Academic Editor: Alain Berthod Received: 11 June 2016; Accepted: 26 August 2016; Published: 22 September 2016

Transcription

1 separations Article Improved Separations Protes and Sugar Derivatives Usg Small-Scale Cross-Axis Coil Planet Centrifuge with Locular Multilayer Coiled Columns Kazufusa Shomiya 1, *, Kazumasa Zaima 1, Naoki Harikai 1 and Yoichiro Ito 2 1 School Pharmacy, Nihon University, 7-7-1, Narashodai, Funabashi-shi, Chiba , Japan; zaima.kazumasa@nihon-u.ac.jp (K.Z.); harikai.naoki@nihon-u.ac.jp (N.H.) 2 Laboratory Bioseparation Technology, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes Health, 10 Center Drive, Buildg 10, Room 8N230, Besda, MD 20892, USA; itoy@nhlbi.nih.gov * Correspondence: shomiya.kazufusa@nihon-u.ac.jp; Tel.: Academic Editor: Ala 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 usg a solid support matrix. This technique requires furr improvement peak resolution and shorteng separation time. (2) Methods: The long-pressed multilayer coils with and without mixer s were developed for separation protes and 4-methylumbelliferyl sugar derivatives usg a small-scale cross-axis coil planet centrifuge. (3) Results: Protes were separated from or and separation improved when flow rate mobile decreased from 0.8 to 0.4 ml/m. On or hand, 4-methylumbelliferyl sugar derivatives were separated at resolution almost over 1.5 short separation time under satisfactory retention when flow rate mobile creased from to 1.4 ml/m. (4) Conclusion: Better peak resolutions over previous results were achieved usg long-pressed multilayer coil for protes with aqueous two- systems (ATPS) and for 4-methylumbelliferyl sugar derivatives with organic-aqueous two- solvent systems by sertg a to locule. Keywords: countercurrent chromatography; cross-axis coil planet centrifuge; multilayer coil; partition efficiency; protes; 4-methylumbelliferyl sugar derivatives 1. Introduction Countercurrent chromatography (CCC) is liquid-liquid partition chromatography which elimates solid support matrix for [1 4]. In past, various types CCC struments have been developed to achieve satisfactory retention liquid column. Among m, type-j multilayer coil planet centrifuge (type-j CCC) and cross-axis coil planet centrifuge (cross-axis CCC) have proven most useful for effective CCC separations. The type-j CCC is useful for separations maly usg organic-aqueous two- solvent systems [1 4], while cross-axis CCC can be used for separation biopolymers with aqueous two- systems (ATPS) havg low-terfacial tension and high viscosity. The cross-axis CCC has a distctive feature that coiled column revolves around vertical central axis centrifuge while it rotates about its horizontal axis at same angular velocity [5,6]. This centrifuge strument permits flow tubes to rotate without twistg so that rotatg column can be contuously eluted with mobile without a risk leakage which is ten observed Separations 2016, 3, 29; doi: /separations

2 Separations 2016, 3, centrifuge struments with a rotary seal. In our laboratory, floor model cross-axis CCC has been built and successfully applied to prote separation usg ATPS [7]. Recently, a new small-scale cross-axis CCC has been furr developed our laboratory to improve oretical plates and peak resolution [8,9]. This small-scale cross-axis CCC is a compact model where four coiled Separations 2016, 3, columns similar weight are mounted symmetrically around rotary frame to achieve stable balancg observed centrifuge struments even under with a high a rotary revolution seal. In our speed. laboratory, floor model crossaxis peak CCC resolution has been built and and successfully applied retention to prote this separation cross-axis usg CCC ATPS varies [7]. Recently, dependg on The a new small-scale cross-axis CCC has been furr developed our laboratory to improve configuration separation column such as conventional multilayer coil [8 10], eccentric oretical plates and peak resolution [8,9]. This small-scale cross-axis CCC is a compact model where coil [7,11,12], four coiled toroidal columns coil similar [11,12], weight