small molecular compounds with less polar solvent systems, and this is of great significance for fast analysis and preparation of small bioactive metabolites from plant and natural resources. Good resolution can also be obtained for polar peptides and proteins using a polar organic–aqueous solvent system and ATPS, and this makes it possible for the separation of macromolecules, although it still needs to be modified to improve the retention and mass transfer in highly viscous ATPSs.
[1] Y. Ito, CRC Crit. Rev. Anal. Chem. 17 (1986) 65. [2] Y. Ito, W.D. Conway (Eds.), High-Speed Countercurrent Chromatography (Chemical Analysis, vol. 132), Wiley-Interscience, New York, 1995. [3] A. Berthod (Ed.), Countercurrent Chromatography (Wilson & Wilson’s Comprehensive Analytical Chemistry, vol. 38), Elsevier, Amsterdam, 2002. [4] Y.W. Lee, in: J.-M. Menet, D. Thiebaut (Eds.), Countercurrent Chromatography (Chromatographic Science Series, vol. 82), Marcel Dekker, New York, 1999, p. 149. [5] Y. Ito, F.-Q. Yang, P.E. Fitze, J.V. Sullivan, J. Liq. Chromatogr. Rel. Technol. 26 (2003) 1355. [6] Y. Ito, F.-Q. Yang, P.E. Fitze, J. Powell, D. Ide, J. Chromatogr. A 1017 (2003) 71. [7] Y. Ito, L. Qi, J. Powell, F. Sharpnack, H. Metger, J. Yost, X.-L. Cao, Y.-M. Dong, L.-S. Huo, X.-P. Zhu, T. Li, J. Chromatogr. A 1151 (2007) 108. [8] A.C. Awade, S. Moreau, D. Moll, G. Brul, J.L. Maubois, J. Chromatogr. A 677 (1994) 279. [9] Y. Shibusawa, S. Iino, H. Shindo, Y. Ito, J. Liq. Chromatogr. Rel. Technol. 24 (2001) 2007.
Journal of Chromatography A, 1188 (2008) 164–170
Stationary phase retention and preliminary application of a spiral disk assembly designed for high-speed counter-current chromatography Xueli Cao a , Guanghui Hu a , Liangsheng Huo b , Xiaoping Zhu b , Ting Li a , Jimmie Powell c , Yoichiro Ito d,∗ a
Beijing Technology and Business University, Beijing Key Lab of Plant Resource Research, Beijing 100037, China b Beijing Technology and Business University, Mechanical Automation College, Beijing 100037, China c Machine Instrumentation Design and Fabrication, National Institutes of Health, Bethesda, MD 20892, USA d Bioseparation Technology, Biochemistry and Biophysics Center, Bldg. 10, Room 8N230, National Heart, Lung, and Blood Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA Received 23 November 2007; received in revised form 2 February 2008; accepted 6 February 2008 Available online 4 March 2008
Abstract A spiral disk assembly composed of five single-channel units was designed for high-speed counter-current chromatography (HSCCC). The retention of different solvent systems ranging from moderately polar to polar organic–aqueous systems to aqueous two-phase systems (ATPs) was investigated under different elution modes. The results indicated that the spiral disk assembly can produce excellent retention of stationary phase for moderately polar organic–aqueous solvent systems, such as chloroform–methanol–water (4:3:2) and hexane–ethyl acetate–methanol–water (1:1:1:1) by pumping lower mobile phase from head (H) to tail (T), and upper mobile phase from tail (T) to head (H) even at a high flow-rate (8 mL/min, Sf > 70%), regardless of whether the inlet is at the inner or outer terminal of the channel. This makes it possible for fast analysis of some small molecular compounds. This has been proved in the separation of mixtures of three flavones, including isorhamnetin, kaempferol, and quercetin. The spiral disk assembly can also provide satisfactory retention for polar to ATPS such as 1-butanol–acetic acid–water (4:1:5) (<3 mL/min, Sf > 70%), 12.5% poly(ethylene glycol) (PEG) 1000–12.5% K2 HPO4 –75% water (≤1 mL/min, Sf > 70%) and 4% PEG 8000–5% Dextran T500–91% water (≤0.5 mL/min, Sf > 50%) by pumping lower mobile phase from inner terminal (I) to outer terminal (O), and upper mobile phase from outer terminal (O) to inner terminal (I) at a low flow-rate, while this is not possible with the multilayer coil column. Acceptable resolutions were achieved when it was used for the separation of peptides such as Leu-Tyr and Val-Tyr, and proteins including cytochrome c and myoglobin, lysozyme and myoglobin, and fresh chicken egg-white proteins. © 2008 Elsevier B.V. All rights reserved. Keywords: High-speed counter-current chromatography; Spiral disk assembly; Retention of stationary phase; Two-phase solvent system; Aqueous two-phase system; Protein separation
