Improved Separations of Proteins and Sugar Derivatives Using the Small-Scale Cross-Axis Coil Planet Centrifuge with Locular Multilayer Coiled Columns

Abstract

1) Background

Countercurrent chromatography (CCC) is liquid-liquid partition chromatography without using a solid support matrix. This technique requires further improvement of partition efficiency and shortening theseparation time.

2) Methods

The locular multilayer coils modified with and without mixer glass beads were developed for the separation of proteins and 4-methylumbelliferyl (MU) sugar derivatives using the small-scale cross-axis coil planet centrifuge.

3) Results

Proteins were well separated from each other and the separation was improved at a low flow rate of the mobile phase. On the other hand, 4-MU sugar derivatives were sufficiently resolved with short separation time at a highflow rate of the mobile phase under satisfactory stationary phase retention.

4) Conclusion

Effective separations were achieved using the locular multilayer coil for proteins with aqueous-aqueous polymer phase systems and for 4-MU sugar derivatives with organic-aqueous two-phase solvent systems by inserting a glass bead into each locule.

1. Introduction

Countercurrent chromatography (CCC) is one of liquid-liquid partition chromatography which eliminates the solid support matrix for stationary phase [1–4]. In the past, various types of CCC instruments have been developed to achieve satisfactory retention of the liquid stationary phase in the column. Among them, the type-J multilayer coil planet centrifuge (CPC) and the cross-axis CPC have proven most useful for effective CCC separations. The type-J multilayer CPC is useful for the separation mainly using organic-aqueous two-phase solvent systems, while the cross-axis CPC is suitabale for the separation of biopolymers with aqueous-aqueous polymer phase systems having low-interfacial tension and high viscosity.

The cross-axis CPC has a distinctive feature that the coiled column revolves around the vertical central axis of the centrifuge while it rotates about its horizontal axis at the same angular velocity [5, 6]. This centrifuge system permits flow tubes to rotate without twisting so that the rotating column can be continuously eluted with the mobile phase without a risk of leakage which is often observed in the conventional centrifuge system with a rotary seal. In our laboratory, the floor model of the cross-axis CPC has been built and successfully applied to the protein separation using aqueous-aqueous polymer phase system [7]. Recently, a new-small-scale cross-axis CPC has been further developed in our laboratory to improve the partition efficiency [8, 9]. This small-scale cross-axis CPC is a compact model where four coiled columns of similar weight are mounted symmetrically around the rotary frame to achieve stable balancing of the centrifuge system even under a high revolution speed.

The partition efficiency of this cross-axis CPC varies depending on the geometry of the separation column such as conventional multilayer coil [8–10], eccentric coil [7, 11, 12], toroidal coil [11, 12], spiral coil [13] and so forth. Our previous studies revealed that the locular multilayer coil prepared by compressing a long piece of polytetrafluoroethylene (PTFE) tubing with a pair of hemostats at regular intervals was useful for protein separation with aqueous-aqueous polymer phase systems [14]. It has also been reported that the spiral disk mounted by the cross-pressed tubing for the type-J planet centrifuge was useful for separation of proteins with aqueous-aqueous polymer phase systems [15]. The present paper describes the improved separations of proteins with aqueous-aqueous polymer phase systems for the locular multilayer coil and of 4-methylumbelliferyl sugar derivatives for the locular multilayer coil containing a glass bead mixer in each locule using the cross-axis CPC.

2. Materials and Methods

2.1. Apparatus

The small-scale cross-axis CPC employed in the present study was constructed at the Machining Technology Center of Nihon University (Chiba, Japan). The design and fabrication of the apparatus and the column configuration were previously described in detail [8, 9].

2.2. Preparation of locular tubing and locular beads tubing

The newly designed locular tubing was prepared from a single piece of 1.5 mm I.D., 2.5 mm O.D. PTFE (polytetrafluoroethylene) tubing (Flon Kogyo, Tokyo, Japan) by compressing it with a pair of tongs (0.8 cm wide) for pressing at 1.2 cm intervals to form a number of segments called locules. The locular beads tubing was prepared by adding one glass ball bead (1 mm I.D., Toshinriko, Inc., Tokyo, Japan) in each locule for enhancing the mixing of two phases. Figure 1 illustrates the schematic drawing of the locular tubing with and without glass beads.

Figure 1.

Schematic illustration of the locular tubing with and without a glass bead mixer.

