separations Article
Improved Separations of Proteins and Sugar Derivatives Using the Small-Scale Cross-Axis Coil Planet Centrifuge with Locular Multilayer Coiled Columns Kazufusa Shinomiya 1, *, Kazumasa Zaima 1 , Naoki Harikai 1 and Yoichiro Ito 2 1 2
*
School of Pharmacy, Nihon University, 7-7-1, Narashinodai, Funabashi-shi, Chiba 274-8555, Japan;
[email protected] (K.Z.);
[email protected] (N.H.) Laboratory of Bioseparation Technology, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, 10 Center Drive, Building 10, Room 8N230, Bethesda, MD 20892, USA;
[email protected] Correspondence:
[email protected]; Tel.: +81-47-465-6137
Academic Editor: Alain Berthod Received: 11 June 2016; Accepted: 26 August 2016; Published: 22 September 2016
Abstract: (1) Background: Countercurrent chromatography (CCC) is liquid-liquid partition chromatography without using a solid support matrix. This technique requires further improvement of peak resolution and shortening of separation time. (2) Methods: The long-pressed locular multilayer coils with and without mixer glass beads were developed for the separation of proteins and 4-methylumbelliferyl sugar derivatives using a small-scale cross-axis coil planet centrifuge. (3) Results: Proteins were separated from each other and the separation was improved when the flow rate of the mobile phase was decreased from 0.8 to 0.4 mL/min. On the other hand, 4-methylumbelliferyl sugar derivatives were separated at the resolution of almost over 1.5 in short separation time under satisfactory stationary phase retention when the flow rate of the mobile phase was increased from 1.0 to 1.4 mL/min. (4) Conclusion: Better peak resolutions over the previous results were achieved using the long-pressed locular multilayer coil for proteins with aqueous two-phase systems (ATPS) and for 4-methylumbelliferyl sugar derivatives with organic-aqueous two-phase solvent systems by inserting a glass bead into each locule. Keywords: countercurrent chromatography; cross-axis coil planet centrifuge; locular multilayer coil; partition efficiency; proteins; 4-methylumbelliferyl sugar derivatives
1. Introduction Countercurrent chromatography (CCC) is 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 (type-J CCC) and the cross-axis coil planet centrifuge (cross-axis CCC) have proven most useful for effective CCC separations. The type-J CCC is useful for separations mainly using organic-aqueous two-phase solvent systems [1–4], while the cross-axis CCC can be used for the separation of biopolymers with aqueous two-phase systems (ATPS) having low-interfacial tension and high viscosity. The cross-axis CCC 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 instrument 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
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in centrifuge instruments with a rotary seal. In our laboratory, the floor model of the cross-axis CCC has been built and successfully applied to the protein separation using ATPS [7]. Recently, a new small-scale cross-axis CCC has been further developed in our laboratory to improve the theoretical plates and peak resolution [8,9]. This small-scale cross-axis CCC is a compact model where2 offour coiled Separations 2016, 3, 29 10 columns of similar weight are mounted symmetrically around the rotary frame to achieve stable observed centrifuge instruments a rotary seal. In our laboratory, the floor model of the crossbalancing of theincentrifuge even underwith a high revolution speed. axis CCC has been built and successfully applied to the protein separation usingCCC ATPSvaries [7]. Recently, The peak resolution and the stationary phase retention of this cross-axis depending on a new small-scale cross-axis CCC has been further developed in our laboratory to improve the the configuration of the separation column such as conventional multilayer coil [8–10], eccentric theoretical plates and peak resolution [8,9]. This small-scale cross-axis CCC is a compact model where coil [7,11,12], toroidal [11,12], spiral coil [13], and so forth. Our previous revealed four coiled columnscoil of similar weight are mounted symmetrically around the rotary framestudies to achieve that thestable locular multilayer coil prepared by compressing a long piece of polytetrafluoroethylene balancing of the centrifuge even under a high revolution speed. The with peak resolution the stationary phase retention of this CCC varies depending (PTFE) tubing a pair ofand hemostats at regular intervals wascross-axis