1. Introduction The commonly used high-speed counter-current chromatography (type-J HSCCC) instrument (also known as coil planet centrifuge, CPC) usually uses a multilayer coil as a separation column. The column is made simply by winding a single piece of polytetrafluoroethylene (PTFE) tubing in several layers on a spool [1]. The HSCCC centrifuge based on multilayer coil can produce a highly efficient separation with good retention
∗
Corresponding author. Tel.: +1 30149 61210; fax: +1 30140 23404. E-mail address: [email protected] (Y. Ito).
0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.02.073
of stationary phase for a variety of two-phase solvent systems, especially for those organic–aqueous systems widely used for the separation of small molecular compounds in natural products [2,3]. However, it often fails to retain a satisfactory amount of the stationary phase for highly viscous, low interfacial tension aqueous two-phase systems (ATPSs), that are useful for the separation of biological macromolecules [4]. The cross-axis CCC has been investigated and applied for separation of macromolecules using ATPS [2,4], but due to its complex design, lower stationary phase retention and lower peak resolution, the cross-axis CCC was not commercialized until now. In the conventional type J high-speed CCC centrifuge, the retention of stationary phase almost entirely depends upon the Archimedean
X. Cao et al. / J. Chromatogr. A 1188 (2008) 164–170
screw force produced by the planetary motion of coil, which can be improved by forming a spiral tube configuration to increase the radially acting centrifugal force gradient. However, in the traditional spiral column design, the spiral pitch is limited by the outer diameter of the PTFE tubing. During recent years, a spiral disk column design was devised to increase the pitch of the spiral [5–7]. In this paper, using a multiple spiral disk assembly designed and manufactured in our laboratory, we have investigated the retention of a series of solvent systems including both typical organic–aqueous systems and ATPSs. In addition, its preliminary application in the separation of small molecular flavones, peptides, and macromolecular proteins was demonstrated. 2. Experimental 2.1. Design and development of the apparatus A type-J coil planet centrifuge with a 9.7 cm revolution radius was designed as seen in Fig. 1A, the dimension is 66 cm long, 50 cm wide, and 58 cm high. A spiral disk assembly was mounted on one side and a counterweight on the other side to balance the planet centrifuge system as shown in Fig. 1B, which is a top view of the inside without the upper cover. The β values
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range from 0.24 to 0.78. The column consists of five single-spiral disks, each of them sandwiched by a pair of PTFE septum, they are held between a pair of stainless-steel flanges using a multiple set of screws around the inner and outer edges. The top flange is equipped with a plastic gear, which engages to an identical stationary gear mounted on the central shaft of the CPC machine. Each disk made of polymonochlorotrifluoroethylene (Kel-F) has a single-spiral groove (2.5 mm wide, 2.0 mm deep, 4.0 mm pitch), which starts at the inner terminal (I) and ends at the outer terminal (O) (see Fig. 1C). It forms a spiral channel when sealed with a PTFE septum equipped with a transfer hole (see Fig. 1D). When a liquid is introduced from a flying lead and a hole made on the upper flange, it flows straight through the hole on a septum to reach the inner terminal (I) of the spiral channel on the first disk, then to the outer terminal (O), and through a hole inside the outer terminal (O) to reach the other side of the disk, and then it follows through a radial narrow groove (1 mm wide and 1 mm deep) to reach the point just behind the inner terminal (I) and then through the transfer hole on the septum to reach the inner terminal (I) of the next disk. In this way, the spiral channels in different disks are connected in series. Finally, the liquid exits the disk column through the hole made on the lower flange and flows out of the machine through another flying lead. The liquid
Fig. 1. Photos of the prototype J coil planet centrifuge equipped with a spiral disk column. Dimension: 66 cm × 50 cm × 58 cm. (A) The front appearance of the machine. (B) Top view of the inside without upper cover. (C) The spiral disks with a single-spiral channel. I, inner terminal; O, outer terminal. (D) The septums equipped with a transfer hole.