spiral are mounted coil [13], symmetrically and so forth. around Our rotary previous frame studies to achieve revealed that stable balancg multilayer centrifuge coil prepared even under by compressg a high revolution a speed. long piece polytetrafluoroethylene (PTFE) tubg The with peak resolution a pair and hemostats regular retention tervals this cross-axis useful for CCC prote varies dependg separation with ATPS [14]. on It has configuration also been reported separation thatcolumn spiral such disk as conventional mounted by multilayer cross-pressed coil [8 10], eccentric tubg for coil [7,11,12], toroidal coil [11,12], spiral coil [13], and so forth. Our previous studies revealed that type-j planet centrifuge useful for separation protes with ATPS [15]. Recently, Englert et al. multilayer coil prepared by compressg a long piece polytetrafluoroethylene (PTFE) reported tubg modifications usg crimpg tool to improve separation efficiency for tubg with a pair hemostats at regular tervals useful for prote separation with ATPS [14]. CCC separation It has also been organic-aqueous reported that spiral two- disk mounted solvent by system cross-pressed by type-j tubg CCC for [16,17]. type-j planet As described above, centrifuge cross-axis useful CCCfor is useful separation for protes separation with ATPS various [15]. Recently, compounds Englert havg et al. reported a wide range hydrophobicity tubg modifications from fattyusg acids tocrimpg protes tool with to improve organic-aqueous separation and efficiency aqueous-aqueous for CCC separation two- solvent systems, organic-aqueous respectively. two- Insolvent order system to enhance by type-j mixg CCC [16,17]. As two described s above, locule cross-axis CCC is useful for separation various compounds havg a wide range tubg, extension compression length to crease lear velocity hydrophobicity from fatty acids to protes with organic-aqueous and aqueous-aqueous two- mobile and sertion a to locule long-pressed tubg solvent systems, respectively. In order to enhance mixg two s locule may lead to improved tubg, separations extension over compression previous length studies to crease [14]. The lear present velocity paper mobile describes improved separations and sertion protes a with to ATPS locule for long-pressed tubg multilayer may lead coil to and 4-methylumbelliferyl improved separations sugarover derivatives previous for studies long-pressed [14]. The present paper multilayer describes coil contag improved a mixer separations locule protes usg with ATPS small-scale for cross-axis long-pressed CCC. multilayer coil and 4- methylumbelliferyl sugar derivatives for long-pressed multilayer coil contag a 2. Materials mixer and Methods locule usg small-scale cross-axis CCC. 2. Materials and Methods 2.1. Apparatus The 2.1. small-scale Apparatus cross-axis CCC employed present study constructed at Machg TechnologyThe Center small-scale Nihon cross-axis University CCC employed (Chiba, Japan). present Thestudy design and constructed fabricationat Machg apparatus and Technology column configuration Center Nihon were University previously (Chiba, Japan). described The design detail and fabrication [8,9]. apparatus 1 illustrates and column configuration were previously described detail [8,9]. 1 illustrates schematic drawg small-scale cross-axis CCC used present study. Four coiled columns schematic drawg small-scale cross-axis CCC used present study. Four coiled columns are distributed at a constant distance from central revolution axis. The neighborg columns rotate are distributed at a constant distance from central revolution axis. The neighborg columns opposite rotate direction opposite todirection or to while or revolvg while revolvg a horizontal a horizontal plane plane around around vertical axis centrifuge axis ascentrifuge dicatedas by dicated a set by arrows. a set arrows. Revolution Axis Column 3 Rotation Axis Column 2 Column 4 Column 1 1. Schematic illustration small-scale cross-axis CCC used present study. 1. Schematic illustration small-scale cross-axis CCC used present study.

