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Mixer-settler counter-current chromatography with a barricaded spiral disk assembly with glass beads
Journal of Chromatography A, 1151 (2007) 108–114
Mixer-settler counter-current chromatography with a barricaded spiral disk assembly with glass beads Yoichiro Ito a,∗ , Lin Qi b , Jimmie Powell c , Frank Sharpnack c , Howard Metger e , James Yost d , Xue-Li Cao e , Yin-Mao Dong e , Liang-Sheng Huo e , Xiao-Ping Zhu e , Ting Li e a
Center for Biochemistry and Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 50, Room 3334, 50 South Drive, Bethesda, MD 20892-8014, USA b Office of New Drug Quality Assessment, OPS/CDER/FDA, 10903 New Hampshire Avenue, Room 3525, Silver Spring, MD 20993-0002, USA c Machine Instrumentation Design and Fabrication, Building 13, National Institutes of Health, Bethesda, MD 20892, USA d NIST, Shady Grove, MD 20982, USA e Beijing Technology and Business University, College of Chemistry and Enviromental Engineering, Beijing Key Laboratory of Plant Resource Research, Beijing 100037, China Available online 21 February 2007
Abstract A novel spiral disk is designed by placing barricades at 6 mm intervals in the middle of the spiral channel to divide the channel into multiple sections. Glass beads are placed in every other section so that the planetary motion produces repetitive mixing and settling of polymer phase systems. Performance of this mixer-settler spiral disk assembly was examined for separation of lysozyme and myoglobin with a polymer phase system. The best results were obtained with a spiral disk equipped with barricades with openings ranging from 1.2 to 0.4 mm on each side at a high revolution speed up to 1200 rpm. © 2006 Elsevier B.V. All rights reserved. Keywords: Mixer-settler counter-current chromatography; Barricaded spiral disk; Assembly; Coil planet centrifuge; Purification of biopolymers
1. Introduction During the past 2 decades, type-J high-speed counter-current chromatography (HSCCC) has been widely used for separation of various natural and synthetic products [1]. However, the multiplayer-coiled separation column used for HSCCC fails to retain a sufficient amount of polymer phase systems [2] which are useful for separation of biopolymers such as proteins and nucleic acids. In order to overcome this problem, a new separation column called spiral disk assembly has been introduced [3]. In this column design, the spiral configuration of the channel facilitates the retention of the polymer phase systems by utilizing the radially acting centrifugal force field. The performance of the spiral disk assembly has been tested using a polymer phase system composed of polyethylene glycol 1000 and dibasic potassium phosphate. The result clearly demonstrated that, although the spiral channel retains a satisfactory
∗
Corresponding author. Tel.: +1 3014961210; fax: +1 3014023404. E-mail address: [email protected] (Y. Ito).
0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.11.078
amount of the stationary phase, the method yielded low partition efficiency for the protein separation apparently due to a lack of mixing of viscous polymer phases within the spiral channel. The effort has been made to improve the partition efficiency of the spiral disk by modifying the channel configuration by dividing it into multiple round partition units or compartments which are serially connected with narrow transfer ducts [4]. This bead-chain spiral disk has substantially improved the partition efficiency over the original spiral disk [4], while the estimated theoretical plate numbers range from 10 to 30 for a spiral disk which has 1430 partition units. This clearly indicates that there is an ample room to further improve the partition efficiency. Our previous studies on mass transfer of various molecules showed that the mass transfer rate of molecule through the interface of the two-phase solvent system is highly correlated with the molecular weight of the compound, i.e., the higher the molecular weight, the lower the transfer rate [5]. The results of these studies suggest that the partition efficiency of protein samples could be improved by increasing the interface area between the two phases. This can be achieved by actively mixing the
Y. Ito et al. / J. Chromatogr. A 1151 (2007) 108–114
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two phases to form numerous minute droplets of one phase into the other as in the conventional mixer-settler extraction system. In the present study, we developed a new design of separation column called a mixer-settler spiral disk which repeats alternately mixing and settling of the polymer phases in multiple pairs of two elongated sections divided by barricades. In each pair of sections one contains a glass bead to mix the contents while the other is left empty to settle the two phases. The performance of this new spiral disk is examined for separation of myoglobin and lysozyme with a polymer phase system composed of polyethylene glycol 1000 and dibasic potassium phosphate at various flow rates and revolution speeds. 2. Experimental 2.1. Apparatus The apparatus used in the present studies is a J-type coil planet centrifuge manufactured by P.C. Inc., Potomac, MD, USA. It holds a spiral disk assembly and a counterweight mass symmetrically at a distance of 10 cm from the centrifuge axis. The revolution speed of the apparatus is regulated with a speed controller (Bodine Electric Company, Chicago, IL, USA) up to 1500 rpm. The spiral disks were fabricated at the NIH machine shop from Kel-F (monochlorotrifluoroethylene) plates measuring 17.5 cm in diameter and 0.4 cm in thickness (Piedmont Plastics, Beltsville, MD, USA). The designs of locular and barricaded spiral disks are illustrated in Fig. 1. Several different designs of the spiral disk (see Table 1) are fabricated and each examined the performance for separation of the test protein samples. All disks are equipped with a set of four spiral grooves (16 mm pitch) which are connected in series through transfer ducts made on the opposite side of the disk as in the original design [3]. The locular disk has multiple elongated pits called “locules” (608 each, 2.8 mm wide, 6 mm long and 2 mm deep) connected with narrow ducts (1.2 mm wide, 0.5 mm long, 2 mm deep) with a total capacity of about 21 ml. The barricaded disks are made by placing multiple central barricades along each spiral groove (2.8 mm wide, 2 mm deep) at 6 mm intervals, thus dividing each groove into 152 sections or a total of 608 sections for each spiral disk. Three different widths of the barricade are tested, 0.4 mm (1.2 mm), 1.2 mm (0.8 mm), and 2.0 mm (0.4 mm) where the figures in the parenthesis indicate the opening of both sides of the barricade. The length of the barricades measures ca 0.5 mm for all disks. In order to produce thorough mixing of the contents, glass beads (1.5 mm diameter) are placed in every other section of the spiral groove while the rest are left empty to facilitate settling of the two phases. Each spiral disk is sandwiched between a pair of Teflon septa and pressed with a pair of metal flanges which are tightly bolted with multiple screws so as to form four spiral channels connected in series as described earlier [3]. The column is mounted on one side of the rotary frame of the J-type planetary centrifuge through a pair of bearing blocks while a counterweight mass is placed on the other side to balance the centrifuge.