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can also be introduced in a reversed way. The total capacity of the above-mentioned column is 74 mL. 2.2. Reagents Hexane, ethyl acetate, methanol, chloroform, 1-butanol, acetic acid, potassium phosphates (K2 HPO4 and KH2 PO4 ) were purchased from Beijing Chemical Reagent Co. (Beijing, China) in analytical grade. Poly(ethylene glycol)s (PEGs) 1000 and 8000, Dextran T500, and test samples including leucyl-tyrosine (Leu-Tyr), valyl-tyrosine (Val-Tyr), myoglobin, cytochrome c, lysozyme, conalbumin, and ovalbumin were all obtained from Sigma–Aldrich (St. Louis, MO, USA). Isorhamnetin, kaempferol, and quercetin were prepared in our laboratory. Reagents used for electrophoresis include acrylamide, N,N methylene-bisacryl amide, sodium dodecyl sulfate (SDS), ammonium persulfate, bromophenol blue, -mercaptoethanol (Sigma–Aldrich). 2.3. Preparation of solvent systems and sample solution The following two-phase solvent systems were prepared: chloroform–methanol–water (4:3:2, v/v), hexane–ethyl acetate–methanol–water (1:1:1:1, v/v), 1-butanol–acetic acid–water (4:1:5, v/v), 12.5% PEG 1000–12.5% K2 HPO4 –75% water (w/w), 4% PEG 8000–5% Dextran T500–91% water (w/w). Each solvent system was prepared by thoroughly equilibrating it in a separatory funnel at room temperature and two-phases separated shortly before use. The sample solutions were prepared by dissolving the sample mixture in the lower or upper mobile phase used for the separation. The sample solution for chicken egg-white was prepared according to the method described by Awade et al. [8]. One volume of fresh chicken egg-white was mixed with two volumes of 0.05 M Tris–HCl (pH 9.0) containing 0.4 M NaCl and 10 mM -mercaptoethanol, and the mixture was gently stirred overnight. The solvent system composed of 16% PEG 1000–12.5% (K2 HPO4 + KH2 PO4 )–71.5% water, pH 8.0 was employed for the separation of chicken egg-white protein according to Shibusawa et al. [9]. The sample solution for CCC was then prepared by adding 0.8 g of PEG 1000 and 0.6 g of potassium phosphate (mixture of monobasic and dibasic potassium phosphates) to 3.6 g of the egg-white solution, to fit with the composition of the solvent system used for the separation. 2.4. Studied on the retention of different solvent systems A series of experiments was performed to evaluate the retention of different solvent systems in different rotation directions, different mobile phases, different flow rates and different flow ways. The experiments were carried out as follows: the column was firstly filled entirely with the stationary phase, and then the apparatus was rotated at 800 rpm in a certain direction while the mobile phase was pumped into the column at a given flow-rate and flow direction. A graduated cylinder was used to collect the stationary phase eluted out from the column. After the
dynamic equilibrium was established without carryover of the stationary phase, the volume of stationary phase pushed out from the column was measured to determine the percentage retention of stationary phase or Sf . Then the flow-rate was increased to another value, more stationary phase was pushed out, and the Sf was calculated again. In this way, the retention of stationary phase at different flow rates was measured. The experiments were performed in two rotation directions: clockwise and counter clockwise viewing from the tube flying lead side which was opposite to the channel grooved side of the disk. In other words, viewing Fig. 1C from