3 Separations 2016, 3, Separations 2016, 3, Preparation Long-Pressed Locular Tubg and Bead Embedded Locular Tubg 2.2. Preparation Long-Pressed Locular Tubg and Bead Embedded Locular Tubg The newly designed long-pressed tubg prepared from a sgle piece 1.5 mm The newly designed long-pressed tubg prepared from a sgle piece 1.5 mm I.D., 2.5 mm O.D. PTFE (polytetrafluoroethylene) tubg (Flon Kogyo, Tokyo, Japan) by compressg I.D., 2.5 mm O.D. PTFE (polytetrafluoroethylene) tubg (Flon Kogyo, Tokyo, Japan) by compressg it with a pair tongs (0.8 cm wide) for pressg at 1.2 cm tervals to form a number segments it with a pair tongs (0.8 cm wide) for pressg at 1.2 cm tervals to form a number segments called called locules. locules. The The embedded embedded tubg prepared preparedby byaddg addg a a ball ball (1 mm (1 mm I.D., Toshriko, I.D., Toshriko, Inc., Inc., Tokyo, Tokyo, Japan) Japan) locule for enhancg mixg mixg two two s. s. 2 2 illustrates illustrates schematic drawg long-pressed tubg with and and without s. s. A. Normal tubg B. Locular tubg clamped 2 cm 2 cm terval clamped C. Long-pressed tubg 1.2 cm terval and 0.8 cm pressed 0.8 cm 1.2 cm D. Bead embedded tubg mm 2. Schematic 2. illustration tubg with and without a a mixer. mixer Preparation Coiled Column Assembly 2.3. Preparation Coiled Column Assembly Each multilayer coil prepared by tightly wdg a long piece aforementioned PTFE Each multilayer coil prepared by tightly wdg a long piece aforementioned PTFE tubg around holder hub 3 cm diameter, formg five tight coiled layers between a pair tubg flanges around spaced holder 5 cm apart. hub Each 3 coiled cm column diameter, formg prepared five accordg tight coiled to layers followg between procedure: a pair flanges The spaced tubg 5 cmdirectly apart. Each wound coiled onto column holder hub prepared startg on accordg proximal to side, followg which is close procedure: to The tubg center revolution. directly wound After ontocoil layer holder completed, hub startg layer on proximal wrapped with side, an which adhesive is close to tape center and revolution. tubg straightly After returned coil to layer or side completed, to resume wdg layer same wrapped direction. with an adhesive It results tape and a multilayer tubgcoil sembly straightly composed returned eir to entirely or side right- toor resume left-handed wdg coils, which same direction. is different It results from that a multilayer commonly coil used assembly type-j composed CCC. Two eir columns entirely left-handed right- or left-handed coils were coils, whichsubjected is different to from forward that commonly rotation; and used right-handed type-j coils, CCC. backward Two columns rotation. left-handed Two pairs coils right- were and left-handed coil assemblies were connected series with flow tubes such a way that distal subjected to forward rotation; and right-handed coils, backward rotation. Two pairs rightand left-handed coil assemblies were connected series with flow tubes such a way that distal termal first column assembly is connected to proximal termal, which is close to center revolution, second column assembly, and so on. Four coil assemblies were termal symmetrically first mounted column assembly on rotary is connected frame for balancg to proximal centrifuge termal, strument. which is close to center revolution, second column assembly, and so on. Four coil assemblies were symmetrically mounted 2.4. Reagents on rotary frame for balancg centrifuge strument. Polyethylene glycol (PEG) 1000 (MW 1000), cytochrome C (horse heart) (MW 12,384), myoglob 2.4. Reagents (horse skeletal muscle) (MW 17,800), and lysozyme (chicken egg) (MW 13,680) were purchased from Polyethylene Sigma (St. Louis, glycol MO, (PEG) USA). Dibasic 1000 (MW potassium 1000), phosphate cytochrome C obtaed (horse heart) from Wako (MW Pure 12,384), Chemicals myoglob (horse(osaka, skeletal Japan). muscle) (MW 17,800), and lysozyme (chicken egg) (MW 13,680) were purchased from Sigma (St. 4-Methylumbelliferyl Louis, MO, USA). Dibasic sugar derivatives potassiumused phosphate as test samples obtaed were purchased from Wakoas Pure follows: Chemicals 4- Methylumbelliferyl-α-L-fucopyranoside (), 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: (), and 4-methylumbelliferyl-β-D-cellobioside () were purchased from BIOSYNTH AG (Staad, 4-Methylumbelliferyl-α-L-fucopyranoside (), 4-methylumbelliferyl-β-D-fucopyranoside (β-d-fuc), 4-methylumbelliferyl-β-D-glucopyranoside (Glc), 4-methylumbelliferyl-β-D-galactopyranoside (), and 4-methylumbelliferyl-β-D-cellobioside () were purchased from BIOSYNTH AG