Fig. 1. Design of the locular and barricaded spiral disks. (a) Locular spiral disk with four channels; (b) barricaded spiral disk with four channels.
2.2. Reagents Polyethylene glycol (PEG)-1000, dibasic potassium phosphate, myoglobin (horse skeletal muscle) and lysozyme (chicken egg) are all obtained from Sigma, St. Louis, MO, USA. 2.3. Preparation of two-phase solvent systems and sample solution Separation of proteins was performed with a polymer phase system composed of 12.5% (w/w) PEG1000 and 12.5% (w/w) dibasic potassium phosphate in distilled water which was thor-
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Table 1 Designs of four spiral disks used in the present studies Name
Number of spirals
Compartment Length/width/depth (mm)
Barricades Width (mm)
Connecting ducta Length/width/depth (mm)
Total capacity (ml)
Locular Barricaded I Barricaded II Barricaded III
4 4 4 4
6.0/2.8/2.0 6.0/2.8/2.0 6.0/2.8/2.0 6.0/2.8/2.0
– 0.4 0.8 1.2
0.5/1.2/2.0 0.5/1.2/2.0 0.5/0.8/2.0 0.5/0.4/2.0
21 22 22 22
a
For barricaded disk, opening width on each side of the barricade. Number of locules or barricades are 608 in all channels.
oughly equilibrated in a plastic bottle at room temperature. Sample solution was prepared by dissolving 100 mg each of lysozyme and myoglobin in 20 ml of upper phase of the above polymer phase system, and 1 ml was loaded in each separation. 2.4. Experimental procedure The experiment is initiated by filling the column entirely with the stationary phase of either upper or the lower phase. Trapping air bubbles in the channel is avoided by first replacing the air in the channel with carbon dioxide which is easily dissolved in the stationary phase. Then, the sample solution is injected into the inlet of the column through a sample loop of 1 ml capacity while the column is rotated at a desired revolution speed and the mobile phase is pumped through the inlet of the column at a desired flow rate (Perkin-Elmer Series 200 lc pump, Perkin Elmer, Norwalk, CT, USA). In order to retain a sufficient amount of the stationary phase in the spiral channel, the upper phase is eluted from the outer terminal and the lower phase from the inner terminal of the spiral channel. The effluent from the outlet of the column is continuously monitored with a UV detector (Uvicord s, LKB, Stockholm, Sweden) and fractionated into test tubes using a fraction collector (LKB). The elution curve was recorded with a strip-chart recorder (REC 102, Pharmacia LKB, Stockholm, Sweden). In order to improve tracing of the elution curve, a hollow fiber filter (MicroKros, 30 cm, Spectrum, New Brunswick, NJ, USA) is placed on the flow line between the column outlet and the monitor so that the small droplets of the stationary phase carried over in the mobile phase is dissolved before entering the flow cell in the monitor. When the first separation is completed, the column rotation is stopped and the following experiment is started by directly introducing the stationary phase into the column at a flow rate of 1 or 2 ml/min to quickly displace the column contents. For the present study it is not necessary to empty the column contents and completely displace with the stationary phase after each experiment, since the separation is performed with a mixture of two sample proteins, and the retention of the stationary phase is computed from the chromatogram.
stationary phase retained in the column assuming that most of the solvent is collected from the column. It has been found, however, that the locular or barricaded channel containing glass beads tends to hold a fairly large amount of polymer stationary phase against a gas flow, producing a large error in computation of the stationary phase retention. In order to solve this problem, the stationary phase retention is computed from the chromatogram using the following method. The amount of the stationary phase retained in the column Vs is estimated from the retention volume of each peak computed from the chromatogram and the difference in K values between two protein samples according to the following equation: Vs =
R2 − R 1 K2 − K 1
(1)
where R is the retention volume and K, the partition coefficient for each specified peak. The K value of lysozyme in the present polymer phase system is Kup/lp = 1.69 or Klp/up = 0.59, and that of myoglobin is Kup/lp = 0.51or Klp/up = 1.96. These K values are obtained by a conventional test tube experiment by mixing a small amount of each standard sample in a test tube containing preequilibrated two-phase solvent system and diluting an aliquot of each phase with water to measure the absorbance at 280 nm with a spectrophotometer (Genesys 10 V, Thermo Spectronic,
2.5. Measurement of stationary phase retention (SF ) and peak resolution (RS ) The retention of the stationary phase in HSCCC is usually measured by emptying the column contents into a graduated cylinder after the separation and measuring the volume of the
Fig. 2. Mechanism of mixer-settler counter-current chromatography with locular and barricaded spiral disks. Upper diagram: (a) locular channel; lower diagram: (b) barricaded channel.