useful for protein separation with on the configuration the separation suchdisk as conventional multilayer coil [8–10], eccentric ATPS [14]. It has also beenofreported that column the spiral mounted by the cross-pressed tubing for the coil [7,11,12], toroidal coil [11,12], spiral coil [13], and so forth. Our previous studies revealed that the type-J planet centrifuge was useful for separation of proteins with ATPS [15]. Recently, Englert et al. locular multilayer coil prepared by compressing a long piece of polytetrafluoroethylene (PTFE) reported the tubing modifications using the crimping tool to improve the separation efficiency for tubing with a pair of hemostats at regular intervals was useful for protein separation with ATPS [14]. CCC separation of organic-aqueous two-phase solventbysystem by the type-J CCC [16,17]. As described It has also been reported that the spiral disk mounted the cross-pressed tubing for the type-J planet above, the cross-axis CCC for is useful for of the separation of various compounds a wide centrifuge was useful separation proteins with ATPS [15]. Recently, Englert having et al. reported therange of tubing modifications theto crimping toolwith to improve the separationand efficiency for CCC separation hydrophobicity from fattyusing acids proteins organic-aqueous aqueous-aqueous two-phase organic-aqueous two-phase by the the type-J CCC [16,17]. described above, the locule solventofsystems, respectively. Insolvent order system to enhance mixing of theAstwo phases in each cross-axis CCC is useful for the separation of various compounds having a wide range of of the locular tubing, the extension of the compression length to increase the linear velocity of hydrophobicity from fatty acids to proteins with organic-aqueous and aqueous-aqueous two-phase the mobile phase and the insertion of a bead into each locule of the long-pressed locular tubing solvent systems, respectively. In order to enhance the mixing of the two phases in each locule of the may lead to improved over the previous [14]. present describes the locular tubing, the separations extension of the compression lengthstudies to increase theThe linear velocitypaper of the mobile improved separations of proteins with for the long-pressed loculartubing multilayer coil phase and the insertion of a bead into ATPS each locule of the long-pressed locular may lead to and of improved separations the previous studies [14]. The present describes thecontaining improved a glass 4-methylumbelliferyl sugarover derivatives for the long-pressed locularpaper multilayer coil separations proteins with for thecross-axis long-pressed locular multilayer coil and of 4bead mixer in eachoflocule using theATPS small-scale CCC. methylumbelliferyl sugar derivatives for the long-pressed locular multilayer coil containing a glass
bead mixer each locule using the small-scale cross-axis CCC. 2. Materials and in Methods 2. Materials and Methods
2.1. Apparatus
2.1. Apparatus cross-axis CCC employed in the present study was constructed at the Machining The small-scale small-scale cross-axis CCC employed the present was and constructed at the Machining TechnologyThe Center of Nihon University (Chiba,inJapan). Thestudy design fabrication of the apparatus Technology Center of Nihon University (Chiba, Japan). The design and fabrication of the and the column configuration were previously described in detail [8,9]. Figure apparatus 1 illustrates the and the column configuration were previously described in detail [8,9]. Figure 1 illustrates the schematic drawing of the small-scale cross-axis CCC used in the present study. Four coiled columns schematic drawing of the small-scale cross-axis CCC used in the present study. Four coiled columns are distributed at a constant distance from the central revolution axis. The neighboring columns rotate are distributed at a constant distance from the central revolution axis. The neighboring columns in the opposite direction each other while in a horizontal plane around rotate in the oppositetodirection to each otherrevolving while revolving in a horizontal plane aroundthe the vertical vertical axis of the centrifuge as indicated by a set of arrows. axis of the centrifuge as indicated by a set of arrows.
Revolution Axis
Column 3 Column 2
Rotation Axis
Column 4 Column 1
Figure 1. Schematic illustration of the small-scale cross-axis CCC used in the present study.
Figure 1. Schematic illustration of the small-scale cross-axis CCC used in the present study.