the reversed side of the drawing. When the column is rotated clockwise, the outer channel terminal (O) in each disk was the head (H), and inner terminal (I) was the tail (T), and vice versa. The following eight different flow modes were examined. In this notation, the phase pumped (the mobile phase) is indicated by the first letter (L or U for lower or upper phase). The second letter indicates the terminal the mobile phase enters (I or O for the inner or outer terminal of the channel). The third letter indicates the head (H) or tail (T) nature of the inlet terminal. Clockwise rotation was used for modes 1–4 and counter clockwise rotation for modes 5–8. (1) L–I–T: lower mobile phase pumped from inner tail to outer head; (2) L–O–H: lower mobile phase pumped from outer head to inner tail; (3) U–I–T: upper mobile phase pumped from inner tail to outer head; (4) U–O–H: upper mobile phase pumped from outer head to inner tail; (5) L–I–H: lower mobile phase pumped from inner head to outer tail; (6) L–O–T: lower mobile phase pumped from outer tail to inner head; (7) U–I–H: upper mobile phase pumped from inner head to outer tail; (8) U–O–T: upper mobile phase pumped from outer tail to inner head. In the present study, the above eight elution modes were examined for each solvent system, except for the PEG–Dextran system in which four elution modes were tested. 2.5. Preliminary separation procedures Based on the above retention experiments, preliminary application of the spiral disk assembly was performed on the separation of both small molecular compounds and macromolecules, including typical flavones (isorhamnetin, kaempferol, and quercetin), dipeptides (Leu-Tyr, Val-Tyr), and proteins (cytochrome c, myoglobin, etc.). After a certain elution mode was chosen, each separation was carried out as follows: the column was first filled with the stationary phase, and then the mobile phase was pumped through the column while the machine was rotated at 800 rpm. Once the equilibrium was established, the sample solution was loaded through an injector valve.
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The effluent was continuously monitored at 280 nm using a UV detector and fractions collected.
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3. Results and discussion 3.1. Study on the retention of stationary phase
2.6. SDS-PAGE analysis Chicken egg-white proteins were analyzed by SDS-PAGE (polyacrylamide gel electrophoresis) which was carried out with 15% separation and 4% stacking gels. The gels were stained with 0.1% (w/v) Coomassie Brilliant Blue R 250 in 24% (v/v) ethanol containing 8% glacial acetic acid, and destained with 25% ethanol containing 8% glacial acetic acid.
Although the design and development of the above spiral column was intended to improve the retention of the highly viscous, low interfacial ATPS, and finally to be used for separation of bio-macromolecules such as proteins, a series of additional solvent systems including moderately polar and, polar organic–aqueous solvent systems were also investigated to evaluate its versatility for future applications. The retention
Fig. 2. Stationary phase retention of different solvent systems under different elution modes. *(A) Chloroform–methanol–water (4:3:2, v/v); (B) ethyl acetate–nhexane–methanol–water (1:1:1:1, v/v); (C) n-butanol–acetic acid–water (4:1:5, v/v); (D) PEG 1000–K2 HPO4 –water (12.5:12.5:75, w/w); (E) PEG 8000–Dextran T500–water (4:5:91, w/w). The triangle line present the results obtained by Dr. Ito [5].