2.3. Preparation of coiled column assembly

Each multilayer coil was prepared by tightly winding a long piece of the aforementioned PTFE tubing around the holder hub of 3 cm in diameter, forming five tight coiled layers between a pair of flanges spaced 5 cm apart. Each coiled column was prepared according to the following procedure: The tubing was directly wound onto the holder hub starting on the proximal side, which is close to the center of revolution. After each coil layer was completed, the layer was wrapped with an adhesive tape and the tubing was straightly returned to the other side to resume winding in the same direction. It results in a multilayer coil assembly composed of either entirely right- or left-handed coils, which is different from that commonly used in the type-J multilayer CPC. Two columns of left-handed coils were subjected to the forward rotation; and right-handed coils, the backward rotation. Two pairs of right- and left-handed coil assemblies were connected in series with flow tubes in such a way that the distal terminal of the first column assembly is connected to the proximal terminal, which is close to the center of revolution, of the second column assembly, and so on. Four coil assemblies were symmetrically mounted on the rotary frame for balancing the centrifuge system.

2.4. Reagents

Polyethylene glycol (PEG) 1000 (MW 1,000), cytochrome C (horse heart) (MW 12,384), myoglobin (horse skeletal muscle) (MW 17,800) and lysozyme (chicken egg) (MW 13,680) were purchased from Sigma (St. Louis, MO, USA). Dibasic potassium phosphate was obtained from Wako Pure Chemicals (Osaka, Japan).

4-Methylumbelliferyl sugar derivatives used as test samples were purchased as follows: 4-Methylumbelliferyl-α-L-fucopyranoside (α-L-Fuc), 4-methylumbelliferyl-β-D-fucopyranoside (β-D-Fuc), 4-methylumbelliferyl-β-D-glucopyranoside (Glc), 4-methylumbelliferyl-β-D-galactopyranoside (Gal) and 4-methylumbelliferyl-β-D-cellobioside (Cel) were purchased from BIOSYNTH AG (Staad, Switzerland). 4-Methylumbelliferyl-α-D-mannopyransoide (Man) was obtained from Wako Pure Chemicals.

All other reagents were of reagent grade.

2.5. Preparation of two-phase solvent systems and sample solutions

An aqueous-aqueous polymer phase system composed of 12.5% (w/w) PEG 1000 and 12.5% (w/w) dibasic potassium phosphate was prepared by dissolving 125 g of PEG 1000 and 125 g of dibasic potassium phosphate (anhydrous) in 750 g of distilled water.

The organic-aqueous two-phase solvent system used in the present studies was composed of ethyl acetate/1-butanol/water (3 : 2 : 5, v/v) for lower phase mobile and (1 : 4 : 5, v/v) for upper phase mobile [16].

Each solvent mixture was thoroughly equilibrated in a separatory funnel at room temperature and the two phases were separated after the two layers were formed.

The sample solutions were prepared by dissolving the standard sample mixture with equal volumes of each phase of the two-phase solvent system used for separation.

2.6. CCC separation of proteins

Each separation was initiated by completely filling the column with the stationary phase, followed by injection of the sample solution through the flow tube leading into the head of CCC column by a syringe. Then, the mobile phase was pumped into the column at a suitable flow rate using a reciprocating pump (Model LC-6A, Shimadzu Corporation, Kyoto, Japan), while the column was rotated at 1000 rpm of revolution speed. The effluent from the column outlet was collected into test tubes using a fraction collector (Model CHF 100AA, Advantec, Tokyo, Japan).

2.7. Analysis of CCC fractions

Each collected fraction was diluted with an aliquot of distilled water for proteins and of methanol for 4-methylumbelliferyl sugar derivatives and the absorbance measured at 280 nm for proteins, 540 nm for myoglobin, and 318 nm for 4-methylumbelliferyl sugar derivatives with a spectrophotometer (Model UV-1600, Shimadzu).

2.8. Evaluation of partition efficiency

The efficiencies in separations in the present studies were computed from the chromatogram and expressed in terms of theoretical plate number (N) and peak resolution (Rs) each according to the conventional formula.

3. Results

3.1. Proteins

Figure 2 illustrates the CCC separation of stable proteins obtained using the locular multilayer coil with the lower mobile phase of the 12.5% (w/w) PEG 1000 – 12.5% (w/w) dibasic potassium phosphate system. The decrease of the flow rate produced better resolution of cytochrome C, myoglobin and lysozyme peaks apparently due to the increased stationary phase retention in the column. Table 1 (upper side) summarizes the analytical values calculated from each chromatogram shown in Figure 2. Remarkable increase in the peak resolution was observed by a lower flow rate with increased retention of the stationary phase while the theoretical plate number calculated from the myoglobin peak was almost unchanged.

Figure 2.