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2.2. Preparation of the Long-Pressed Locular Tubing and the Bead Embedded Locular Tubing 2.2. Preparation of the Long-Pressed Locular Tubing and the Bead Embedded Locular Tubing
The newly designed long-pressed locular tubing was prepared from a single piece of 1.5 mm The newly designed long-pressed 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 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 it with a pair of tongs (0.8 cm wide) for pressing at 1.2 cm intervals to form a number of segments calledcalled locules. TheThe bead embedded was prepared preparedbyby adding a glass (1 mm locules. bead embeddedlocular loculartubing tubing was adding a glass ballball beadbead (1 mm I.D., Toshinriko, Inc., Tokyo, Japan) in each locule for enhancing the mixing of two phases. Figure 2 I.D., Toshinriko, Inc., Tokyo, Japan) in each locule for enhancing the mixing of two phases. Figure 2 illustrates the schematic drawing ofof the tubingwith with and without glass beads. illustrates the schematic drawing thelong-pressed long-pressed locular locular tubing and without glass beads. A. Normal tubing
B. Locular tubing clamped
2 cm
2 cm interval clamped C. Long-pressed locular tubing 1.2 cm interval and 0.8 cm pressed 0.8 cm 1.2 cm D. Bead embedded locular tubing
1.0 mm
Figure 2. Schematic illustrationofofthe thelocular locular tubing tubing with a glass bead mixer. Figure 2. Schematic illustration withand andwithout without a glass bead mixer.
2.3. Preparation of Coiled Column Assembly 2.3. Preparation of Coiled Column Assembly Each multilayer coil was prepared by tightly winding a long piece of the aforementioned PTFE
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 tubing around the holder hubEach of 3 coiled cm in column diameter, tight coiled between a pair of flanges spaced 5 cm apart. wasforming preparedfive according to the layers following procedure: flanges cmdirectly apart. Each coiled column to the procedure: Thespaced tubing 5was wound onto the holderwas hubprepared starting onaccording the proximal side,following which is close to The tubing was directly wound onto the holder hub starting on the side,an which is close the center of revolution. After each coil layer was completed, the layer wasproximal wrapped with adhesive to thetape center of revolution. After each coil to layer was side completed, layerinwas wrapped with an and the tubing was straightly returned the other to resumethe winding the same direction. It results in a multilayer coil assembly composed of either entirely rightor left-handed coils, which adhesive tape and the tubing was straightly returned to the other side to resume winding in the same is different from commonly used in the type-J CCC.of Two columns of left-handed coils werecoils, direction. It results in that a multilayer coil assembly composed either entirely right- or left-handed subjected to the forward rotation; and right-handed coils, the backward rotation. Two pairs of coils right-were which is different from that commonly used in the type-J CCC. Two columns of left-handed and left-handed coil assemblies were connected in series with flow tubes in such a way that the distal subjected to the forward rotation; and right-handed coils, the backward rotation. Two pairs of rightterminal of the first column assembly is connected to the proximal terminal, which is close to the and left-handed coil assemblies were connected in series with flow tubes in such a way that the distal center of revolution, of the second column assembly, and so on. Four coil assemblies were terminal of the firstmounted column assembly is connected to the proximal terminal, which is close to the center symmetrically on the rotary frame for balancing the centrifuge instrument. of revolution, of the second column assembly, and so on. Four coil assemblies were symmetrically 2.4. Reagents mounted on the rotary frame for balancing the centrifuge instrument. Polyethylene glycol (PEG) 1000 (MW 1000), 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,(PEG) USA). Dibasic potassium was from Wako Chemicals Polyethylene glycol 1000 (MW 1000), phosphate cytochrome C obtained (horse heart) (MW Pure 12,384), myoglobin Japan). (horse(Osaka, skeletal muscle) (MW 17,800), and lysozyme (chicken egg) (MW 13,680) were purchased from sugar derivatives as test samples were purchased asPure follows: 4Sigma (St.4-Methylumbelliferyl Louis, MO, USA). Dibasic potassiumused phosphate was obtained from Wako Chemicals Methylumbelliferyl-α-L-fucopyranoside (α-L-Fuc), 4-methylumbelliferyl-β-D-fucopyranoside (β-D(Osaka, Japan). Fuc), 4-methylumbelliferyl-β-D-glucopyranoside (Glc), 4-methylumbelliferyl-β-D-galactopyranoside 4-Methylumbelliferyl sugar derivatives used as test samples were purchased as follows: (Gal), and 4-methylumbelliferyl-β-D-cellobioside (Cel) were purchased from BIOSYNTH AG (Staad,