4 Separations 2016, 3, (Staad, Switzerland). 4-Methylumbelliferyl-α-D-mannopyransoide () obtaed from Wako Pure Chemicals. All or reagents were reagent grade Preparation Two-Phase Solvent Systems and Sample Solutions An ATPS composed 12.5% (w/w) PEG 1000 and 12.5% (w/w) dibasic potassium phosphate prepared by dissolvg 125 g PEG 1000 and 125 g dibasic potassium phosphate (anhydrous) 750 g distilled water. The organic-aqueous two- solvent system used present studies composed ethyl acetate/1-butanol/water (3:2:5, v/v) for separation by lower mobile and (1:4:5, v/v) for upper mobile [18]. Each solvent mixture thoroughly equilibrated a separatory funnel at room temperature. The two s were separated after two layers were formed. The sample solutions were prepared by dissolvg standard sample mixture with equal volumes two- solvent system used for separation CCC Separation Each separation itiated by completely fillg column with, followed by jection sample solution through flow tube leadg to head CCC column by a syrge. Then, mobile pumped to column at a suitable flow rate usg a reciprocatg pump (Model LC-6A, Shimadzu Corporation, Kyoto, Japan), while column rotated at 1000 rpm revolution speed. The effluent from column outlet collected to test tubes usg a fraction collector (Model CHF 100AA, Advantec, Tokyo, Japan) Analysis CCC Fractions Each collected fraction diluted with an aliquot distilled water for protes and methanol for 4-methylumbelliferyl sugar derivatives and absorbance measured at 280 nm for protes, 540 nm for myoglob, and 318 nm for 4-methylumbelliferyl sugar derivatives with a spectrophotometer (Model UV-1600, Shimadzu) Evaluation Theoretical Plate Number and Peak Resolution The efficiencies separations present studies were computed from chromatogram and expressed terms oretical plate number (N) and peak resolution (Rs) accordg to followg conventional formula. 3. Results 3.1. Protes N = ( ) 2 4tR W Rs = 2(t R2 t R1 ) W 1 + W 2 3 illustrates CCC separation stable protes obtaed usg long-pressed multilayer coil with lower mobile 12.5% (w/w) PEG % (w/w) dibasic potassium phosphate system. The decrease flow rate produced better resolution cytochrome C, myoglob, and lysozyme peaks apparently due to creased retention column. Table 1 (upper side) summarizes analytical values calculated from chromatogram shown 3. Remarkable crease peak resolution observed by a lower flow rate

5 ABSORBANCE (280 nm) Separations 2016, 3, Separations 2016, 3, with creased retention while while oretical oretical plate plate number number calculated calculated from from myoglob myoglob peak peak almost almost unchanged. unchanged. A. 0.8 ml/m B. 0.6 ml/m C. 0.4 ml/m 2.0 Cytochrome C Cytochrome C 1.5 Myoglob 0.5 SF SF Myoglob Cytochrome C SF Myoglob TIME (m) 3. CCC separation protes obtaed by long-pressed multilayer coiled columns (without s) s) with with lower lower mobile mobile at threeat different three different flow rates. flow Experimental rates. Experimental conditions: apparatus: conditions: small-scale apparatus: cross-axis small-scale CCCcross-axis with four long-pressed CCC with four long-pressed multilayer coil assemblies, multilayer 1.5 mm coil I.D. assemblies, and 90 ml 1.5 total mm capacity; I.D. and sample: 90 ml total cytochrome capacity; C sample: (2 mg), myoglob cytochrome (8C mg), (2 mg), andmyoglob lysozyme (10 (8 mg); mg), solvent and lysozyme system: ( % mg); (w/w) solvent PEGsystem: % 12.5% (w/w) (w/w) dibasic PEG % potassium phosphate; (w/w) dibasic mobile potassium : lower phosphate; mobile (outward : elution); lower revolution: (outward 1000 rpm; elution); revolution revolution: direction: 1000 counterclockwise; rpm; revolution direction: fractions collected counterclockwise; at 2 m/tube. fractions SF = collected solvent front. at 2 m/tube. SF = solvent front. Table Analytical values obtaed by by CCC separations protes with long-pressed multilayer columns at at three three different flow flow rates. rates. Theoretical Stationary Flow rate Elution volume (ml) Peak resolution (Rs) plate (ml/m) Elution volume (ml) Peak resolution (Rs) plate (ml/m) number (N) retention (%) number (N) retention (%) Lower mobile Lower mobile Cyt C Myo Lys Cyt C/Myo Myo/Lys 0.8 Cyt 70 C Myo 83 Lys 110 Cyt C/Myo 1.3 Myo/Lys Upper 0.6 mobile Lys Myo Lys/Myo Upper mobile Lys Myo 128 Lys/Myo Stationary retention (Sf) 119 calculated accordg to0.8 conventional formula: Sf 387 = (Vc Vm)/Vc} , where Vc and Vm dicate column capacity and retention volume solvent front (volume mobile column), respectively. 126 The plate number 1.2 calculated from 497 myoglob (Myo) peak 17.3 for lower mobile and lysozyme (Lys) peak for upper mobile Stationary retention (Sf) calculated accordg to conventional formula: Sf = (Vc 4 similarly illustrates CCC separation protes obtaed usg long-pressed Vm)/Vc} 100, where Vc and Vm dicate column capacity and retention volume solvent multilayer coil (without s) with upper mobile. The decrease front (volume mobile column), respectively. The plate number calculated from flow rates also improved resolution between lysozyme and myoglob peaks while ir myoglob (Myo) peak for lower mobile and lysozyme (Lys) peak for upper separation times were creased. As summarized Table 1 (lower side), both oretical plate mobile.