CCC separation of proteins obtained by the locular multilayer coiled columns (without glass beads) with lower phase mobile at three different flow rates. Experimental conditions: apparatus: small-scale cross-axis CPC with four locular multilayer coil assemblies, 1.5 mm I.D. and 90 mL total capacity; sample: cytochrome C (2 mg), myoglobin (8 mg) and lysozyme (10 mg); solvent system: 12.5% (w/w) PEG 1000 – 12.5% (w/w) dibasic potassium phosphate; mobile phase: lower phase (outward elution); revolution: 1000 rpm; revolution direction: counterclockwise; fractionate: 2min/tube. SF = solvent front.

Table 1.

Analytical values obtained by CCC separations of proteins with long-pressed locular multilayer coiled columns at three different flow rates

Flow rate (mL/min)	Elution volume (mL)			Peak resolution (Rs)		Theoretical plate number (N)	Theoretical plate number per column capacity (N/mL)	Stationary phase retention (%)
Lower phase mobile
	Cyt C	Myo	Lys	Cyt C/Myo	Myo/Lys
0.8	70	83	110	1.3	1.2	599	6.7	28.9
0.6	60	76	111	1.8	1.5	472	5.2	36.0
0.4	55	72	113	2.2	2.0	568	6.3	39.6
Upper phase mobile
	Lys	Myo		Lys/Myo
0.8	96	119		0.8		387	4.3	9.3
0.6	94	126		1.2		497	5.5	17.3
0.4	89	128		1.8		685	7.6	27.1

Stationary phase retention (Sf) was calculated according to the conventional formula: Sf ={(Vc − Vsf)/Vc}×100, where Vc and Vsf indicate the column capacity and the retention volume of the solvent front (volume of the mobile phase in the column), respectively. The plate number was calculated from the myoglobin (Myo) peak for lower phase mobile and the lysozyme (Lys) peak for upper phase mobile.

Figure 3 similarly illustrates the CCC separation of proteins obtained using the locular multilayer coil (without glass beads) with upper phase mobile. The decrease of the flow rates also improved the resolution between the lysozyme and the myoglobin peaks while their separation times were increased. As summarized in Table 1 (lower side), both of theoretical plate number of the lysozyme peak and stationary phase retention were increased by decreased flow rate of the upper mobile phase.

Figure 3.

CCC separation of proteins obtained by the locular multilayer coiled columns with upper phase mobile at three different flow rates. Experimental conditions: apparatus: small-scale cross-axis CPC with four locular multilayer coil assemblies, 1.5 mm I.D. and 90 mL total capacity; sample: lysozyme (10 mg) and myoglobin (8 mg); solvent system: 12.5% (w/w) PEG 1000 – 12.5% (w/w) dibasic potassium phosphate; mobile phase: upper phase (inward elution); revolution: 1000 rpm; revolution direction: clockwise; fractionate: 2min/tube. SF = solvent front.

3.2. Sugar derivatives

In order to achieve further increase of partition efficiency in protein separation, one glass bead is inserted into each locule of the locular tubing as shown in Figure 1D. It is expected to enhance mixing of two phases in the rotating column during the revolution. However, at the flow rate of 0.6 and 0.4 mL/min the retention of stationary phase substantially decreased with sharp peak resolution and at the flow rate of 0.8 mL/min no stationary phase was retained in the column. This result suggests that vigorous mixing of two phases is produced by glass beads in the locule. It is predicted that improved separation is obtained using an organic-aqueous two-phase solvent system with higher interfacial tension than of the aqueous-aqueous two-phase solvent system. Figure 4 illustrates the CCC separation of five 4-methylumbelliferyl sugar derivatives obtained using the locular beads multilayer coil with lower phase mobile. Under retaining the satisfactory volume of stationary phase in CCC separation, faster flow rate revealed the completed peak resolution between each sugar derivative peak. At the flow rate of 1.4 mL/min, five sugar derivatives were separated in almost 150 min with excellent peak resolution.

Figure 4.

CCC separation of 4-methylumbelliferyl sugar derivatives obtained by the locular beads multilayer coiled columns with lower phase mobile at three different flow rates. Experimental conditions: apparatus: small-scale cross-axis CPC with four locular multilayer coil assemblies, 1.5 mm I.D. and 90 mL total capacity; sample: 4-methylumbelliferyl derivative of β-D-cellobioside (1 mg), β-D-glucopyranoside (1 mg), α-D-mannopyranoside (1 mg), β-D-fucopyranoside (1 mg) and α-L-fucopyranoside (1 mg); solvent system: ethyl acetate/1-butanol/water (3 : 2 : 5, v/v); mobile phase: lower phase (outward elution); revolution: 1000 rpm; revolution direction: counterclockwise; fractionate: 2min/tube. SF = solvent front.