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of stationary phase of different solvent systems under different operating conditions was summarized in Fig. 2, in which the retention of stationary phase was expressed as Sf (%). The results were compared with those reported by Ito et al. [5]. For moderately polar solvent systems such as chloroform– methanol–water (4:3:2) and hexane–ethyl acetate–methanol– water (1:1:1:1), better retention was obtained in the following four elution modes: U–I–T; L–O–H; U–O–T; L–I–H. For these moderately polar organic–aqueous systems, better retention of stationary phase can be achieved only by pumping lower mobile phase in the head (H) to tail (T) direction, or upper mobile phase in the tail (T) to head (H) direction, regardless of whether the inlet is located at the inner (I) or outer (O) terminals. This was consistent with that of the classical multilayer column on the Jtype CPC [2]. In addition, relatively high retention of stationary phase can be maintained under these four conditions even at a higher flow-rate of mobile phase, especially when lower phase was used as mobile phase where, retention of the stationary phase of over 70% can be attained in the column as the flow-rate increases even to 8 mL/min. More than 50% of stationary phase can still be retained under a higher flow-rate of 16 mL/min. This may due to the big cross-section area of the rectangular channel. This result suggests that the above spiral disk column could be used for fast analysis or preparative separation of some small molecular compounds, and this has been proved by our later experiments. For polar solvent systems such as 1-butanol–acetic acid– water (4:1:5, v/v/v) with lower interfacial tension, the retention value of stationary phase was lower compared to the above less polar systems. The best retention was obtained in the following modes: L–I–T; U–O–H; L–I–H. It seems that for the polar solvent system, higher retention of stationary phase can be achieved by pumping lower mobile phase from inner terminal (I) to outer terminal (O), and upper mobile phase from outer terminal (O) to inner terminal (I), no matter that when the terminal is the head or the tail. This result agrees with the results reported by Ito et al. using a similar spiral disk assembly [5] with an exception that for when the upper mobile phase was pumped from the outer terminal (O) in the tail to head (U–O–T) elution mode, steady retention of stationary phase could not be achieved, although this phenomenon cannot be explained at this moment. A similar phenomenon was also observed for the PEG 1000–12.5% K2 HPO4 –75% water system as discussed below. Unlike the less polar solvent system, increased flow-rate of mobile phase reduces the retention of stationary phase and Sf can be reduced to less than 20%. However, at a lower flow-rate (<3 mL/min), Sf can still maintain a high value (>70%) in L–I–T and U–O–H elution modes. The retention trends for 12.5% PEG 1000–12.5% K2 HPO4 –75% water were similar to that of 1-butanol–acetic acid–water (4:1:5). In this solvent system the increase of flow-rate results in more serious loss of the stationary phase from the column, reaching the retention lower than 10% at a flow-rate of 5–7 mL/min. However, a high retention level of about 70% can still be obtained at a low flow-rate of less than 1 mL/min, or even higher retention of over 80% can be attained
in the L–I–T mode. For the 4% PEG 8000–5% Dextran T500–91% water (w/w) system, the following four conditions were tested: L–I–T, U–O–H, L–I–H, and U–O–T. Among them, the upper mobile phase can produce relatively high retention of stationary phase under a lower flow-rate (<2.0 mL/min), where nearly 50% of lower stationary phase can be retained at a flow-rate of 0.5 mL/min. The retention value this dextran solvent system was much lower than that of above-mentioned systems due to its high viscosity and small difference in density between the two phases. As compared in Fig. 2, the retention values for these solvent systems including 1-butanol–acetic acid–water (4:1:5, v/v/v),
Fig. 3. Separation of a mixture of isorhamnetin, kaempferol, and quercetin using chloroform–methanol–water (4:3:2) system in L–O–H mode. Rotation speed: 800 rpm; sample: 2 mg mixture in 5 mL mixture of stationary and mobile phase; flow-rate: 2 mL/min (A), 4 mL/min (B), 8 mL/min (C); detection: 280 nm.
Fig. 4. Separation of two peptide leucyl-tyrosine (Leu-Tyr), valyl-tyrosine (ValTyr) using 1-butanol–acetic acid–water (4:1:5, v/v) system in U–O–H mode. Rotation speed: 800 rpm; sample: Leu-Tyr (7 mg) + Val-Tyr (5 mg) in 1 mL mobile phase; flow-rate: 2 mL/min (A), 4 mL/min (B); detection: 280 nm.
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12.5% PEG 1000–12.5% K2 HPO4 –75% water (w/w), and 4% PEG 8000–5% Dextran T500–91% water (w/w) were similar to those reported by Ito et al. [5]. The overall results suggested that the spiral disk column can produce excellent retention of stationary phase for moderately polar organic–aqueous solvent systems even at high flow-rate, and this makes it possible for fast separation of some small molecular compounds. Meanwhile, the spiral disk column can also provide satisfactory retention for polar to aqueous twophase systems at lower flow-rate, and this makes it possible to be used for separation of some polar to water soluble small molecular compounds and macromolecules such as proteins.