6 ABSORBANCE (280 nm, 540 nm ) Separations 2016, 3, similarly illustrates CCC separation protes obtaed usg long-pressed multilayer coil (without s) with upper mobile. The decrease flow Separations 2016, 3, rates also improved resolution between lysozyme and myoglob peaks while ir separation times were creased. As summarized Table 1 (lower side), both oretical plate number lysozyme peak and retention were creased by by decreased flow flow rate rate upper upper mobile.. A. 0.8 ml/m B. 0.6 ml/m C. 0.4 ml/m SF Myoglob Myoglob 0.5 SF 0.5 SF Myoglob TIME (m) CCC CCC separation separation protes protes obtaed obtaed by by long-pressed long-pressed multilayer multilayer coiled coiled columns columns with with upper uppermobile mobile at three at three different different flow flow rates. rates. Experimental Experimental conditions: conditions: apparatus: apparatus: smallscale small-scale cross-axis cross-axis CCC with CCC four with long-pressed four long-pressed multilayer multilayer coil assemblies, coil assemblies, 1.5 mm I.D. 1.5 and mm 90 ml I.D. total and 90 capacity; ml total sample: capacity; lysozyme sample: (10 lysozyme mg) and (10 myoglob mg) and (8 myoglob mg); solvent (8 mg); system: solvent 12.5% system: (w/w) 12.5% PEG % (w/w) PEG % (w/w) dibasic (w/w) potassium dibasic phosphate; potassium phosphate; mobile : mobile upper : upper (ward elution); (ward revolution: elution); revolution: 1000 rpm; 1000 revolution rpm; revolution direction: clockwise; direction: fractions clockwise; collected fractions at 2 collected m/tube. at SF 2 m/tube. = solvent front. SF = solvent front Sugar Sugar Derivatives Derivatives In In order order to to achieve achieve furr furr crease crease peak peak resolution resolution prote prote separation, separation, a serted serted to to locule locule long-pressed long-pressed tubg tubg as as shown shown 2D. 2D. The The clusion clusion a to to locule locule expected expected to to enhance enhance mixg mixg two two s s rotatg rotatg column. column. However, However, at at flow flow rate rate and and ml/m, ml/m, retention retention substantially substantially decreased decreased along along with with peak peak resolution. resolution. At At flow flow rate rate ml/m, ml/m, no no retaed retaed column. column. It It seems seems that that enhances enhances mixg mixg but but does does not not allow allow enough enough settlg settlg to to provide provide for for significant significant retention. retention. It predicted It predicted that improved that separation improved separation would be obtaed would usg be obtaed an organic-aqueous usg an organic-aqueous two- solvent two- system with solvent higher system terfacial with tension higher terfacial than ATPS. tension than 5 illustrates ATPS. CCC separation 5 illustrates five 4-methylumbelliferyl CCC separation sugar derivatives five 4- methylumbelliferyl obtaed usg sugar embedded derivatives obtaed multilayer usg coil with embedded lower mobile. multilayer While coil retag with satisfactory lower mobile volume. While retag CCC satisfactory separation, volume faster flow rate revealed basele CCC separation, peak resolution faster between flow rate revealed sugar derivative basele peak peak. resolution At flow between rate 1.4 sugar ml/m, derivative five sugar peak. At derivatives flow rate were separated 1.4 ml/m, almost five sugar 150 m derivatives with basele were separated peak resolution. almost 150 m with basele peak resolution.