Figure 5 illustrates the CCC separation of three 4-methylumbelliferyl sugar derivatives obtained using the locular beads multilayer coil with upper phase mobile. Increased flow rates produced shorter separation times within 100 min at the flow rate of 1.4 mL/min where three sugar derivatives completely separated from each other at peak resolution ratio of over 2.0 as summarized in Table 2.

Figure 5.

CCC separation of 4-methylumbelliferyl sugar derivatives obtained by the locular beads multilayer coiled columns with upper phase mobile at three different flow rates. Experimental conditions: apparatus: small-scale cross-axis CPC with four locular multilayer coil assemblies, 1.5 mm I.D. and 90 mL total capacity; sample: 4-methylumbelliferyl derivative of α-L-fucopyranoside (1 mg), β-D-galactopyranoside (1 mg) and β-D-cellobioside (1 mg); solvent system: ethyl acetate/1-butanol/water (1 : 4 : 5, v/v); mobile phase: upper phase (inward elution); revolution: 1000 rpm; revolution direction: clockwise; fractionate: 2min/tube. SF = solvent front.

Table 2.

Analytical values obtained by CCC separations of 4-methylumbelliferyl sugar derivatives with long-pressed locular beads multilayer coiled columns at three different flow rates

Flow rate (mL/min)		Elution volume (mL)				Peak resolution (Rs)				Theoretical plate number (N)	Theoretical plate number per column capacity (N/mL)	Stationary phase retention (%)
Lower phase mobile
	Cel	Glc	Man	β-D-Fuc	α-L-Fuc	Cel/Glc	Glc/Man	Man/β-D-Fuc	β-D-Fuc/α-L-Fuc
1.0	64	93	118	144	180	3.2	1.9	1.5	1.6	1103	12.3	42.5
1.2	68	98	122	149	185	4.0	1.8	1.3	1.4	1525	16.9	40.8
1.4	70	98	122	146	183	3.1	1.8	1.4	1.5	1111	12.3	39.7
Upper phase mobile
			α-L-Fuc	Gal	Cel		α-L-Fuc/Gal	Gal/Cel
1.0			75	96	134		2.6	2.8		1462	16.2	34.2
1.2			66	86	122		2.2	2.6		900	10.0	32.0
1.4			77	97	132		2.3	2.7		1137	12.6	30.5

Stationary phase retention (Sf) was calculated according to the conventional formula: Sf ={(Vc − Vsf)/Vc}×100, where Vc and Vsf indicate the column capacity and the retention volume of the solvent front (volume of the mobile phase in the column), respectively. The theoretical plate number was calculated from the 4-methylumbelliferyl β-D-glucopyranoside (Glc) peak for lower phase mobile and the 4-methylumbelliferyl β-D-galactopyranoside (Gal) peak for upper phase mobile.

4. Discussion

The cross-axis CPC is useful for separation of hydrophilic and hydrophobic compounds using both aqueous-aqueous and organic-aqueous two-phase solvent systems at a wide range of hydrophobicity. In the present study, improved separations of proteins and 4-methylumbelliferyl sugar derivatives were performed using the locular tubing with with or without glass beads for the cross-axis CPC.

As shown in Figure 2, sufficient partitioning of proteins between aqueous two immiscible phases in the rotating column was achieved even at a relatively high flow rate of the lower mobile phase of 0.8 mL/min, while the separation was further increased by a lower flow rate but with a longer separation time. Figure 3 also suggested that more sufficient partitioning of proteins is accomplished at a lower flow rate of the upper mobile phase.

Using the organic-aqueous two-phase solvent system with lower phase mobile, 4-methylumbelliferyl sugar derivatives were completely separated from each other at high theoretical plate number obtained from the second β-D-glucopyranoside peak (Figure 3). The results obtained from the upper phase mobile shown in Figure 4 also showed similar to those obtained from the lower phase mobile, indicating that a glass bead inserted into each locule promotes the partitioning of analytes between two phases.

The locular tubing used in the present CCC system successfully contributed to the improvement of CCC separation. It suggests that the compressed portion of the tubing increases the linear velocity of the mobile phase to enhance the mixing of the two phases in each locule.

5. Conclusions

The cross-axis CPC can be used for the effective separation and purification of various compounds at a wide range of hydrophobicity. Improved separation is accomplished using the locular multilayer coil for highly hydrophilic compounds such as proteins with aqueous-aqueous polymer phase systems, and the locular multilayer coil inserted with a glass bead into each locule for hydrophobic compounds with organic-aqueous two-phase solvent systems.