2.4. Reagents
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
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(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 ATPS 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 the separation by the lower mobile phase and (1:4:5, v/v) for the upper mobile phase [18]. Each solvent mixture was thoroughly equilibrated in a separatory funnel at room temperature. 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 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 Theoretical Plate Number and Peak Resolution 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 following conventional formula. N= Rs =
4tR W
2
2(tR2 − tR1 ) W1 + W2
3. Results 3.1. Proteins Figure 3 illustrates the CCC separation of stable proteins obtained using the long-pressed 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 3. Remarkable increase in the peak resolution was observed by a lower flow rate
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withincreased increasedretention retentionofofthe thestationary stationary phase while theoretical plate number calculated with phase while thethe theoretical plate number calculated fromfrom the the myoglobin almost unchanged. myoglobin peakpeak was was almost unchanged. A. 0.8 mL/min
Cytochrome C
ABSORBANCE (280 nm)
2.0
1.5
C. 0.4 mL/min
B. 0.6 mL/min
Cytochrome C 1.5 Myoglobin
3.0 Myoglobin
Lysozyme
Lysozyme
1.0
1.0
Cytochrome C
2.5 2.0
SF Myoglobin Lysozyme
1.5 SF
0.5
0.5
1.0
SF
0.5 100
0
200 0
200
100
0
100
200
300
400
TIME (min) Figure Figure 3. 3. CCC CCC separation separation of of proteins proteins obtained obtained by by the the long-pressed long-pressed locular locular multilayer multilayer coiled coiled columns columns (without with the lower mobile phase at threeat different flow rates.flow Experimental conditions: (withoutglass glassbeads) beads) with the lower mobile phase three different rates. Experimental apparatus: CCCcross-axis with four long-pressed locular multilayer coil assemblies, 1.5 mm conditions:small-scale apparatus:cross-axis small-scale CCC with four long-pressed locular multilayer coil I.D. and 90 mL cytochrome (2 mg), myoglobin andmyoglobin lysozyme (10 assemblies, 1.5total mm capacity; I.D. and sample: 90 mL total capacity;Csample: cytochrome(8Cmg), (2 mg), (8 mg); mg), solvent system: (10 12.5% (w/w) PEGsystem: 1000–12.5% potassium phosphate; mobile phase: and lysozyme mg); solvent 12.5%(w/w) (w/w)dibasic PEG 1000–12.5% (w/w) dibasic potassium lower phase (outward elution); revolution: 1000 rpm; revolution direction: counterclockwise; fractions phosphate; mobile phase: lower phase (outward elution); revolution: 1000 rpm; revolution direction: collected at 2 min/tube. SF =collected solvent front. counterclockwise; fractions at 2 min/tube. SF = solvent front. Table Table1.1. Analytical Analytical values values obtained obtained by by CCC CCC separations separations of of proteins proteins with with the the long-pressed long-pressed locular locular multilayer columns at three different flow rates. multilayer columns at three different flow rates. Flow Flowrate rate (mL/min)
(mL/min)
Theoretical Theoretical Elution volume (mL)
Elution volume (mL)
Peak resolution (Rs)
Peak resolution (Rs)
0.6 0.8 0.4
60 5570
76 83 72
0.6mobile phase 60 Upper
76
0.4 55 Lys 0.8 96 Upper mobile phase 0.6 94 0.4 89 Lys
Lys
Cyt C/Myo
110 Cyt C/Myo 1.3 Lys
Stationary Stationary phase phase retention (%)
number (N)
retention (%)
1.2 Myo/Lys 1.5 1.2 2.0
599 472 599 568
28.9 36.0 28.9 39.6
Lower mobile phase
Lower mobile phase Cyt C Myo 0.8 70 83 Cyt C Myo
plate plate number (N)
Myo/Lys
111 110 113
1.8 1.3 2.2
111
1.8
1.5
472
36.0
72 Myo113
2.2
Lys/Myo2.0 0.8 1.2 1.8 Lys/Myo
568
39.6
119 126 128 Myo
387 497 685
9.3 17.3 27.1
Stationary phase96 retention (Sf) was the conventional formula: Sf = (Vc − Vm)/Vc} × 100, 0.8 119calculated according to0.8 387 9.3 where Vc and Vm indicate the column capacity and the retention volume of the solvent front (volume of the 0.6 phase in94 126 1.2was calculated from the 497 17.3for mobile the column), respectively. The plate number myoglobin (Myo) peak the lower mobile phase and the lysozyme (Lys) peak for the upper mobile phase.