7 ABSORBANCE ABSORBANCE (318 (318 nm) nm) ABSORBANCE ABSORBANCE (318 (318 nm) nm) Separations 2016, 3, Separations 2016, 3, Separations 2016, 3, A. ml/m B. 1.2 ml/m C. 1.4 ml/m A. ml/m B. 1.2 ml/m 16 C. 1.4 ml/m Glc 10 Glc Glc 108 Glc β-d-fuc Glc 6 β-d-fuc 86 β-d-fuc β-d-fuc SF SF SF SF SF SF TIME (m) TIME (m) 5. CCC 5. CCC separation 4-methylumbelliferyl sugar derivatives obtaedby by embedded multilayer coiled with at three different flow rates. multilayer 5. CCC separation coiled columns 4-methylumbelliferyl with lower sugar mobile derivatives obtaed at three by different embedded flow rates. Experimental Experimental conditions: apparatus: CCC with four embedded multilayer conditions: coiled apparatus: columns with small-scale lower cross-axis mobile CCC with at three four different embedded flow rates. multilayer coil assemblies, 1.5 mm I.D. and 90 ml total capacity; sample: 4-methylumbelliferyl multilayer Experimental coil assemblies, conditions: apparatus: 1.5 mm I.D. small-scale and 90 cross-axis ml total CCC capacity; with four sample: 4-methylumbelliferyl embedded derivative β-d-cellobioside (1 mg), β-d-glucopyranoside (1 mg), α-d-mannopyranoside (1 mg), β- derivative multilayer coil β-d-cellobioside assemblies, 1.5 mm (1 mg), I.D. and β-d-glucopyranoside 90 ml total capacity; (1sample: mg), 4-methylumbelliferyl α-d-mannopyranoside D-fucopyranoside (1 mg), and α-l-fucopyranoside (1 mg); solvent system: ethyl acetate/1- (1 mg), derivative β-d-fucopyranoside β-d-cellobioside (1 mg), β-d-glucopyranoside and α-l-fucopyranoside (1 mg), α-d-mannopyranoside (1 mg); solvent system: (1 mg), β- ethyl butanol/water (3:2:5, v/v); mobile : lower (outward elution); revolution: 1000 rpm; acetate/1-butanol/water D-fucopyranoside (1 mg), (3:2:5, and v/v); α-l-fucopyranoside mobile : (1 lower mg); solvent (outward system: elution); ethyl acetate/1- revolution: revolution direction: counterclockwise; fractions collected at 2 m/tube. SF = solvent front. 1000butanol/water rpm; revolution (3:2:5, direction: v/v); mobile counterclockwise; : lower fractions (outward collectedelution); at 2 m/tube. revolution: SF = 1000 solvent rpm; front. revolution direction: counterclockwise; fractions collected at 2 m/tube. SF = solvent front. 6 illustrates CCC separation three 4-methylumbelliferyl sugar derivatives obtaed usg 6 illustrates embedded CCC multilayer coil with upper mobile. sugar Increased derivatives flow obtaed rates 6 illustrates CCC separation three 4-methylumbelliferyl sugar derivatives obtaed usgproduced shorter separation times with 100 m at flow rate 1.4 ml/m. where Increased three flow sugar embedded multilayer coil with upper mobile. Increased flow rates rates produced derivatives shorter basele separated from or at peak resolution ratio 1.4 over 2.0 as where summarized three sugar produced shorter separation times with 100 m at flow rate 1.4 ml/m where three sugar Table 2. derivatives basele separated from or at peak resolution ratio over over as summarized as Table Table A. ml/m B. 1.2 ml/m C. 1.4 ml/m Glc β-d-fuc β-d-fuc A. ml/m B. 1.2 ml/m 10 C. 1.4 ml/m SF 4 2 SF 4 2 SF 2 SF 2 SF 2 SF TIME (m) TIME (m) 6. CCC separation 4-methylumbelliferyl sugar derivatives obtaed by embedded multilayer coiled columns with upper mobile at three different flow rates. 