Y. Ito et al. / J. Chromatogr. A 1151 (2007) 108–114
Rochester, NY, USA). SF is obtained by the following equation: SF (%) =
100Vs Vc
(2)
where Vc is the total column capacity. The peak resolution (RS ) is obtained from the conventional equation: RS =
2(R2 − R1 ) , W1 + W 2
(3)
where W is the width of each specified peak. When the two peaks are only partially separated, Rs is approximated by the following modified equation using the front half of the first peak (W1 ) and the rear half of the second peak (W2 ): RS =
R2 − R 1 W1 + W2
(4)
3. Results and discussion 3.1. Design and mechanism of mixer-settler spiral disk Fig. 2 shows portions of two types of mixer-settler spiral disks, one is called locular spiral disk and the other, barricaded spiral disk. In each disk glass beads are placed in every other
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compartment as shown in the figure where the upper phase is stationary and the lower phase is mobile in both channels [6]. In the locular channel (Fig. 2a), the channel is first filled with the upper stationary phase and the lower mobile phase is eluted through the channel. Then the mobile lower phase in the right hand settling-locule flows into the mixing-locule where it is mixed with the stationary upper phase by a vibrating glass bead, while a mixture of the two phases leaves the mixing-locule for the next settling-locule to settle into two layers as shown in the figure. This indicates that the mixing-locule receives only the lower phase from the right settling-locule while it gives up a mixture of the two phases to the left settling locule. Consequently, the mixing-locule gradually loses the stationary upper phase, which would result in a loss of partition capability. This problem is eliminated by a barricaded channel described below. In the barricaded channel (Fig. 2b), the spiral channel is divided into multiple sections by placing barricades along the center of the channel at given intervals so that the liquid can freely flow through the opening on the top and the bottom of each barricade. From the right settling-section the lower phase flows into the mixing-section where it is mixed with the upper stationary phase, and the mixture of the two phases moves into the left settling-section where two phases are separated as shown in the figure as in the locular column above. However, because
Fig. 3. Performance of the original, locular and barricaded spiral disks in separation of protein samples. Left panel: original spiral disk; second panel from left: locular spiral disk without glass bead; third panel from left: locular spiral disk with glass beads in every other locule; right panel: barricaded disk with 1.2 mm opening on both sides. Experimental conditions: apparatus: J-type coil planet centrifuge with 10 cm revolution radius; solvent system: 12.5% (w/w) PEG1000-12.5% (w/w) dibasic potassium phosphate; sample: lysozyme (chicken egg) and myoglobin (horse skeletal muscle) each 5 mg in 1 ml of upper phase; flow rate: indicated on the top of each panel; elution mode: indicated on the left (L-I-T: lower phase introduced from inner tail terminal), L-I-H: lower phase introduced from inner head end); U-O-T: upper phase introduced from outer tail terminal; U-O-H: upper phase introduced from outer head terminal); revolution speed: 800 rpm. Retention of the stationary phase: indicated on each chromatogram.
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the barricaded channel provides an opening at the top and the bottom of the barricade, the upper phase in the left settling-section can steadily flow back into the mixing-section. Consequently, the mixing section can always maintain a sufficient amount of the stationary phase by this counter-current process through the top opening of the channel. As demonstrated later in this article, the amount of the stationary phase retained in the channel is highly dependent upon the centrifugal force field acting perpendicularly across the channel. A greater force field will improve the retention of the stationary phase by facilitating both phase separation in the settling-section and the counter-current flow of the stationary phase back into the mixing-section. 3.2. Comparison in protein separation between four different spiral disks Fig. 3 shows the performance of these two spiral disks compared with that obtained from the original simple spiral disk (left panel) for separation of a pair of stable protein samples, lysozyme and myoglobin, with a polymer phase system composed of 12.5% (w/w) PEG1000–12.5% (w/w) dibasic potassium phosphate [6]. The 4 different elution modes were
applied to these separations as indicated on the left: L-I-T is lower phase pumped from the inner tail terminal toward the outer head terminal; L-I-H; lower phase pumped from the inner head terminal toward the outer tail terminal; U-O-T, upper phase pumped from the outer tail terminal toward the inner head terminal and U-O-H; the upper phase pumped from the outer head terminal toward the inner tail terminal. The reversed elution mode such as L-O-H, L-O-T, U-I-T and U-I-H produced low retention of the stationary phase due to the adversely acting centrifugal force field. Various flow rates (indicated on the top of the diagram) were applied all at a revolution speed of 800 rpm. In the original spiral disk (left panel) two protein samples were poorly resolved despite the satisfactory retention of the stationary phase. When the spiral channel is modified into a locular configuration (second panel from the left) the peak resolution is substantially improved in all elution modes. When glass beads are placed in every other locule throughout the channel, the partition efficiency is further improved (third panel from the left). Finally, the separation using the barricaded spiral disk with mixing beads (right panel) yielded better peak resolution than that of the mixer-settler locular disk at high flow rates of the mobile phase mainly due to higher retention of the stationary
Fig. 4. Effect of width of barricades of barricaded spiral disks on protein separation. Spiral disk: three disks with different width of the barricade as indicated at the top of each panel; also see Table 1. Other experimental conditions are described in Fig. 3.
Y. Ito et al. / J. Chromatogr. A 1151 (2007) 108–114
phase. The barricaded spiral disk used in this experiment has short barricades of 0.4 mm in width (1.2 mm opening on both sides, see Table 1). As explained earlier (Fig. 2a), the locular disk with glass beads gradually loses the stationary phase from the mixing locules. This is visualized by comparing the chromatograms obtained from locular and barricaded mixer settler disks (third and fourth panels from left). In all operating conditions, retention of the stationary phase in the barricaded disk exceeds that in the locular disk under the same experimental conditions. This lower retention in the locular disk is mainly due to the carryover of the stationary phase in the mobile phase since the solvent front emerges at the similar elution times. The amount of carryover of the stationary phase may be evaluated from these chromatograms. For example, comparing the L-I-T elution mode (top of the panel), the locular disk loses the stationary phase at a rate of 5.8% (61.6–55.8%) of the column volume or1.2 ml during 1.5 h (from the solvent front to the second peak) at a 0.25 ml/min flow rate and 11.8% (50.1–38.3%) or 2.5 ml during 40 min at a 0.5 ml/min flow rate. The loss of the stationary phase by carryover in the locular disk becomes much greater in the upper phase elution mode. It is interesting to note that in the original and locular spiral disk with no glass beads the lower mobile phase produced better separation than the upper mobile phase whereas in the mixer-settler spiral disk in both locular and barricaded types this relationship is reversed. In practice, the lower phase is more conveniently used for the separation, since the fraction contains a much less amount of PEG which is not easily removed.