0.4
89
128
1.8
685
27.1
Stationary phase retention (Sf) was calculated according to the conventional formula: Sf = (Vc −
Figure 4 similarly illustrates the CCC separation of proteins obtained using the long-pressed Vm)/Vc} × 100, where Vc and Vm indicate the column capacity and the retention volume of the solvent locular multilayer coil (without glass beads) with the upper mobile phase. The decrease of the front (volume of the mobile phase in the column), respectively. The plate number was calculated from flow rates also improved the resolution between the lysozyme and the myoglobin peaks while their the myoglobin (Myo) peak for the lower mobile phase and the lysozyme (Lys) peak for the upper separation times were increased. As summarized in Table 1 (lower side), both of theoretical plate mobile phase.
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ABSORBANCE (280 nm
, 540 nm
)
Figure 4 similarly illustrates the CCC separation of proteins obtained using the long-pressed locular multilayer coil (without glass beads) with the upper mobile phase. The decrease of the 6flow Separations 2016, 3, 29 of 10 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 decreased flow rate of number of the lysozyme lysozyme peak peak and andstationary stationaryphase phaseretention retentionwere wereincreased increasedbyby decreased flow rate thethe upper mobile phase. of upper mobile phase.
B. 0.6 mL/min
A. 0.8 mL/min 2.5
Lysozyme
2.0
1.5
1.5
1.0
1.0
SF
0.5 100
200
0
3.0
Lysozyme
2.5 2.0
1.5 Myoglobin
Myoglobin
0.5 0
Lysozyme
2.5
2.0
C. 0.4 mL/min
SF 100
1.0 0.5
200
300 0
Myoglobin
SF 100
200
300
400
TIME (min)
Figure Figure 4. 4. CCC CCC separation separation of of proteins proteins obtained obtained by by the the long-pressed long-pressed locular locular multilayer multilayer coiled coiled columns columns with at three different flowflow rates. Experimental conditions: apparatus: smallwith the theupper uppermobile mobilephase phase at three different rates. Experimental conditions: apparatus: scale cross-axis CCC with four long-pressed locular multilayer coil assemblies, 1.5 mm I.D. and 90 mL small-scale cross-axis CCC with four long-pressed locular multilayer coil assemblies, 1.5 mm I.D. total capacity; sample: lysozyme (10 mg) and myoglobin (8 mg); solvent system: 12.5% (w/w) PEG and 90 mL total capacity; sample: lysozyme (10 mg) and myoglobin (8 mg); solvent system: 12.5% 1000–12.5% (w/w) dibasic potassium mobile phase: upper phase (inward (w/w) PEG 1000–12.5% (w/w) dibasic phosphate; potassium phosphate; mobile phase: upper phase elution); (inward revolution: 1000 rpm; 1000 revolution direction: clockwise; collected at 2collected min/tube. = solvent elution); revolution: rpm; revolution direction: fractions clockwise; fractions at SF 2 min/tube. front. SF = solvent front.
3.2. 3.2. Sugar Sugar Derivatives Derivatives In In order order to to achieve achieve further further increase increase of of peak peak resolution resolution in in protein protein separation, separation, aa glass glass bead bead was was inserted into each locule of the long-pressed locular tubing as shown in Figure 2D. The inclusion inserted into each locule of the long-pressed locular tubing as shown in Figure 2D. The inclusion of of aa glass glass bead bead into into each each locule locule was was expected expected to to enhance enhance mixing mixing of of two two phases phasesin inthe therotating rotatingcolumn. column. However, flow rate rate of of 0.6 0.6 and and 0.4 0.4 mL/min, mL/min, the However, at at the the flow the retention retention of of stationary stationary phase phase substantially substantially decreased along with peak resolution. At the flow rate of 0.8 mL/min, no stationary decreased along with peak resolution. At the flow rate of 0.8 mL/min, no stationary phase phase was was retained in the column. It seems that the glass bead enhances mixing but does not allow retained in the column. It seems that the glass bead enhances mixing but does not allow enough enough settling toprovide provide significant stationary retention. It was predicted that separation improved settling to forfor significant stationary phasephase retention. It was predicted that improved separation would using be obtained using an organic-aqueous two-phase solvent system with tension higher would be obtained an organic-aqueous two-phase solvent system with higher interfacial interfacial tension than of the ATPS. Figure 5 illustrates the CCC separation of five 4than of the ATPS. Figure 5 illustrates the CCC separation of five 4-methylumbelliferyl sugar derivatives methylumbelliferyl sugar derivatives obtained using the bead embedded locular multilayer coil with obtained using the bead embedded locular multilayer coil with the lower mobile phase. While retaining the lower mobile phase. While retaining the satisfactory volume of stationary phase CCC the satisfactory volume of stationary phase in CCC separation, faster flow rate revealed the in baseline separation, faster flow rate revealed the baseline peak resolution between each sugar derivative peak. peak resolution between each sugar derivative peak. At the flow rate of 1.4 mL/min, five sugar At the flow rate 1.4 mL/min, five sugar derivatives were separated in almost 150 min with baseline derivatives wereofseparated in almost 150 min with baseline peak resolution. peak resolution.