6. CCC 6. CCC separation 4-methylumbelliferyl sugar derivatives obtaed by by embedded Experimental conditions: apparatus: small-scale cross-axis CCC with four embedded multilayer coiled columns with upper mobile at at three three different different flow flow rates. rates. multilayer coil assemblies, 1.5 mm I.D. and 90 ml total capacity; sample: 4-methylumbelliferyl Experimental derivative conditions: α-l-fucopyranoside apparatus: (1 mg), small-scale CCC with four embedded β-d-galactopyranoside cross-axis CCC (1 with mg) and four β-d-cellobioside embedded (1 mg); multilayer multilayer coil assemblies, 1.5 mm I.D. and 90 sample: 4-methylumbelliferyl solvent coil system: assemblies, ethyl acetate/1-butanol/water 1.5 mm I.D. and (1:4:5, 90 mlv/v); total mobile capacity; : sample: upper 4-methylumbelliferyl (ward elution); derivative α-l-fucopyranoside (1 mg), β-d-galactopyranoside (1 mg) and β-d-cellobioside (1 mg); derivative revolution: α-l-fucopyranoside 1000 rpm; revolution (1direction: mg), β-d-galactopyranoside clockwise; fractions collected (1 mg) and at 2 m/tube. β-d-cellobioside SF = solvent (1 mg); solvent system: ethyl acetate/1-butanol/water (1:4:5, v/v); mobile : upper (ward elution); solvent front. system: ethyl acetate/1-butanol/water (1:4:5, v/v); mobile : upper (ward revolution: 1000 rpm; revolution direction: clockwise; fractions collected at 2 m/tube. SF = solvent elution); revolution: 1000 rpm; revolution direction: clockwise; fractions collected at 2 m/tube. front. SF = solvent front.

8 Separations 2016, 3, Table 2. Analytical values obtaed by CCC separations 4-methylumbelliferyl sugar derivatives with embedded multilayer coiled column at three different flow rates. Flow rate (ml/m) Lower mobile Elution volume (ml) Peak resolution (Rs) Theoretical plate number (N) Stationary retention (%) Glc β-d-fuc /Glc Glc/ / β-d-fuc β-d-fuc/ Upper mobile / / Stationary retention (Sf) calculated accordg to conventional formula: Sf = {(Vc Vm)/Vc} 100, where Vc and Vm dicate column capacity and retention volume solvent front (volume mobile column), respectively. The oretical plate number calculated from 4-methylumbelliferyl β-d-glucopyranoside (Glc) peak for lower mobile and 4-methylumbelliferyl β-d-galactopyranoside () peak for upper mobile. 4. Discussion The cross-axis CCC is useful for separation hydrophilic and hydrophobic compounds usg both aqueous-aqueous and organic-aqueous two- solvent systems at a wide range hydrophobicity. In present study, improved separations protes were performed usg long-pressed tubg without s and basele separations 4-methylumbelliferyl sugar derivatives were achieved usg embedded tubg for small-scale cross-axis CCC. As shown 3, sufficient partitiong protes at resolution over 1.2 between aqueous two immiscible s rotatg column achieved even at a relatively high flow rate lower mobile 0.8 ml/m, while separation furr creased by a lower flow rate but with a longer separation time. 4 also suggested that more sufficient partitiong protes at resolution over 1.5 is accomplished at a lower flow rate 0.4 ml/m with upper mobile. As summarized Table 1, retention is versely proportional to flow rate. Decreased flow rate mobile produced crease retention and peak resolution. In general, higher N value does not follow higher Rs values. Faster flow rate decreases separation time analyte due to decrease retention. As described conventional formula, apparently both retention time and W decrease at faster flow rates. If N creases or decreases depend on ir rate decrease, smaller W value calculates higher N value while all analytes elute with shorter separation time. Usg organic-aqueous two- solvent system with lower mobile, 4-methylumbelliferyl sugar derivatives were basele separated from or at high oretical plate number obtaed from second β-d-glucopyranoside peak ( 5). The results obtaed usg upper mobile shown 6 also appear similar to those obtaed usg lower mobile, dicatg that a serted to locule promotes partitiong analytes between two s. As described above, creased flow rate generally decreases retention and peak resolution. However, present studies, basele separations at ir resolutions almost over 1.5 were achieved by faster flow rates whereas retention gradually decreased as summarized Table 2. In a series se experiments, long-pressed tubg serted with a to every or locule and long-pressed tubg serted with a to every third locule were also applied to similar separations 4-methylumbelliferyl sugar derivatives with almost equivalent column volumes. Better peak resolution with shorter separation time obtaed usg long-pressed tubg serted with a to locule than or two kds embedded tubg. This suggests that s creased mixg at