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3.3. Effect of width of barricades Fig. 4 similarly shows three panels of HSCCC chromatograms of lysozyme and myoglobin obtained from the barricaded spiral disks with three different barricades measuring 0.4, 0.8 and 1.2 mm in width (see Table 1) where the left panel (0.4 mm-wide barricade) is the same as the right panel of the chromatograms shown in Fig. 3. All separations were performed at 800 rpm under various flow rates as indicated on the top of each panel. As shown in these diagrams, the peak resolution between two proteins and retention of the stationary phase are not substantially altered as the width of the barricades is increased from 0.4 to 1.2 mm. When the size of the barricades was further increased to 2.2 mm (opening 0.2 mm), however, the retention of the stationary phase became below 30% at a flow rate of 0.25 ml/min resulting in poor peak resolution (not shown in the figure). 3.4. Effect of flow rate and revolution speed As mentioned earlier, the process of both mixing and settling of the two phases in the channel can be enhanced by higher revolution rates. Fig. 5 shows the effect of revolution speed and flow rate on the peak resolution of lysozyme and myoglobin obtained from a barricaded disk with 0.4 mm opening (1.2 mm width of barricade). Four panels of chromatograms were obtained from the left to right at revolution speeds of 600, 800, 1000 and 1200 rpm. As expected, the peak resolution is significantly improved by higher revolution speed, where at 1200 rpm the res-
Fig. 5. Effect of revolution speed and flow-rate on protein separation. Spiral disk: barricaded disk with 1.2 mm wide barricades placed at every 6 mm intervals; revolution speed: varied from 600 to 1200 rpm. Other experimental conditions are described in Fig. 3.
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olution between the two peaks (Rs ) exceeds 1.6 at a flow rate of 0.25 ml/min and the two peaks are still partially resolved at a higher flow rate of 1 ml/min. 3.5. Comparison of partition process between mixer-settler HSCCC and the conventional partition system Here it may be worthwhile to compare the efficiency of the present system with other partition systems such as Craig counter-current distribution apparatus [7] and Albertsson’s thin layer counter-current distribution instrument [2]. These existing systems repeat a partition cycle of mixing, settling and transferring the mobile phase to the next unit at regular intervals of 2–5 min/cycle depending upon the settling time of the two-phase solvent system. In the present mixer-settler HSCCC system using a barricaded spiral disk, vibrating glass beads continuously mix two solvent phases in the mixing section at an enormously high frequency of 13–20 times/s while the mobile phase travels through the channel at flow rates of 0.25–1 ml/min. The capacity of each set of mixer-settler unit is 6 mm × 2.8 mm × 2.0 mm ×2 − 1.32 mm3 (volume of glass beads) = 66 mm3 or 0.066 ml. Since the stationary phase always occupies about 50% of the space, the volume of the mobile phase present in one partition unit is around 0.033 ml. If the mobile phase flows through one partition unit at a rate of 0.5 ml/min, a unit partition time or the time for the mobile phase to spend in each partition unit is 0.066 min/cycle compared with 2–5 min/cycle in the existing discontinuous partition systems. However, the present system is a nonequilibrium partition system yielding around 100 theoretical plates for 360 mixer-settler partition units in a barricaded spiral disk. Therefore, the partition efficiency of the present system is ca. 28% per unit which gives a unit partition time of about 0.24 min/cycle as an equilibrium partition system. From the above computation it may be concluded that the present mixersettler HSCCC is 10-times more efficient than the conventional counter-current distribution system.
4. Conclusion A novel design of the separation column called a barricaded spiral disk is introduced to perform mixer-settler HSCCC. The performance of the barricaded spiral disk is compared with those of the original and locular spiral disks on the separation of test protein samples (lysozyme and myoglobin) with a polymer phase system composed of 12.5% (w/w) PEG1000 and 12.5% (w/w) dibasic potassium phosphate under a given set of experimental conditions. The results revealed that peak resolution between two test proteins is remarkably improved by the mixer-settler spiral disk with multiple-barricaded sections each 6 mm long and containing glass beads at every other section. The peak resolution is improved by higher revolution speed up to 1200 rpm. A comparative study on the cyclic partition time revealed that the present system is 10-times more efficient than the conventional counter-current distribution system. The overall results of our studies obtained from a single disk indicate that a mixer-settler spiral disk assembly with several disk units connected in series will perform highly efficient chromatographic separation of protein samples within a reasonable elution time. References [1] Y. Ito, CRC Crit. Rev. Anal. Chem. 17 (1986) 65. ˚ Albertsson, Partition of Cells and Macromolecules, Wiley[2] P.-A. Interscience, New York, 1986. [3] Y. Ito, F.-Q. Yang, P.E. Fitze, J.V. Sullivan, J. Liq. Chromatogr. Rel. Technol. 26 (2003) 1355–1732. [4] Y. Ito, F.-Q. Yang, P.E. Fitze, J. Powell, D. Ide, J. Chromatogr. A 1017 (2003) 71–81. [5] Y. Ito, K. Matsuda, Y. Ma, L. Qi, J. Chromatogr. A 802 (1998) 277– 283. [6] Y. Ito, L. Qi., F.-Q. Yang, J. Powell, F. Sharpnack, D. Ide, Presented at the 3rd International Conference on Counter-current Chromatography, Tokyo, 28–31 August, 2004. [7] L.C. Craig, Comprehensive Biochemistry, vol. 4, Elsevier, Amsterdam, 1962.