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B. 1.2 mL/min
A. 1.0 mL/min
Glc 16 C. 1.4 mL/min
B. 1.2 mL/min
A. 1.0 mL/min
Glc
16 14
Cel
14 12
nm) nm) (318 (318 ABSORBANCE ABSORBANCE
10 Cel
8
Cel
86
Glc Glc
SF
42
SF
2
Cel Glc
64
200
100
0
200
100
Man
108
Man β-D-Fuc
86
α-L-Fuc β-D-Fuc
64
α-L-Fuc
42 SF
SF
2
0
β-D-Fuc
SF
42
Cel
10 12
Man β-D-Fuc α-L-Fuc Man α-L-Fuc
86
β-D-Fuc
Man β-D-Fucα-L-Fuc Man α-L-Fuc
64
Cel Glc
108
2
0
200
100 TIME (min) 100
0
7 of 10
C. 1.4 mL/min
200
SF 0
100
0
100
TIME (min)
Figure 5. CCC separation of 4-methylumbelliferylsugar sugar derivatives derivatives obtained the bead embedded Figure 5. CCC separation of 4-methylumbelliferyl obtainedbyby the bead embedded locular multilayer coiled columns with the lower mobile phase at three different flow rates. locular multilayer coiled columns with the lower mobile phase at three different flow Figure 5. CCC separation of 4-methylumbelliferyl sugar derivatives obtained by the bead embeddedrates. Experimental conditions: apparatus: small-scale cross-axis CCC with four bead embedded locular Experimental conditions: apparatus: small-scale cross-axis four bead embedded locular locular multilayer coiled columns with the lower mobile CCC phasewith at three different flow rates. multilayer coil assemblies, 1.5 mm I.D. and 90 mL total capacity; sample: 4-methylumbelliferyl multilayer coil assemblies, 1.5 mm I.D. and 90 mL total capacity; sample: 4-methylumbelliferyl Experimental conditions: apparatus: small-scale cross-axis CCC with four bead embedded locular derivative of β-D-cellobioside (1 mg), β-D-glucopyranoside (1 mg), α-D-mannopyranoside (1 mg), βmultilayer 1.5 mm I.D. and mL total capacity;(1sample: derivative of coil β-Dassemblies, -cellobioside (1 mg), β-D90 -glucopyranoside mg), 4-methylumbelliferyl α-D-mannopyranoside D-fucopyranoside (1 mg), and α-L-fucopyranoside (1 mg); solvent system: ethyl acetate/1derivative of β-D-cellobioside (1 (1 mg), D-glucopyranoside (1 mg), α-(1 D-mannopyranoside (1 mg), β-ethyl (1 mg), β-D-fucopyranoside mg),β-and α-L-fucopyranoside mg); solvent system: butanol/water (3:2:5, v/v); mobile phase: lower phase (outward elution); revolution: 1000 rpm; D-fucopyranoside (1 mg), andv/v); α-L-fucopyranoside mg);phase solvent system: elution); ethyl acetate/1acetate/1-butanol/water (3:2:5, mobile phase: (1 lower (outward revolution: revolution direction: counterclockwise; fractions collected at 2 min/tube. SF = solvent front. (3:2:5, v/v); mobile phase: lowerfractions phase (outward revolution: rpm; 1000butanol/water rpm; revolution direction: counterclockwise; collectedelution); at 2 min/tube. SF =1000 solvent front. revolution direction: counterclockwise; fractions collected at 2 min/tube. SF = solvent front.