9 Separations 2016, 3, higher flow rates which compensated for decrease retention. Similar studies were furr performed usg ATPS for prote separation. Better peak resolution and retention obtaed long-pressed tubg without sertg with a to locule more than three kds embedded tubg, which different from result obtaed usg organic-aqueous two- solvent systems with present small-scale cross-axis CCC strument. The long-pressed tubg and embedded tubg used present CCC strument successfully contributed to improvement CCC separation. It suggests that compressed portion tubg creases lear velocity mobile to enhance mixg two s locule. 5. Summary The cross-axis CCC can be used for effective separation and purification various compounds at a wide range hydrophobicity. Improved separation is accomplished usg long-pressed multilayer coil for highly hydrophilic compounds such as protes with ATPS, and long-pressed multilayer coil serted with a to locule for hydrophobic compounds with organic-aqueous two- solvent systems. Acknowledgments: The authors would like to thank Motoki Umezawa, ami Seki, and Jun Nitta for ir technical assistance. Author Contributions: Kazufusa Shomiya designed, conducted, performed experiments present study and wrote paper; Kazumasa Zaima and Naoki Harikai analyzed data; Yoichiro Ito discussed about data and checked paper. Conflicts Interest: The authors declare no conflict terest. References 1. Ito, Y. Prciples and Instrumentation Countercurrent Chromatography. In Countercurrent Chromatography: Theory and Practice; dava, N.B., Ito, Y., Eds.; Marcel Dekker: New York, NY, USA, 1988; pp Conway, W.D. The evolution Countercurrent Chromatography. In Countercurrent Chromatography: Apparatus, Theory & Applications; VCH: Weheim, Germany, 1990; pp Ito, Y. Prciple, Application, and Methodology High-Speed Countercurrent Chromatography. In High-Speed Countercurrent Chromatography; Ito, Y., Conway, W.D., Eds.; Wiley-Interscience: New York, NY, USA, 1996; pp Ito, Y.; Menet, J.-M. Coil Planet Centrifuge for High-Speed Countercurrent Chromatography. In Countercurrent Chromatography; Menet, J.-M., Thiebaut, D., Eds.; Marcel Dekker: New York, NY, USA, 1999; pp Ito, Y. Cross-axis synchronous flow-through coil planet centrifuge free rotary seals for preparative countercurrent chromatography. Part I. Apparatus and analysis acceleration. Sep. Sci. Technol. 1987, 22, [CrossRef] 6. Ito, Y. Cross-axis synchronous flow-through coil planet centrifuge free rotary seals for preparative countercurrent chromatography. Part II. Studies on distribution and partition efficiency coaxial coils. Sep. Sci. Technol. 1987, 22, [CrossRef] 7. Shomiya, K.; Muto, M.; Kabasawa, Y.; Fales, H.M.; Ito, Y. Prote separation by improved cross-axis coil planet centrifuge with eccentric coil assemblies. J. Liq. Chromatogr. Relat. Technol. 1996, 19, [CrossRef] 8. Shomiya, K.; Yanagidaira, K.; Ito, Y. New small-scale cross-axis coil planet centrifuge: The design apparatus and its application to counter-current chromatographic separation protes with aqueous-aqueous polymer systems. J. Chromatogr. A 2006, 1104, [CrossRef] [PubMed] 9. Shomiya, K.; Kobayashi, H.; Inokuchi, N.; Kobayashi, K.; Oshima, H.; Kitanaka, S.; Yanagidaira, K.; Sasaki, H.; Muto, M.; Okano, M.; et al. New small-scale cross-axis coil planet centrifuge: Partition efficiency and application to purification bullfrog ribonuclease. J. Chromatogr. A 2007, 1151, [CrossRef] [PubMed]

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