Mixer-settler counter-current chromatography with a barricaded spiral disk assembly with glass beads Yoichiro Ito a,∗ , Lin Qi b , Jimmie Powell c , Frank Sharpnack c , Howard Metger e , James Yost d , Xue-Li Cao e , Yin-Mao Dong e , Liang-Sheng Huo e , Xiao-Ping Zhu e , Ting Li e a
Center for Biochemistry and Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 50, Room 3334, 50 South Drive, Bethesda, MD 20892-8014, USA b Office of New Drug Quality Assessment, OPS/CDER/FDA, 10903 New Hampshire Avenue, Room 3525, Silver Spring, MD 20993-0002, USA c Machine Instrumentation Design and Fabrication, Building 13, National Institutes of Health, Bethesda, MD 20892, USA d NIST, Shady Grove, MD 20982, USA e Beijing Technology and Business University, College of Chemistry and Enviromental Engineering, Beijing Key Laboratory of Plant Resource Research, Beijing 100037, China Available online 21 February 2007
Abstract A novel spiral disk is designed by placing barricades at 6 mm intervals in the middle of the spiral channel to divide the channel into multiple sections. Glass beads are placed in every other section so that the planetary motion produces repetitive mixing and settling of polymer phase systems. Performance of this mixer-settler spiral disk assembly was examined for separation of lysozyme and myoglobin with a polymer phase system. The best results were obtained with a spiral disk equipped with barricades with openings ranging from 1.2 to 0.4 mm on each side at a high revolution speed up to 1200 rpm. © 2006 Elsevier B.V. All rights reserved. Keywords: Mixer-settler counter-current chromatography; Barricaded spiral disk; Assembly; Coil planet centrifuge; Purification of biopolymers
1. Introduction During the past 2 decades, type-J high-speed counter-current chromatography (HSCCC) has been widely used for separation of various natural and synthetic products [1]. However, the multiplayer-coiled separation column used for HSCCC fails to retain a sufficient amount of polymer phase systems [2] which are useful for separation of biopolymers such as proteins and nucleic acids. In order to overcome this problem, a new separation column called spiral disk assembly has been introduced [3]. In this column design, the spiral configuration of the channel facilitates the retention of the polymer phase systems by utilizing the radially acting centrifugal force field. The performance of the spiral disk assembly has been tested using a polymer phase system composed of polyethylene glycol 1000 and dibasic potassium phosphate. The result clearly demonstrated that, although the spiral channel retains a satisfactory
∗
Corresponding author. Tel.: +1 3014961210; fax: +1 3014023404. E-mail address: [email protected] (Y. Ito).
0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.11.078
amount of the stationary phase, the method yielded low partition efficiency for the protein separation apparently due to a lack of mixing of viscous polymer phases within the spiral channel. The effort has been made to improve the partition efficiency of the spiral disk by modifying the channel configuration by dividing it into multiple round partition units or compartments which are serially connected with narrow transfer ducts [4]. This bead-chain spiral disk has substantially improved the partition efficiency over the original spiral disk [4], while the estimated theoretical plate numbers range from 10 to 30 for a spiral disk which has 1430 partition units. This clearly indicates that there is an ample room to further improve the partition efficiency. Our previous studies on mass transfer of various molecules showed that the mass transfer rate of molecule through the interface of the two-phase solvent system is highly correlated with the molecular weight of the compound, i.e., the higher the molecular weight, the lower the transfer rate [5]. The results of these studies suggest that the partition efficiency of protein samples could be improved by increasing the interface area between the two phases. This can be achieved by actively mixing the
Y. Ito et al. / J. Chromatogr. A 1151 (2007) 108–114
109
two phases to form numerous minute droplets of one phase into the other as in the conventional mixer-settler extraction system. In the present study, we developed a new design of separation column called a mixer-settler spiral disk which repeats alternately mixing and settling of the polymer phases in multiple pairs of two elongated sections divided by barricades. In each pair of sections one contains a glass bead to mix the contents while the other is left empty to settle the two phases. The performance of this new spiral disk is examined for separation of myoglobin and lysozyme with a polymer phase system composed of polyethylene glycol 1000 and dibasic potassium phosphate at various flow rates and revolution speeds. 2. Experimental 2.1. Apparatus The apparatus used in the present studies is a J-type coil planet centrifuge manufactured by P.C. Inc., Potomac, MD, USA. It holds a spiral disk assembly and a counterweight mass symmetrically at a distance of 10 cm from the centrifuge axis. The revolution speed of the apparatus is regulated with a speed controller (Bodine Electric Company, Chicago, IL, USA) up to 1500 rpm. The spiral disks were fabricated at the NIH machine shop from Kel-F (monochlorotrifluoroethylene) plates measuring 17.5 cm in diameter and 0.4 cm in thickness (Piedmont Plastics, Beltsville, MD, USA). The designs of locular and barricaded spiral disks are illustrated in Fig. 1. Several different designs of the spiral disk (see Table 1) are fabricated and each examined the performance for separation of the test protein samples. All disks are equipped with a set of four spiral grooves (16 mm pitch) which are connected in series through transfer ducts made on the opposite side of the disk as in the original design [3]. The locular disk has multiple elongated pits called “locules” (608 each, 2.8 mm wide, 6 mm long and 2 mm deep) connected with narrow ducts (1.2 mm wide, 0.5 mm long, 2 mm deep) with a total capacity of about 21 ml. The barricaded disks are made by placing multiple central barricades along each spiral groove (2.8 mm wide, 2 mm deep) at 6 mm intervals, thus dividing each groove into 152 sections or a total of 608 sections for each spiral disk. Three different widths of the barricade are tested, 0.4 mm (1.2 mm), 1.2 mm (0.8 mm), and 2.0 mm (0.4 mm) where the figures in the parenthesis indicate the opening of both sides of the barricade. The length of the barricades measures ca 0.5 mm for all disks. In order to produce thorough mixing of the contents, glass beads (1.5 mm diameter) are placed in every other section of the spiral groove while the rest are left empty to facilitate settling of the two phases. Each spiral disk is sandwiched between a pair of Teflon septa and pressed with a pair of metal flanges which are tightly bolted with multiple screws so as to form four spiral channels connected in series as described earlier [3]. The column is mounted on one side of the rotary frame of the J-type planetary centrifuge through a pair of bearing blocks while a counterweight mass is placed on the other side to balance the centrifuge.