Figure 6 illustrates the CCC separation of three 4-methylumbelliferyl sugar derivatives obtained using the bead embedded locular multilayer coil with the upper mobile phase. Increased flowobtained rates Figure 6 illustrates the CCC three 4-methylumbelliferyl sugar derivatives Figure 6 illustrates the CCCseparation separation of of three 4-methylumbelliferyl sugar derivatives obtained produced shorter separation times within 100 min at the flow rate of 1.4 mL/min where three sugar using the the bead embedded locular withthe theupper uppermobile mobile phase. Increased using bead embedded locularmultilayer multilayer coil coil with phase. Increased flowflow ratesrates derivatives baseline separated from each 100 other at peak resolution ratio of over 2.0 aswhere summarized in produced shorter separation times within min at the flow rate of 1.4 mL/min three sugar produced shorter separation times within 100 min at the flow rate of 1.4 mL/min where three sugar Table 2. derivatives baseline separatedfrom fromeach each other other at peak of of over 2.02.0 as summarized in in derivatives baseline separated peakresolution resolutionratio ratio over as summarized Table Table 2. 2. A. 1.0 mL/min
B. 1.2 mL/min
nm) nm) (318 (318 ABSORBANCE ABSORBANCE
A. 1.0 mL/min
B. 1.2 mL/min
α-L-Fuc
8
8
α-L-Fuc Gal
86
86
Gal
6 4
Cel
42
SF
Cel
SF
2 0 0
42
100
86
Gal Cel Cel
SF
2 100
0 0
α-L-Fuc Gal
Gal α-L-Fuc
SF
mL/min α-L-Fuc 10 C. 1.4 10 8
α-L-Fuc
64
C. 1.4 mL/min
6 4
Cel
4 2
SF
TIME (min) 100
Cel
SF
2 100
Gal
0 0
100 100
TIME (min) Figure 6. CCC separation of 4-methylumbelliferyl sugar derivatives obtained by the bead embedded locular multilayer coiled columns with the upper mobile phase at three different flow rates. Figure 6. CCC separation 4-methylumbelliferyl sugar obtained by by thethe bead embedded Figure 6. CCC separation ofof4-methylumbelliferyl sugarderivatives derivatives obtained bead embedded Experimental conditions: apparatus: small-scale cross-axis CCC with four bead embedded locular locular multilayer coiled columns with the upper mobile phase at three different flow rates.rates. locular multilayer coiled columns with the upper mobile phase at three different flow multilayer coil assemblies, 1.5 mm I.D. and 90 mL total capacity; sample: 4-methylumbelliferyl Experimental conditions: apparatus: small-scale cross-axis CCC with four bead embedded locular Experimental apparatus: small-scale cross-axis CCC(1with fourβ-bead embedded locular derivative conditions: of α-L-fucopyranoside (1 mg), β-D-galactopyranoside mg) and D-cellobioside (1 mg); multilayer coil assemblies,1.5 1.5mm mmI.D. I.D. and and 90 90 mL mL total total capacity; sample: 4-methylumbelliferyl multilayer coil assemblies, capacity; sample: 4-methylumbelliferyl solvent system: ethyl acetate/1-butanol/water (1:4:5, v/v); mobile phase: upper phase (inward elution); derivative of α-L-fucopyranoside (1 mg), β-D-galactopyranoside (1 mg) and β-D-cellobioside (1 mg); derivative of α-1000 L -fucopyranoside mg), β-Dclockwise; -galactopyranoside (1 mg) and β-D-cellobioside (1 mg); revolution: rpm; revolution(1direction: fractions collected at 2 min/tube. SF = solvent solvent system: ethyl acetate/1-butanol/water (1:4:5, v/v); mobile phase: upper phase (inward elution); solvent system: ethyl acetate/1-butanol/water (1:4:5, v/v); mobile phase: upper phase (inward front. revolution: 1000 rpm; revolution direction: clockwise; fractions collected at 2 min/tube. SF = solvent elution); revolution: 1000 rpm; revolution direction: clockwise; fractions collected at 2 min/tube. front.
SF = solvent front.