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phase at different flow rates in the L–O–H mode. As the flowrate was increased from 2 to 8 mL/min, over 70% of stationary phase was retained, and almost the same resolution was achieved while the separation time was reduced over four times from 90 to about 22 min. 3.2.2. Separation of peptides Fig. 4 shows the separation of two peptides including leucyl-tyrosine (Leu-Tyr), valyl-tyrosine (Val-Tyr) using 1butanol–acetic acid–water (4:1:5, v/v) system by pumping the upper mobile phase in the U–O–H mode. The two peptides were separated with the peak resolution of Rs ≈ 1.0 at flow rates of both 2.0 and 4.0 mL/min.
3.2. Preliminary separation applications Based on the above studies, the performance of the spiral disk assembly was examined for the separation of the following compounds, including typical flavones, peptides, and proteins. 3.2.1. Separation of flavones Fig. 3 shows the separation of three flavones, isorhamnetin, kaempferol, and quercetin, using the chloroform– methanol–water (4:3:2) system by pumping the lower mobile
Fig. 5. Separation of protein mixtures using 12.5% PEG 1000–12.5% K2 HPO4 –75% water system. Rotation speed: 800 rpm; flow-rate: 1 mL/min; detection: 280 nm. (A) Sample: 0.3 mg cytochrome c + 0.3 mg myoglobin in 1 mL mobile phase; elution mode: L–I–T. (B) Sample: 0.2 mg lysozyme + 0.2 mg myoglobin in 3 mL mobile phase; elution mode: U–O–H.
3.2.3. Separation of proteins The above spiral disk assembly was applied to the separation of several protein mixtures using the 12.5% PEG 1000–12.5% K2 HPO4 –75% water system (see Fig. 5). Fig. 5A shows the separation of cytochrome c and myoglobin in the L–I–T mode. The two proteins were well separated. Fig. 5B displays the separation of lysozyme and myoglobin in the U–O–H mode. Fig. 6 presents the separation of chicken egg-white using the 16% PEG 1000 and 12.5% potassium phosphates polymer phase system at pH 8 in the L–I–T mode (see Fig. 6A).
Fig. 6. Separation of chicken egg-white using 16% PEG 1000 and 12.5% potassium phosphates aqueous solution at pH 8 in L–I–T mode and followed by U–O–H mode. (A) HSCCC chromatograms. Rotation speed: 800 rpm; sample: 5 g of chicken egg-white solution prepared in above-mentioned method; flow-rate: 1.5 mL/min; detection: 280 nm. (B) SDS-PAGE analysis of chicken egg-white fractions obtained from (A). Lanes: 1, peak 1; 2, peak 2; 3, peak 2 tail; 4, lysozyme; 5, ovalbumin; 6, conalbumin; 7, chicken egg-white.
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SDS-PAGE analysis in Fig. 6B reveals that conalbumin and ovalbumin can be well separated, while lysozyme was not detected in either the CCC chromatogram or the SDS-PAGE analysis due to its low concentration.
Acknowledgements The authors thank the National Natural Science Foundation of China and the Beijing Municipal Natural Science Foundation for the financial supports.
4. Conclusions References Our retention studies indicated that the spiral disk assembly can produce higher retention of stationary phase for moderately polar solvent systems even at a high flow-rate of the mobile phase, and it can also provide satisfactory retention for polar to aqueous two-phase systems at a lower flow-rate, which is not possible with the conventional multilayer coil column. The preliminary separation of test samples proved that the spiral disk column can produce efficient separation for small molecular compounds with less polar solvent systems, and this is of great significance for fast analysis and preparation of small bioactive metabolites from plant and natural resources. Good resolution can also be obtained for polar peptides and proteins using a polar organic–aqueous solvent system and ATPS, and this makes it possible for the separation of macromolecules, although it still needs to be modified to improve the retention and mass transfer in highly viscous ATPSs.
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