Fig. 1. Design of the locular and barricaded spiral disks. (a) Locular spiral disk with four channels; (b) barricaded spiral disk with four channels.
2.2. Reagents Polyethylene glycol (PEG)-1000, dibasic potassium phosphate, myoglobin (horse skeletal muscle) and lysozyme (chicken egg) are all obtained from Sigma, St. Louis, MO, USA. 2.3. Preparation of two-phase solvent systems and sample solution Separation of proteins was performed with a polymer phase system composed of 12.5% (w/w) PEG1000 and 12.5% (w/w) dibasic potassium phosphate in distilled water which was thor-
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Table 1 Designs of four spiral disks used in the present studies Name
Number of spirals
Compartment Length/width/depth (mm)
Barricades Width (mm)
Connecting ducta Length/width/depth (mm)
Total capacity (ml)
Locular Barricaded I Barricaded II Barricaded III
4 4 4 4
6.0/2.8/2.0 6.0/2.8/2.0 6.0/2.8/2.0 6.0/2.8/2.0
– 0.4 0.8 1.2
0.5/1.2/2.0 0.5/1.2/2.0 0.5/0.8/2.0 0.5/0.4/2.0
21 22 22 22
a
For barricaded disk, opening width on each side of the barricade. Number of locules or barricades are 608 in all channels.
oughly equilibrated in a plastic bottle at room temperature. Sample solution was prepared by dissolving 100 mg each of lysozyme and myoglobin in 20 ml of upper phase of the above polymer phase system, and 1 ml was loaded in each separation. 2.4. Experimental procedure The experiment is initiated by filling the column entirely with the stationary phase of either upper or the lower phase. Trapping air bubbles in the channel is avoided by first replacing the air in the channel with carbon dioxide which is easily dissolved in the stationary phase. Then, the sample solution is injected into the inlet of the column through a sample loop of 1 ml capacity while the column is rotated at a desired revolution speed and the mobile phase is pumped through the inlet of the column at a desired flow rate (Perkin-Elmer Series 200 lc pump, Perkin Elmer, Norwalk, CT, USA). In order to retain a sufficient amount of the stationary phase in the spiral channel, the upper phase is eluted from the outer terminal and the lower phase from the inner terminal of the spiral channel. The effluent from the outlet of the column is continuously monitored with a UV detector (Uvicord s, LKB, Stockholm, Sweden) and fractionated into test tubes using a fraction collector (LKB). The elution curve was recorded with a strip-chart recorder (REC 102, Pharmacia LKB, Stockholm, Sweden). In order to improve tracing of the elution curve, a hollow fiber filter (MicroKros, 30 cm, Spectrum, New Brunswick, NJ, USA) is placed on the flow line between the column outlet and the monitor so that the small droplets of the stationary phase carried over in the mobile phase is dissolved before entering the flow cell in the monitor. When the first separation is completed, the column rotation is stopped and the following experiment is started by directly introducing the stationary phase into the column at a flow rate of 1 or 2 ml/min to quickly displace the column contents. For the present study it is not necessary to empty the column contents and completely displace with the stationary phase after each experiment, since the separation is performed with a mixture of two sample proteins, and the retention of the stationary phase is computed from the chromatogram.
stationary phase retained in the column assuming that most of the solvent is collected from the column. It has been found, however, that the locular or barricaded channel containing glass beads tends to hold a fairly large amount of polymer stationary phase against a gas flow, producing a large error in computation of the stationary phase retention. In order to solve this problem, the stationary phase retention is computed from the chromatogram using the following method. The amount of the stationary phase retained in the column Vs is estimated from the retention volume of each peak computed from the chromatogram and the difference in K values between two protein samples according to the following equation: Vs =
R2 − R 1 K2 − K 1
(1)
where R is the retention volume and K, the partition coefficient for each specified peak. The K value of lysozyme in the present polymer phase system is Kup/lp = 1.69 or Klp/up = 0.59, and that of myoglobin is Kup/lp = 0.51or Klp/up = 1.96. These K values are obtained by a conventional test tube experiment by mixing a small amount of each standard sample in a test tube containing preequilibrated two-phase solvent system and diluting an aliquot of each phase with water to measure the absorbance at 280 nm with a spectrophotometer (Genesys 10 V, Thermo Spectronic,
2.5. Measurement of stationary phase retention (SF ) and peak resolution (RS ) The retention of the stationary phase in HSCCC is usually measured by emptying the column contents into a graduated cylinder after the separation and measuring the volume of the
Fig. 2. Mechanism of mixer-settler counter-current chromatography with locular and barricaded spiral disks. Upper diagram: (a) locular channel; lower diagram: (b) barricaded channel.