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Table 2. Analytical values obtained by CCC separations of 4-methylumbelliferyl sugar derivatives with the bead embedded locular multilayer coiled column at three different flow rates. Elution volume (mL)
Flow rate (mL/min)
Peak resolution (Rs)
Theoretical plate number (N)
Stationary phase retention (%)
β-D-Fuc/ α-L-Fuc 1.6 1.4 1.5
1103 1525 1111
42.5 40.8 39.7
Gal/Cel 2.8 2.6 2.7
1462 900 1137
34.2 32.0 30.5
Lower mobile phase
1.0 1.2 1.4
Cel
Glc
Man
β-D-Fuc
α-L-Fuc
Cel/Glc
Glc/Man
64 68 70
93 98 98
118 122 122
144 149 146
180 185 183
3.2 4.0 3.1
1.9 1.8 1.8
Man/ β-D-Fuc 1.5 1.3 1.4
Upper mobile phase 1.0 1.2 1.4
α-L-Fuc 75 66 77
Gal 96 86 97
Cel 134 122 132
α-L-Fuc/Gal 2.6 2.2 2.3
Stationary phase retention (Sf) was calculated according to the conventional formula: Sf = {(Vc − Vm)/Vc} × 100, where Vc and Vm 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 the lower mobile phase and the 4-methylumbelliferyl β-D-galactopyranoside (Gal) peak for the upper mobile phase.
4. Discussion The cross-axis CCC 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 were performed using the long-pressed locular tubing without glass beads and baseline separations of 4-methylumbelliferyl sugar derivatives were achieved using the bead embedded locular tubing for the small-scale cross-axis CCC. As shown in Figure 3, sufficient partitioning of proteins at the resolution of over 1.2 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 4 also suggested that more sufficient partitioning of proteins at the resolution of over 1.5 is accomplished at a lower flow rate of 0.4 mL/min with the upper mobile phase. As summarized in Table 1, the stationary phase retention is inversely proportional to flow rate. Decreased flow rate of the mobile phase produced the increase of stationary phase retention and peak resolution. In general, higher N value does not follow higher Rs values. Faster flow rate decreases separation time of each analyte due to the decrease of the stationary phase retention. As described in the conventional formula, apparently both retention time and W decrease at faster flow rates. If N increases or decreases depend on their rate of decrease, smaller W value calculates higher N value while all analytes elute within shorter separation time. Using the organic-aqueous two-phase solvent system with the lower mobile phase, 4-methylumbelliferyl sugar derivatives were baseline separated from each other at high theoretical plate number obtained from the second β-D-glucopyranoside peak (Figure 5). The results obtained using the upper mobile phase shown in Figure 6 also appear similar to those obtained using the lower mobile phase, indicating that a glass bead inserted into each locule promotes the partitioning of analytes between two phases. As described above, increased flow rate generally decreases stationary phase retention and peak resolution. However, in the present studies, baseline separations at their resolutions of almost over 1.5 were achieved by the faster flow rates whereas the stationary phase retention was gradually decreased as summarized in Table 2. In a series of these experiments, the long-pressed locular tubing inserted with a bead into every other locule and the long-pressed tubing inserted with a bead into every third locule were also applied to similar separations of 4-methylumbelliferyl sugar derivatives with almost equivalent column volumes. Better peak resolution with shorter separation time was obtained using the long-pressed locular tubing inserted with a bead into each locule than the other two kinds of the bead embedded locular tubing. This suggests that the beads increased mixing at
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higher flow rates which compensated for decrease in stationary phase retention. Similar studies were further performed using ATPS for protein separation. Better peak resolution and stationary phase retention was obtained in the long-pressed locular tubing without inserting with a bead into each locule more than three kinds of the bead embedded locular tubing, which was different from the result obtained using organic-aqueous two-phase solvent systems with the present small-scale cross-axis CCC instrument. The long-pressed locular tubing and the bead embedded locular tubing used in the present CCC instrument 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. Summary The cross-axis CCC can be used for the effective separation and purification of various compounds at a wide range of hydrophobicity. Improved separation is accomplished using the long-pressed locular multilayer coil for highly hydrophilic compounds such as proteins with ATPS, and the long-pressed locular multilayer coil inserted with a glass bead into each locule for hydrophobic compounds with organic-aqueous two-phase solvent systems. Acknowledgments: The authors would like to thank Motoki Umezawa, Manami Seki, and Jun Nitta for their technical assistance. Author Contributions: Kazufusa Shinomiya designed, conducted, performed the experiments in the present study and wrote the paper; Kazumasa Zaima and Naoki Harikai analyzed the data; Yoichiro Ito discussed about the data and checked the paper. Conflicts of Interest: The authors declare no conflict of interest.
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