Y. Ito et al. / J. Chromatogr. A 1151 (2007) 108–114
Rochester, NY, USA). SF is obtained by the following equation: SF (%) =
100Vs Vc
(2)
where Vc is the total column capacity. The peak resolution (RS ) is obtained from the conventional equation: RS =
2(R2 − R1 ) , W1 + W 2
(3)
where W is the width of each specified peak. When the two peaks are only partially separated, Rs is approximated by the following modified equation using the front half of the first peak (W1 ) and the rear half of the second peak (W2 ): RS =
R2 − R 1 W1 + W2
(4)
3. Results and discussion 3.1. Design and mechanism of mixer-settler spiral disk Fig. 2 shows portions of two types of mixer-settler spiral disks, one is called locular spiral disk and the other, barricaded spiral disk. In each disk glass beads are placed in every other
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compartment as shown in the figure where the upper phase is stationary and the lower phase is mobile in both channels [6]. In the locular channel (Fig. 2a), the channel is first filled with the upper stationary phase and the lower mobile phase is eluted through the channel. Then the mobile lower phase in the right hand settling-locule flows into the mixing-locule where it is mixed with the stationary upper phase by a vibrating glass bead, while a mixture of the two phases leaves the mixing-locule for the next settling-locule to settle into two layers as shown in the figure. This indicates that the mixing-locule receives only the lower phase from the right settling-locule while it gives up a mixture of the two phases to the left settling locule. Consequently, the mixing-locule gradually loses the stationary upper phase, which would result in a loss of partition capability. This problem is eliminated by a barricaded channel described below. In the barricaded channel (Fig. 2b), the spiral channel is divided into multiple sections by placing barricades along the center of the channel at given intervals so that the liquid can freely flow through the opening on the top and the bottom of each barricade. From the right settling-section the lower phase flows into the mixing-section where it is mixed with the upper stationary phase, and the mixture of the two phases moves into the left settling-section where two phases are separated as shown in the figure as in the locular column above. However, because
Fig. 3. Performance of the original, locular and barricaded spiral disks in separation of protein samples. Left panel: original spiral disk; second panel from left: locular spiral disk without glass bead; third panel from left: locular spiral disk with glass beads in every other locule; right panel: barricaded disk with 1.2 mm opening on both sides. Experimental conditions: apparatus: J-type coil planet centrifuge with 10 cm revolution radius; solvent system: 12.5% (w/w) PEG1000-12.5% (w/w) dibasic potassium phosphate; sample: lysozyme (chicken egg) and myoglobin (horse skeletal muscle) each 5 mg in 1 ml of upper phase; flow rate: indicated on the top of each panel; elution mode: indicated on the left (L-I-T: lower phase introduced from inner tail terminal), L-I-H: lower phase introduced from inner head end); U-O-T: upper phase introduced from outer tail terminal; U-O-H: upper phase introduced from outer head terminal); revolution speed: 800 rpm. Retention of the stationary phase: indicated on each chromatogram.
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the barricaded channel provides an opening at the top and the bottom of the barricade, the upper phase in the left settling-section can steadily flow back into the mixing-section. Consequently, the mixing section can always maintain a sufficient amount of the stationary phase by this counter-current process through the top opening of the channel. As demonstrated later in this article, the amount of the stationary phase retained in the channel is highly dependent upon the centrifugal force field acting perpendicularly across the channel. A greater force field will improve the retention of the stationary phase by facilitating both phase separation in the settling-section and the counter-current flow of the stationary phase back into the mixing-section. 3.2. Comparison in protein separation between four different spiral disks Fig. 3 shows the performance of these two spiral disks compared with that obtained from the original simple spiral disk (left panel) for separation of a pair of stable protein samples, lysozyme and myoglobin, with a polymer phase system composed of 12.5% (w/w) PEG1000–12.5% (w/w) dibasic potassium phosphate [6]. The 4 different elution modes were
applied to these separations as indicated on the left: L-I-T is lower phase pumped from the inner tail terminal toward the outer head terminal; L-I-H; lower phase pumped from the inner head terminal toward the outer tail terminal; U-O-T, upper phase pumped from the outer tail terminal toward the inner head terminal and U-O-H; the upper phase pumped from the outer head terminal toward the inner tail terminal. The reversed elution mode such as L-O-H, L-O-T, U-I-T and U-I-H produced low retention of the stationary phase due to the adversely acting centrifugal force field. Various flow rates (indicated on the top of the diagram) were applied all at a revolution speed of 800 rpm. In the original spiral disk (left panel) two protein samples were poorly resolved despite the satisfactory retention of the stationary phase. When the spiral channel is modified into a locular configuration (second panel from the left) the peak resolution is substantially improved in all elution modes. When glass beads are placed in every other locule throughout the channel, the partition efficiency is further improved (third panel from the left). Finally, the separation using the barricaded spiral disk with mixing beads (right panel) yielded better peak resolution than that of the mixer-settler locular disk at high flow rates of the mobile phase mainly due to higher retention of the stationary
Fig. 4. Effect of width of barricades of barricaded spiral disks on protein separation. Spiral disk: three disks with different width of the barricade as indicated at the top of each panel; also see Table 1. Other experimental conditions are described in Fig. 3.
Y. Ito et al. / J. Chromatogr. A 1151 (2007) 108–114
phase. The barricaded spiral disk used in this experiment has short barricades of 0.4 mm in width (1.2 mm opening on both sides, see Table 1). As explained earlier (Fig. 2a), the locular disk with glass beads gradually loses the stationary phase from the mixing locules. This is visualized by comparing the chromatograms obtained from locular and barricaded mixer settler disks (third and fourth panels from left). In all operating conditions, retention of the stationary phase in the barricaded disk exceeds that in the locular disk under the same experimental conditions. This lower retention in the locular disk is mainly due to the carryover of the stationary phase in the mobile phase since the solvent front emerges at the similar elution times. The amount of carryover of the stationary phase may be evaluated from these chromatograms. For example, comparing the L-I-T elution mode (top of the panel), the locular disk loses the stationary phase at a rate of 5.8% (61.6–55.8%) of the column volume or1.2 ml during 1.5 h (from the solvent front to the second peak) at a 0.25 ml/min flow rate and 11.8% (50.1–38.3%) or 2.5 ml during 40 min at a 0.5 ml/min flow rate. The loss of the stationary phase by carryover in the locular disk becomes much greater in the upper phase elution mode. It is interesting to note that in the original and locular spiral disk with no glass beads the lower mobile phase produced better separation than the upper mobile phase whereas in the mixer-settler spiral disk in both locular and barricaded types this relationship is reversed. In practice, the lower phase is more conveniently used for the separation, since the fraction contains a much less amount of PEG which is not easily removed.
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3.3. Effect of width of barricades Fig. 4 similarly shows three panels of HSCCC chromatograms of lysozyme and myoglobin obtained from the barricaded spiral disks with three different barricades measuring 0.4, 0.8 and 1.2 mm in width (see Table 1) where the left panel (0.4 mm-wide barricade) is the same as the right panel of the chromatograms shown in Fig. 3. All separations were performed at 800 rpm under various flow rates as indicated on the top of each panel. As shown in these diagrams, the peak resolution between two proteins and retention of the stationary phase are not substantially altered as the width of the barricades is increased from 0.4 to 1.2 mm. When the size of the barricades was further increased to 2.2 mm (opening 0.2 mm), however, the retention of the stationary phase became below 30% at a flow rate of 0.25 ml/min resulting in poor peak resolution (not shown in the figure). 3.4. Effect of flow rate and revolution speed As mentioned earlier, the process of both mixing and settling of the two phases in the channel can be enhanced by higher revolution rates. Fig. 5 shows the effect of revolution speed and flow rate on the peak resolution of lysozyme and myoglobin obtained from a barricaded disk with 0.4 mm opening (1.2 mm width of barricade). Four panels of chromatograms were obtained from the left to right at revolution speeds of 600, 800, 1000 and 1200 rpm. As expected, the peak resolution is significantly improved by higher revolution speed, where at 1200 rpm the res-
Fig. 5. Effect of revolution speed and flow-rate on protein separation. Spiral disk: barricaded disk with 1.2 mm wide barricades placed at every 6 mm intervals; revolution speed: varied from 600 to 1200 rpm. Other experimental conditions are described in Fig. 3.
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olution between the two peaks (Rs ) exceeds 1.6 at a flow rate of 0.25 ml/min and the two peaks are still partially resolved at a higher flow rate of 1 ml/min. 3.5. Comparison of partition process between mixer-settler HSCCC and the conventional partition system Here it may be worthwhile to compare the efficiency of the present system with other partition systems such as Craig counter-current distribution apparatus [7] and Albertsson’s thin layer counter-current distribution instrument [2]. These existing systems repeat a partition cycle of mixing, settling and transferring the mobile phase to the next unit at regular intervals of 2–5 min/cycle depending upon the settling time of the two-phase solvent system. In the present mixer-settler HSCCC system using a barricaded spiral disk, vibrating glass beads continuously mix two solvent phases in the mixing section at an enormously high frequency of 13–20 times/s while the mobile phase travels through the channel at flow rates of 0.25–1 ml/min. The capacity of each set of mixer-settler unit is 6 mm × 2.8 mm × 2.0 mm ×2 − 1.32 mm3 (volume of glass beads) = 66 mm3 or 0.066 ml. Since the stationary phase always occupies about 50% of the space, the volume of the mobile phase present in one partition unit is around 0.033 ml. If the mobile phase flows through one partition unit at a rate of 0.5 ml/min, a unit partition time or the time for the mobile phase to spend in each partition unit is 0.066 min/cycle compared with 2–5 min/cycle in the existing discontinuous partition systems. However, the present system is a nonequilibrium partition system yielding around 100 theoretical plates for 360 mixer-settler partition units in a barricaded spiral disk. Therefore, the partition efficiency of the present system is ca. 28% per unit which gives a unit partition time of about 0.24 min/cycle as an equilibrium partition system. From the above computation it may be concluded that the present mixersettler HSCCC is 10-times more efficient than the conventional counter-current distribution system.
4. Conclusion A novel design of the separation column called a barricaded spiral disk is introduced to perform mixer-settler HSCCC. The performance of the barricaded spiral disk is compared with those of the original and locular spiral disks on the separation of test protein samples (lysozyme and myoglobin) with a polymer phase system composed of 12.5% (w/w) PEG1000 and 12.5% (w/w) dibasic potassium phosphate under a given set of experimental conditions. The results revealed that peak resolution between two test proteins is remarkably improved by the mixer-settler spiral disk with multiple-barricaded sections each 6 mm long and containing glass beads at every other section. The peak resolution is improved by higher revolution speed up to 1200 rpm. A comparative study on the cyclic partition time revealed that the present system is 10-times more efficient than the conventional counter-current distribution system. The overall results of our studies obtained from a single disk indicate that a mixer-settler spiral disk assembly with several disk units connected in series will perform highly efficient chromatographic separation of protein samples within a reasonable elution time. References [1] Y. Ito, CRC Crit. Rev. Anal. Chem. 17 (1986) 65. ˚ Albertsson, Partition of Cells and Macromolecules, Wiley[2] P.-A. Interscience, New York, 1986. [3] Y. Ito, F.-Q. Yang, P.E. Fitze, J.V. Sullivan, J. Liq. Chromatogr. Rel. Technol. 26 (2003) 1355–1732. [4] Y. Ito, F.-Q. Yang, P.E. Fitze, J. Powell, D. Ide, J. Chromatogr. A 1017 (2003) 71–81. [5] Y. Ito, K. Matsuda, Y. Ma, L. Qi, J. Chromatogr. A 802 (1998) 277– 283. [6] Y. Ito, L. Qi., F.-Q. Yang, J. Powell, F. Sharpnack, D. Ide, Presented at the 3rd International Conference on Counter-current Chromatography, Tokyo, 28–31 August, 2004. [7] L.C. Craig, Comprehensive Biochemistry, vol. 4, Elsevier, Amsterdam, 1962.